WI0800475_DEEMED FILES_20170203

Permit Numbei·
Program Category
Deemed Ground Water
Permit Type
WI0800475
Injection Deemed In-situ Groundwater Remediation Well
Primary Reviewer
shristi.shrestha
Coastal SWRule
Permitted Flow
Facility
Facility Name
GoGas#2
Location Address
1116 S College Rd
Wilmington
Owner
Owner Name
Quality Oil Company LLC
Dates/Events
NC
Orig Issue
2/2/2017
App Received
1/26/2017
Re g ulated Activities
Groundwater remediation
Outfall
Waterbody Name
28403
Draft Initiated
Scheduled
Issuance Public Notice
Central Files: APS SWP
2/3/2017
Permit Tracking Slip
Status
Active
Version
1.00
Project Type
New Project
Permit Classification
Individual
Permit Contact Affiliation
Major/Minor
Minor
Region
Wilmington
County
New Hanover
Facility Contact Affiliation
PO Box2736
Winston Salem
Owner Type
Non-Government
Ow_ner Affiliation
Bradley Snover
PO Box2736
Winston Salem
Issue
2/2/2017
Effective
2/2/2017
NC 27102
NC 27102273
Expiration
Requested /Received Events
Streamlndex Number Current Class Subbasin
Shrestha, Shristi R
From:
Sent:
To:
Cc:
Subject:
Attachments:
Importance:
Dr. Rudo,
Shrestha, Shristi R
Thursday, February 02, 2017 11:43 AM
Rudo, Ken
King, Morella s; Gregson, Jim; Rogers, Michael
WI0800475 Go Gas #2Fw: Progress Environmental NOi and Sampling Plan
Progress Env_2017 GoGas #2_In-Situ GW Remediation Notification.pdf; Progress
Env_GoGas#2_2017 Sampling Plan.pdf
High
Please find the attached application for approval of new additive to inject underground. I am forwarding this
to you on mike's behalf.
Best Regards,
Shristi
Shristi R. Shrestha
Hydro geologist
Water Quality Regional Operations Section
Animal Feeding Operations & Groundwater Protection Branch
North Carolina Department of Environmental Quality
919 807-6406 office
[[e-mail]][e-mail]
512N . Salisbury Street
1636 Mail Service Center
Raleigh, NC 27699 1636
£::mail correspondence to and from this address is subject to the
North Carolina Public Records Law and may be disclosed to third parti~s.
From: Rogers, Michael
Sent: Wednesday, January 25, 2017 3:10 PM
To: Shrestha, Shristi R
Subject: FW: Progress Environmental NOi and Sampling Plan
FYI.
From: Jay Lawson [mailto:[e-mail]]
Sent: Tuesday, January 24, 2017 4:40 PM
To: Rogers, Michael <[e-mail]>; Rudo, Ken <[e-mail]>
Cc: [e-mail]
Subject: Progress Environmental NOi and Sampling Plan
Importance: High
Gentlemen:
I have attached a Notification of Intent (NOi) and associated Sampling Plan per our conversation in October. The site
is located in a developed area of Wilmington serviced by municipal water (no water supply wells). Please let us know
if you have an questions.
I will also submit hard copies. Should I send everything to the DWR-UIC Program address?
Just let me know.
Thanks for your consideration!
Jay Lawson
Director oi' Ecoiof,ica l Serviws
Pro gre::.s Enviro nmental, Inc:.
P.O. Box 5884
Winston-Salem, NC 27113-58R4
Office 3.36. 722 . 9999
Fax [phone]
Sh r estha, Shristi R
From:
Sent:
To:
Cc:
Subject:
Shrestha, Shristi R
Thursday, February 02, 2017 11:39 AM
Rogers, Michael; [e-mail]; Rudo, Ken;
[e-mail]
King, Morella s; Gregson, Jim
WI0800475 NOi Go Gas #2 Re : Progress Environmental NOi and Sampling Plan
Thank you for submitting the Notice of Intent to Construct or Operate Injection Wells (NOi} for the above referenced
site. Your application has been given a UIC tracking number of WI0800475 and has been forwarded to DHHS for
approval of the new additive.
Please remember to submit the following regarding this injection activity:
1) Well Construction Records (GW-1) and Abandonment Records (GW-30) when completed. Please provide copies
of the GW-ls and GW-30s if not already submitted (originals go the address printed on the form). NOTE: Direct
push or Geoprobe wells are considered wells and require construction (GW-1) and abandonment forms (GW-
30). If well construction/abandonment information is the same for the wells, only one form needs to be
completed-just indicate total number of injection points in the Comments/Remarks section of form. These forms
can be found on our website at
http :ljde q .nc.gov/about/divisions /water-resources /water-resources-permits/wastewater-branch /ground-water-
p rotection /g round-water-re porting-forms
2) Injection Event Records (IER). All injections, including air and passive systems require an IER. The IER can be
modified for air sparge wells (e .g., air flow 'continuous' for date or rate of injection, etc.).
You can scan and send these forms directly to me at Shristi.shrestha @ncdenr.gov or via regular mail to address
below. When submitting the above forms, you will need to enter the nine-digit alpha -numeric number on the form
(i.e., WIOXXXXXX) that has been assigned to the injection activity at this site. This number is also referenced in the
subject line of this email. You may if you wish, scan and send back as attachments in re p ly to this emai l, as it will
already have the assigned deemed permit number in the subject line.
Thank you for your cooperation .
Shristi
Shrlsti R. Shrestha
Hydrogeologist
Water Quality Regional Operations Section
Animal Feeding Operations & Groundwater Protection Branch
North Carolina Department of Environmental Quality
919 807-6406 office
[shristi [e-mail]][e-mail]
512N. Salisbury Street
1636 Mail Service Center
Raleigh, NC 27699 1636
Email correspondence to and from this address is subject to the
/1.inrlh ~:::,rnNn,::, P11h/i,-. Q,:,,..r,r,-1., I !:>IA/ :::inrl rn,:w ha ,-lj.,,-./r,<,ar/ fr, fhirrl n::.rfiac,
From: Jay Lawson [mailto:jlawson @p rog ressenv.com]
Sent: Tuesday, January 24, 2017 4:40 PM
To: Rogers, Michael <michael.rogers @ncdenr.gov>; Rudo, Ken <ken.rudo @dhhs.nc.gov>
Cc: [e-mail]
Subject: Progress Environmental NOi and Sampling Plan
Importance: High
Gentlemen:
I have attached a Notification of Intent (NOi) and associated Sampling Plan per our conversation in
October. The site is located in a developed area of Wilmington serviced by municipal water (no water
supply wells). Please let us know if you have an questions.
I will also submit hard copies. Should I send everything to the DWR-UIC Program address?
Just let me know .
Thanks for your consideration!
Jay Lawson
Director of Ecological Services
Progress Environmental, Inc.
P. 0. Box 5884
Winston-Salem, NC 27113-5884
Office 336. 722.9999
Fax [phone]
North Carolina Department of Environmental Quality-Division of Water Resources
NOTIFICATION OF INTENT (NOi) TO CONSTRUCT OR OPERATE INJECTION WELLS
The following are ''permitted by rule" and do not require an individual permit when constructed in accordance
with the rules of 15A NCAC 02C .0200. This fo rm shall be submitted at least 2 WEEKS prior to in iection.
AQUIFER TEST WELLS f1 5A NCAC 02C .0220 )
These wells are used to inject uncontaminated fluid into an aquifer to determine aquifer hydraulic characteristics.
IN SITU REMEDIATION (1 5A NCAC 02C .0225 ) or TRACER WELLS (1 5A NCAC 02c .0229):
1) Passive Injection S vstems -In-well delivery systems to diffuse injectants into the subsurface. Examples include
ORC socks, iSOC systems, and other gas infusion methods.
2) Small-Scale In jection O perations -Injection wells located within a land surface area not to exceed 10,000
square feet for the purpose of soil or groundwater remediation or tracer tests. An individual permit shall be required
for test or treatment areas exceeding 10,000 square feet.
3) Pilot Tests -Preliminary studies conducted for the purpose of evaluating the technical feasibility of a
remediation strategy in order to develop a full scale remediation plan for future implementation, and where the
surface area of the injection zone wells are located within an area that does not exceed five percent of the land
surface above the known extent of groundwater contamination. An individual permit shall be required to conduct
more than one pilot test on any separate groundwater contaminant plume.
4) Air Injection Wells -Used to inject ambient air to enhance in-situ treatment of soil or groundwater.
Print Clearly or Type Information. Illegible Submittals Will Be Returned As Incomplete.
DATE: Januarv 24 , 2017 PERMIT NO. w 1D g 004:?Js=cto be filled ~e~nWR)
IVED/NCDEQlDWR
A. WELL TYPE TO BE CONSTRUCTED OR OPERATED JAN J,6 2017
(1)
(2)
(3)
(4)
(5)
(6)
___ .Air Injection Well ...................................... Complete sections B through F, K, W ater Quality
___ .Aquifer Test Well ....................................... Complete sections B throug:~~paraticins S~®
___ .Passive Injection System ............................... Complete sections B through F, H-N
___ .Small-Scale Injection Operation ...................... Complete sections B through N
X Pilot Test ................................................. Complete sections B through N
___ Tracer Injection Well ................................... Complete sections B through N
B. STATUS OF WELL OWNER: Business/Organization
C. WELL OWNER(S) -State name of Business/Agency, and Name and Title of person delegated authority to
sign on behalf of the business or agency:
Name(s): Ouali tv Oil Com panv. LLC
Mailing Address: P.O. Box 2736
City: Winston-Salem State: NC Zip Code:_2=-7""'1'""0=2 ____ County: Forsyth
Day Tele No.: (336 ) 721-9515 Cell No.: --"""''3'""3~6_._) -'-70""'6'--4-'-7'"'"4"""6 __ _
EMAIL Address: dmciver@q ocnc.com Fax No.: __ .,.{3"""3"""'6_._) -'-7 =-14'--.a..al 6"""8"""0 __ _
Deemed Permitted GW Remediation NOi Rev. 3-1-2016 Page 1
D. PROPERTY OWNER(S) (if different than well owner)
Name and Title: -----'S"-e=e'--'a=b=o'-'v-=-e __________________________ _
Company Name---------------------------------
Mailing Address:---------------------------------
City: _____________ State: __ Zip Code: _______ County: _____ _
DayTeleNo.: ___________ _ Cell No.: _________ _
EMAIL Address: _____________ _ Fax No.: ___________ _
E. PROJECT CONTACT (Typically Environmental Engineering Firm)
Name and Title: -----'J'""'a-)~L=a~w~s'""'o=n~. D~ir'""'e~ct=o~r --=o~f =E=c~ol=o .... g=ic=a~l =S~er'""'v--'-ic=e=s ______________ _
Company Name ---=P~ro ___ gr...,, =es=s~E=n=v=ir=o=nm==e=n=ta=l~. In=c~. ___________________ _
Mailing Address: --~P~.O=.'--'B~o=x~5~8=8~4 _______________________ _
City: Winston-Salem State: __lK;,_ Zip Code:27113 County:--=F-"o""rs"'-'-"th"'--__ _
Day Tele No.: 336-722-9999 Cell No.: 336-782-9726
EMAIL Address: ___ ,=.ila"-w~so=n=·=pr""'o""g"'"re=s=se=n=v""".c""o=m=---Fax No.: 336-722-9998
F. PHYSICAL LOCATION OF WELL SITE
(I) Facility Name & Address: ___,G=o=G=a=s-"#=2 __ ___,a_l l"""'l'""6""'S=o=u=th"'--=C=o=lle""'L""''e'""'R"'"o=a=d'--------------
City: ___ W-'-'-"il=m=i=n=s:t=o=n'---_______ County: New Hanover Zip Code: =28"-4=0=--3 __ _
(2) Geographic Coordinates: Latitude**: ---=34_,_0 _____ll' 52.45" or 0
Longitude**: 77° TI_' 10.92 "or 0
Reference Datum: ________ Accuracy: ________ _
Method of Collection:_G_o_o-g-le_E_a_rt_h _________ _
* *FOR AIR INJECTION AND AQUIFER TEST WELLS ONLY: A FACILITY SITE MAP WITH PROPERTY
BOUNDARIES MAY BE SUBMITTED IN LIEU OF GEOGRAPHIC COORDINATES.
G. TREATMENT AREA
Land surface area of contaminant plume:---"l--'-1.,_.4'""'1=2'---___ square feet
Land surface area ofinj. well network: 314.2 square feet(::: 10,000 ft 2 for small-scale injections)
Percent of contaminant plume area to be treated: 2.75 (must be:::_ 5% of plume for pilot test injections)
H. INJECTION ZONE MAPS -Attach the following to the notification.
(I) Contaminant plume map(s) with isoconcentration lines that show the horizontal extent of the
contaminant plume in soil and groundwater, existing and proposed monitoring wells, and existing and
proposed injection wells; and
(2) Cross-section(s) to the known or projected depth of contamination that show the horizontal and
vertical extent of the contaminant plume in soil and groundwater, changes in lithology, existing and
proposed monitoring wells, and existing and proposed injection wells.
(3) Potentiometric surface map(s) indicating the rate and direction of groundwater movement, plus
existing and proposed wells.
Deemed Permitted GW Remediation NOi Rev. 3-1-2016 Page2
I. DESCRIPTION OF PROPOSED INJECTION ACTIVITIES -Provide a brief narrative regarding the
purpose, scope, and goals of the proposed injection activity. This should include the rate, volume, and
duration of injection over time.
In an effort to facilitate de gradation of persistent dissolved-phase petroleum constituents at the site .
Pro gr ess pro poses to in ject a bioremedial product consistin g of several non-patho genic. naturall v
occurring. non-genetically modified strains of Bacillus s pp . The bioremedial product will be introduced
into existin!!. monitoring well MW-6 via sus pension within a five-foot. one inch Schedule 40 PVC pi pe
with 0.10 slotted screen. A small amount of sugar {a pp roximatel y 1-2% of pi pe volume ) will be added to
the PVC pi pe in order to stimulate microbial activitv. Monitoring well MW-6 has exhibited persistent
dissolved-phase petroleum gr oundwater contamination. The e:oal is to introduce microbes into the
contaminant plume to facilitate petroleum de gr adation. Pro gress will collect a e.roundwater sam ple from
monitoring well MW-6 prior to . and a pp roximate} 30 da s followin i!. in jection activities in order to
evaluate the effectiveness of the remedial product on dissolved-phase petroleum constituents and
influence on bacteriologi cal po pulations under field conditions. The gr oundwater samples will be
anal yzed for volatile organic com pounds (VOCs ) b Standard Method 6200B includin g ethanol. meth yl
tert-butv l ether (MTBE ). and iso pro pv l ether (IPE ). In addition . Pro gr ess will collect groundwater
sam ples for anal vsis via heterotro phic plate count {H PC}. This procedure is used for estimating the
number oflive culturable heterotro phic bacteria in water. Colonies mav arise from pairs . chains . clusters .
or single cells . all of which are included in the term "colony-formin g units" {CFU). Progr ess will collect
geochemical data durin g sam pling activities including measurements of pH . tem perature. conductivit v,
and dissolved ox gen. Pro gress will conduct additional sam pling events (p er above ) at a pp roximate! 30-
day intervals. Sam ple results will be com pared to existing, irr oundwater standards . as a pp ro priate.
J. APPROVED INJECT ANTS -Provide a MSDS for each injectant. Attach additional sheets if necessary.
NOTE: Only injectants approved by the NC Division of Public Health, Department of Health and Human
Services can be iryected. Approved injectants can be found online at htt p://de g.nc.gov/about/divisions/water-
resources/water-resources-permits/wastewater-branch/ ground-water-protection/ ground-water-a pproved-in j ectants.
All other substances must be reviewed by the DHHS prior to use. Contact the UIC Program for more info
(919-807-6496).
Injectant: -----"B=i=o---'-C=a=t'-"M=ic=r=ob=i=a=ls'---'P'--'r'""o.:.Qr""'e=s=s =B=le=n=d=------'-l -"-1-=S=tr=a=in-=------------
Volume of injectant: __ 0=·=2-=g,.,a=ll=on=s"'-------------------------
Concentration at point of injection: -"-1 =lB=--c=C=F'---'U=/-<=g~-------------------
Percent if in a mixture with other injectants: ___________________ _
Injectant: ___ G~r~a~n~u~la~te~d_S~u~g...,_,ar~------------------------
Volume of injectant: __ S~e~e~a=b~o~v~e _______________________ _
Concentration at point of injection:
Percent if in a mixture with other injectants: --~l~-2=o/c~o _______________ _
Injectant: ---------------------------------
Volume ofinjectant: ____________________________ _
Concentration at point of injection:
Percent if in a mixture with other injectants: ___________________ _
K. WELL CONSTRUCTION DATA
(1) Number of injection wells: --~N~/A~~Proposed 1 (MW-6 ) Existing (provide GW-ls)
Deemed Permitted OW Remediation NOI Rev. 3-1-2016 Page3
(2) For Proposed wells or Existing wells not having GW-1 s, provide well construction details for each
injection well in a diagram or table format. A single diagram or line in a table can be used for
multiple wells with the same construction details. Well construction details shall include the
following (indicate if construction is proposed or as-built):
(a) Well type as permanent, Geoprobe/DPT, or subsurface distribution infiltration gallery
(b) Depth below land surface of casing, each grout type and depth, screen, and sand pack
(c) Well contractor name and certification number
Deemed Permitted GW Remediation NOi Rev.3-1-2016 Page4
L. SCHEDULES - Briefly describe the schedule for well construction and injection activities.
Monitoring well MW-6 was _previously installed. Prot Tess will conduct injection activities within
approximately two weeks following approval.
M. MONITORING PLAN -Describe below or in separate attachment a monitoring plan to be used to determine
if violations of groundwater quality standards specified in Subchapter 02L result from the injection activity.
Plan attached
N. SIGNATURE OF APPLICANT AND PROPERTY OWNER
APPLICANT: '?hereby certii51. under penalty of law, that I am familiar with the information submitted in
this document and all attachments thereto and that, based on my inquiry of those individuals immediately
responsible for obtaining said information. I believe that the information is true, accurate and complete. I am
aware that there are significant penalties, including the possibility of fines and imprisonment for submitting
false information. I agree to construct, operate. maintain, repair, and if abandon the injection well
and, 1l related appurtenances in accordance with the ISA NCAC 02C 0200 Rules."
�,_ � '�.•- ,�jcleft( 5- 1.•ci.wSc,A 't BsC lrtv..cfvi s-e5
sit4atnre of pplleant Print or Type Full Name and Title
PROPERTY OWNER [if the property is not owned by the permit applicant):
"As owner of the property on which the injection well(s) are to be constructed and operated, I hereby consent
to allow the applicant to construct each injection well as outlined in this application and agree that it shall be
the responsibility of the applicant to ensure that the injection well(s) conform to the Well Construction
Standards (15A NCAC 02C.0200).'•
"Owner" means any person who holds the fee or other property rights in the well being constructed. A
well is real property and its construction on land shall be deemed to vest ownership in the land owner, in
_he absence of contra agreement in writing.
A/ ‘1\i‘J. tlt",A, Id) 11 C'Z j 56.4, %if
Signature* of Property Owner (if different from applicant) Print or Type Full Name and Title
*An access agreement between the applicant and property owner may be submitted in lieu of a signature on this form.
Submit the completed notification package to:
DWR - LTIC Program
1636 Mail Service Center
Raleigh, NC 27699-1636
Telephone: [phone]
Deemed permitted GW Remediation NO1 Rev. 3-3-2015
Page 5

P.O. Box 5884
Winston-Salem, NC 27113
Telephone: [phone]
Fax:[phone]
wine vrovessenvrranmenrul,cam
Progress
Figure 1
Topographic Site Map
Go Gas #2
1116 South College Road
Wilmington, New Hanover County, North Carolina
USGS
United States Department of the Interior
USGS 7.5 Minute 5enes Topographic Map
Contour Interval: 10 feel
Scale: 1 "=2000'
Wilmington, North Carolina
Date: 2016
Project: Go Gas #2
Client:
Progress Job #:
Date: January 20117
111

;AV 71ns1 r;:
5
My
DRAW
MW-7
/ 1 !'GC
� 7R.HURN!
_ .........=1.6.M:in 1wiTH— 10W—;
�tI ZWORt. RJ G4i PO NSA a IrF1N.: iO4940 {T J
j
AQ'w. • PA11 T / ��
,i 0
3
rr
OW 4
JAN 2017
JSL 441•4 7.017
As SHOWN
Ai MAK
VA 11
how 6
Q8 m
AS—'
BMW N
Inz
SOUTH COLLEGE RD
Progress 4110.
ENVIRONMENTAL, INC.
P.O. 0o.. 5334
WNSTON-SALEM, NORTH CARCIJ NA 27113
❑14ONE 336.7220030 FAX 333.722.900E
FIGURE 2
SITE MAP
1116 SOUTH COLLEGE ROAD
WIL M INGTON, NORTH CAROLINA

ANY/ T1- AS_ t-43
sc A
• 30'
8MO'i0RWi 0CA1f'4
+1111,— r.ROJNDAPATFR Fl.CW
PR[' ON
—911 PO I Ve ZAL- 3!=
CONTc_l
(46.04 GROLNJWA R
E_JIVA*$054 (Pt)
WAS
,7J
07.7$
z
iFealelf
91.
'kW - 3
mug. Km UTE* i OVA)
°A•101,4% I
IZ+
' ! .451,A1: OriVillbil Vi- 4 4_`,'
i f17.115
0 i
'I.' AREA VII
•,,,, .'"1., 1
..,.
, 1 el
....'
i
711
Cr PG.
•
Cf... 'fr.: VT.. , )
.116"04 4,4X110 %, •
• .M.111 •••••••• •••=••••••• •!• 111.• mar • ILEI 1.11 • 1••••• •
Z014417£ LC
SCL. "Li CO, GI
?War
&REM JAB
IAN .2017
1 l' ,JAN 2017
SHOW./ I
Progress
ENNA RONIV1ENTAL, INC_
P.O. Box SSE14.
WINSTON-SALEM, NORTH C.AROLINA 27113
PHONE 336.722, WOE FAX [phone]
FIGURE 3
GROUNDIAATER FLOVV MAP
1116 SOUTH COLLEGE ROAD
VVILMINGTON, NORTH CAROLINA

J
C)
C ;u
I
~
:ii:
Ai
0 :r:
·-!
V} I < I
f17
)>
~
.... EG[\J
!,.;,"t~; ____ . =
o· ':>' ;so·
8 MOt..!,-OR WE. LOCA."!O"l
(158)
a:x.
8E...,Zf"IC COf\CEN-
TRAl CN (ug/c)
Bt.NZ::J,1:. N{H
DCTECTI:D
.--51<--GC, OR ,,:Nt f\
'ug/ l
---·-;,iv,· 2 8 (rla) t/;{"i.t';;}" ~ -< '"' J~
~ y.,b.,:,.,.,,.. '
[I! ~--·~·-·-·-:n-w-;·-rqw::i·-·-·-·-·-·-·-·-·-·-·-·i-,:_.-;:. . .=...:;;;_~~~~%A_._ ~ • ,.'.!Ll>J .tB .... ,,/ol MV.-l :::::=-·-·-·-· ··· -· 1 ~i_ f MW '1 f ;Q\~ ~ (..t.S !-'Oil" ;,rc..;, ~ -· ._ ~J<.:.R~ ..iJ!Ul I{(: r•t, .,. \ "'" 2
~· • {r/~, 1<•./;;) "'I ( !i 1 i L ............J !::: ~
• : j i . ! '
1
1_ /, _.,_,. "ALI :\"VNC ~-; •W ~ •. ••,,.\kh --------~ .~, j __ '7'.,,._-_,.,,.,... ,I
. ~ r
I ,' ,,.<¢ •
! ,'':-~~--
1 -' , I .,, , .. ,, . .. .. --. -----------
I , '\ -~ -----~--..-.-
'Y: .. I ~ ~-t.N: ' i _ ...
..,,...,.-••" ..,-..--.....",u!°',:'U...\m}.S~---
I~. • "' 1 ~ ~ ' ,r~,;t r , ~ . • ,. si ,1 i 11 i . W _ I -= = ----., _ ',"ii ·1 ~II ~ f
l~j •-••·r,.~r : 1 f.<~4"--,.. -.. ~--MW~i "1h ~i/
----
.. ..,. . '...... .. a; ,., ,.
ll
! \ "'"' ~:;.al .. ~-1'\ :"A;"o; "' 10<.;'6) 10 _JJ.Jillil( -
I
I '\ ., .... Ji. Y'a, 8' I I
•,--~ , ~i-0 ~-,: tf ""! \ I I ~ !~ '\ 1/11,-!0°' "' fr--I I I i
''7) · \ '~"'-" 1 I I :: ' ... . \ ..... --
' -.. - -.. (, J,) , , <,1; jffi
I ••••-• -----~~----~=-~,~--r--.--
-"-~AL.,. 'P-AW.~lT
I -~~ ·"-':.;[t'' t ... ... .. .. \ ff ':l;·::l ,' , I ~ fl---
~ 1 ---:------3kL (,',;
...... l ,-----9 , I\. ---i ._l _____ _
'Ri\!I;:-,:'"'• ~ -•-•-•-•-•.-•rr•-•~{~~-•-"~•:11•-•-:.-•~•-•-•-i-•-•-•-•-•-•-•-•-•-•-•-•-•-•-
~:..:, ; -~~f. C I r(, . 100 10 C, .• Cllf • I -----...... Jc ---·
JAB
JSL
SCALE
ASSHO\NN
~
--...:::.,-:;:..:__ I ~-.. __ -::;.! ~-·-.... ·---?}
JAN 2017
IAa..l ">n4"'7
• I J;'QNCRPt (:\~JIG t~At)
SOUTH CCL.EGL HD
Progress -• ENVIRONMENTAL, INC.
P.O. Box 5884
WINSTON-SALEM, NORTii CAROLINAZ7113
PHONE [phone] FAX [phone]
FIGURE4
BENZENE ISOPLETH MAP
1116 SOUTH COLLEGE ROAD
VVILMINGTON, NORTH CAROLINA
I
i
I
i
I I I
I
I
I
i
I
I
I
I
I I
I ~ ;...., I ,., I ;
vi
~-
;j
~ ·-. j
I
(') I
( ,..,
{,J l
l ;..<> .. \J )
.~,!\,.t..
O' 1''' yy
8 ~•:i'lilTOR W· l OCA liOt,
na2) ~~"f X C::Y.\Cf N··
.KA CN (,,c;/ }
:-i ·X ~.Lr
or·;cc . .-..,
r--!11<--B't.X •!'OCONC!'t-.-
fRA T 0:\ {,;<L ·, I
---~--
l ·~:lt:H .••iltt.~C LO~
@""~;),) ,_·/::.~i~~."';:;,/ ~~-' . . ~-:
I ,.,, 1,11,1
I 0 C l"'1<\;,'_.-Jg1/.f;
'i1 I ·-·:1,zr~·-·-·-·=•· ..... •-~-,.--.,..•-·-·-·-·-·-·-·-·-·-·-•.::.~~.~ __ ... _ _ @11,111~11-, . · r-t . M\-'f ··I \h"/·· .......__41.-~~~ •-•-~-•-\" i ( /o} ""°' _i·•Ln} ~~= ---· · · -· ,. . r·• . na: --_____ ....................... , Y_.W·-~ .:::: :::-:;.--=-. -,\.· :::-
:-1 W'N ·8 ;:;c ·,:;1u .. -:.: G"-'S PO~I AM ~• 8 -:U-.~1 ·-r ... 1, -.~ ~ v; ;.,.i..) -~ J
!I ,, /r.' ! -------; (, /o) . ~
~j '' I ~-;'; .,;, • I ~
i j I ,_,,,... WA'"•" . -~, .
6 --------·· \~ ... ..,_, ..,. ---~ ~
i _ --.'--e;('.1!"!'~::-·--·-·· ~"'.,., ~-
• ..,., "' ',...0 f , .,.~ . ~/ • I e, l ~ •s: ""' e., v ·,.:
i .,
~
I , ....... ~~ ••. l .. . . .. .. I ,' 0 .,· t .. .. ----·, l .. _ ...... ---------.......... .
i i : " --~ ~ \ ~ ... --·---
I~ I I ~ BOL rr,-~ ~·1n ,.
,:.. ,-----, J:';1• "/ ~'.~1 I 1 --4--.,,,,-...·.;_ \~il&'J;jj ___ -~•\C"•""'-8 ~ • ' 1 --ti.rw-<--• --ft -.. fiu -i 1 .-, 1 u •<v • ..._ "'fl"·"' I I 8 ' "' "'/'t..Q. MV°':' II. 1 fl a ,'
\ ' " '-Rti6\ ► ' 't:..t.' f 1
I
• \ .... ,,:;, -,:_ • -~ .,.,.,:-,r \' ,1 0 I •Ill 1
,.,.-
' , ~ \ I MW -6 \ \ I I
-----\ '., 8 J C\ '"" 0
• ~,4 I I <o ~ "-.., --S'llli_;C 1 1 (~.\\.,, I I I I i
MAP-r .., , --'-'-~.T I I O
~,\ l ..,_ , ff"t-1,-.s , , "'[ ••
\ -------~ ' :____ -·==-
._,._.,,-.i,.1 Pll.vt¥".:M ~ -,. (Jd: \ \\',[<'i' l ,/ w, I I I ..,
~ ~ J - -... - - _ ......... Ji,ip_ -• , -, , ~ . !l ~ '--•-•-·-·-·-'i---•...LlOi....L"--·-·-•-·-•-·-·-·-·-•-•-·-•-·-·-·-·-·-·-·-·-·-·-
--' ·~. ·'$--•.'!":...ff Ml~,------·-·
•~N~cra,;.-:1 ' o ,, J ,... ----· ---• ---·
........__~ ' I COP. .. f:..t , ,1 10010 ! OQ!f":R('"IT" I f
~~::.-.~ I I 'Q: .• ---.... :.:!!) C:----. --.:/.,,_
DRAYIN
JAB
CHEo<t0
JSL
SCALE
~SSHOV\l'IJ
~
JAI\I 2017
JAN2017
I I . r..ok ,}F f( ¢,.,.:ttt·1rty ( 1r-'Jt.,.· ) -·-
5;QJ" l COi. l .· GF t{)
Progress =--r=
ENVIRONMENTAL, INC.
P .O . Box 5884
V"1NSTON-SALEM, NORTI--1 CAROUNAZT113
PHONE [phone] FAX [phone]
FIGURES
TOTAL BTEX ISOPLETH MAP
1116 SOUTH COLLEGE ROAD
VVILMINGTON, NORTH CAROLINA
%INTER PARK
PRESBYTERIAN
CHURCH
..T'
noun
YIPICHTS•ALL[ FVENUE
ors' &MY
COCAS NO. 2
v
A
JAB
NErtrua7tICAM- I — --
J/N: 2017
L ECE N
CsIS1I DESCH+PTaCN
• TYPE A Ks. TOIE WI_
• t1ft IR 11O MAWS YELL
n 7p
SG4.E M MT
Progress
ENVIRONMENTAL, INC.
PO- 5204
W NSTON-SALEM, NORTH CAROLINA
27113
PHONE [phone] FAX [phone]
FIGURE 6
CROSS-SECTION LOCATION MAP
I116 SOUTH COLLEGE ROAD
WI NORTH CAROLINA

7NTACTS REPRESENT GRADATIONAL CHANGES
:TWfEN UNITS AND ARE APPR07NMATE
ELEVATION IN FEET (ASSUMED)
100
90
70
60
MW-2
EL.99 77
MW-1
EL-100.24
TM W-1
ELm100.00
E)IST1NG
CONCRETE SLAB
ESTIMATED EUTENT
//-
OF UST BASIN
r E7a5TING ASPHALT
MW— 4
EL100.25
0+00
0+SO
HORIZONTAL DISTANCE IH FEET
SCALE: 1-+20. ROW_
1':10. VERT.
1+00
A'
- 110
-100
90
' W
- 80 F
70
60
SW
WELL GRADED 5A1905 OR GRAVELLY SANDS.
LITTLE OR NO TINES
SAl SILTY SAMOS. SAND -SILT MI%TLTRES
CH INORGANIC CLAYS OF HIGH PLAST101TY, FAT CLAYS
0 ELLVAT+21+ N Fitt
11
JAB
r,4lyr r
JAN 2017
02.1.1
AS sER]NTT
IA➢c--
7017
SMOLE INTERVAL
H
ScREEN INTERVAL
WATT max ELTVAi10rl
ASO►7uATDO
MHO
1116 51:411 =UM va
womoul. ILL.
1ti
CROSS SECTION
A -A'
rani
4.2
1111rc1ac KM Emma
w*99152-tO J OCT taw
A5 SNOW LN ,
1+.
sr
Progress
ENVIRONMENTAL, INC.
F.J.LSop( 5884
VNNS7ON-BALE34 NORTH CAROLINA 27113
PHONE 336,722,9099 FAX 338 722,0099
FIGURE 7
CROSS-SECT1 ON A to A'
1116 SOUTH COLLEGE ROAD
1VILMINGTON, NORTH CAROLINA

aTEs:
CuPCTACTS REPRESENT GRADATIONAL C7IANCES
E TAUN owls AND ARF APAR014.1Are
CO M
E-EYATtON IN TEET (A559JIJED)
T10
100
00
70
EK -
r
E10511WG CONCRETE SLAB _ EASING ASPHALT
MW-1
EL-100.2.1
it 00.11 11.00
EL-i00.25
+Ls O► 1 Al
lr
SA
100
90
80
70
00
_ - L
0+00 OtS0 1+00
NOFIYd.TAL DISTANCE 01 HIT
SCA & 1'*•20' NOW.
1-.1G' VERT.
SW WEL! CRAB D SA4G5 0R GRAVELLY SANDS,
'`.? = uT1.E OR NO FINES FL CavATwal kI FtET
SN SILTY SANDS. SAND -SILT Lex!u1ES
INORGANIC MAYS OF KOH PLASTICITY, FAT CLAYS
INORGANIC CLAYS OF LOW TO 41Coan.1 PLASTICITY,
GRAVELLY GAYS, SANDY CLAYS. SILTY CLAYS. LEAD! CLAYS
it
sAYPIE tontRvAL
ELEVATION IN K'EEY (Assr,ki )
SLYtEEN MERV&
WARM TALNLC ELLVA7gN
AS OF 2 HAY m
Krc+d� � bllttxA
Progress
-- ENVIRONMENTAL, INC.
ft
1416 01U (AO.
ILWAw Kt
atoss SECTION B-r8'
"'0 lK4-20 los Oc[ xOo
P_O. Box 5884
WI NSTO N-SAL EM. NORTM CAROLINA 271 T 8
PHONE [phone] FAX 3.14.7zameAs
FIGURE a
CROSS-SECTION B to B'
1116 SOUTH COLLEGE ROAD
VVILMINGTON, NORTH CAROLINA

1 ■ ONRESIDENTIAL WELL. CONSTRUCTION RECORD
North Carolina Deportment of Environment and Natural Resources- Division of Water Quality
WELL CONTRACTOR CERTIFICATION # 3426
1, WELL CONTRACTOR:
Henry Nemargut
Well Contractor (Individual) Name
Henry Nemargut Engineering Services
Well Contractor Company Name
STREET ADDRESS 2211 Chestnut Street
Wilmington NC
28495
City or Town State
910 )- 762-5475
Area code- Phone number
2. WELL INFORMATION:
SITE WELL ID slf(if applicable) MMI-6
STATE WELL PERMIT#(if applicable)
DWQ or OTHER PERMIT #(tf applicable)
WELL USE (Check Applicable Box) Monitoring E{1 Municipal/Public 0
Industrial/Commercial n Agricultural CI
Irrigations Other 0 (list use)
DATE DRILLED 4/8111
Zip Code
Recovery 0 Injection p
TIME COMPLETED 1:40 AM ❑ PM El
3. WELL LOCATION:
C1TY• Wilmington COUNTY New Hanover
1118 8_ College Road, Wilmington, NC 28403
(Street Name, Numbers, Community. Subdrvtsion, Lot No., Parcel. Zip Code)
TOPOGRAPHIC 1 LAND SETTING:
❑Scope ❑Valley KI Fla/ Ridge ❑ Other
(check appropriate box)
LATITUDE 3 34.21543
LONGITUDE 7T.88668
May be in degrees,
minutes, seconds or
in a decimal format
Latitude/longitude source: t iGPS ❑Topographic map
(lacatun of well must be shown on a USG S fopo map and
attached to this form if not using GPS)
4. FACILITY- is the Hama of the pus iness wnar4 the well is ieeaure.
FACILITY IDOff if applicable) 0-022087
NAME OF FACILITY Got3as 112
STREET ADDRESS 1116 5. Collage Road
Wilmington NC
28403
City or Town State Zip Code
CONTACT PERSON Reggie Stanley
MAILING ADDRESS 3301 Bumt Mill Drive
Wilmington NC
28403
City or Town State
310 )- 782-4700
Area code - Phone number
5, WELL DETAILS:
a. TOTAL DEPTH: 14'
Zip Cade
b. DOES WELL REPLACE EXISTING WELL? YES 0 NO
c, WATER LEVEL Below Top of Casing; 6.19 FT.
(Use "+" if Above Top of Casing)
d. TOP OF CASING is 0 FT. Above Land Surface'
'Top of casing terminated at/or below land surface may require
a variance in accordance with 15A NCAC 2C .0118.
e. YIELD (gpm): METHOD OF TEST
f. DISINFECTION: Type Amount
g. WATER ZONES (depth):
From To _ From To
From To From
From To From
6. CASING: Thickness/
To
To
Depth Diameter
From 0' Tod Ft. 2"
From To Ft.
From To_ Ft.
7, GROUT: Depth Malarial
From 0 To 3' Ft. Portland
5741 PVtrial
From To Fi.
From To Ft.
Method
Pour
8. SCREEN: Depth Diameter Slot Size Material
From 4' To 14' Ft.2 in. 0.01 in. PVC
From To Ft, in. In,
From To Ft. in. tn.
8. SAND/GRAVEL FACi[:
Depth Size Material
From 3'
From
From
To f4' Ft. #2 Filter Sand
To FL.
To Ft.
10. DRILLING LOG
From To
0' 6'
Formation Description
pray. Sand wlsilt Ibeckril I
6' 10' It. gray Sand wlsiil
10' 1 4' It. gray. line Sand w/silt
11. REMARKS:
I DO HEREBY CFRTIFY TWIT THIS WELL WAS CONSTRUCTED IN ACC0. nANCE WrT
15A NCAC 2C, WELL CON y''TRUCTION STANDARDS, A.HO TT-tAT A COPY OF MIS
0AC HAS BEEN PrWNiDED TO THE W
5/3/2011
SIGTl1RE OF CAle
RTIFIED WEAL CONTEACTOR DATE
�hr 014.s't
PRINTED NINE OF PERSON C01 STRUCTING THE WELL
Submit the original to the Division of Water Quality within 30 days. Attn: Information Mgt.,
1617 Mali Service Center— Raleigh, NC 27699-1617 Phone No. [phone] ext 566-
Form GW-lb
Rev. 7/05

North Carolina Department of Environmental Quality -Division of Water Resources
NOTIFICATION OF INTENT (NOi) TO CONSTRUCT OR OPERATE INJECTION WELLS
The following are "permitted by rule" and do not require an individual permit when constructed in accordance
with the rules of 15A NCAC 02C .0200. This fo rm shall be submitted at least 2 WEEKS prior to in jection.
AQUIFER TEST WELLS (1 5A NCAC 02c .0220)
These wells are used to inject uncontaminated fluid into an aquifer to determine aquifer hydraulic characteristics.
IN SITU REMEDIATION 0 5A NCAC 02C .0225) or TRACER WELLS (1 5A NCAC 02c .0229 ):
I) Passive ln iection Sv stems -In-well delivery systems to diffuse injectants into the subsurface. Examples include
ORC socks, iSOC systems, and other gas infusion methods.
2) Small-Scale In jection O perations -Injection wells located within a land surface area not to exceed 10,000
square feet for the purpose of soil or groundwater remediation or tracer tests. An individual permit shall be required
for test or treatment areas exceeding 10,000 square feet.
3) Pilot Tests -Preliminary studies conducted for the purpose of evaluating the technical feasibility of a
remediation strategy in order to develop a full scale remediation plan for future implementation, and where the
surface area of the injection zone wells are located within an area that does not exceed five percent of the land
surface above the known extent of groundwater contamination. An individual permit shall be required to conduct
more than one pilot test on any separate groundwater contaminant plume.
4) Air Injection Wells -Used to inject ambient air to enhance in-situ treatment of soil or groundwater.
Print Clearly or Type Information. Illegible Submittals Will Be Returned As Incomplete.
DATE: January 24 , 2017 PERMIT NO. ________ (.to be filled in by DWR)
A. WELL TYPE TO BE CONSTRUCTED OR OPERATED
B.
c.
(1)
(2)
(3)
(4)
(5)
(6)
___ .Air Injection Well ...................................... Complete sections B through F, K, N
--~Aquifer Test Well ....................................... Complete sections B through F, K, N
___ Passive Injection System ............................... Complete sections B through F, H-N
___ Small-Scale Injection Operation ...................... Complete sections B through N
-=X'---'Pilot Test ................................................. Complete sections B through N
___ Tracer Injection Well ................................... Complete sections B through N
STATUS OF WELL OWNER: Business/Organization
RECEIVEDINCDEO/DWR
JAN 2 6 2017
~ELL OWNER(S) -~tate name of Business/Agency, and Name and TWat%f t,fiS.:£!lffi~fberfa f uthority to
sign on behalf of the busmess or agency: Operations Section
Name(s): --~O~u=a=li=t)~' O==il -=C'-'o=m=p~anc=.,,.y,.~L=L~C~----------------------
Mailing Address: --~P~.O~·-=B~o~x~2~7-=3~6 _______________________ _
City: Winston-Salem State: NC Zip Code :_2_7_1_0_2 ____ County: Fors yth
DayTeleNo.: (336 )721-9515 Cell No.: {3 36) 706-4746
EMAIL Address: dmciver(@qocnc.com Fax No.: __ _,_( 3=3=6'-'--) _,__7 "'-14,__-=16=8=-0 __ _
Deemed Permitted GW Remediation NOI Rev . 3-1-2016 Page I
D. PROPERTY OWNER(S) (if different than well owner)
Name and Title: -----'S"'-'ee==--=a::.:bo=-.cv--=e __________________________ _
Company Name ---------------------------------
Mailing Address:--------------------------------
City: ____________ State: __ Zip Code: _______ County: _____ _
Day Tele No.: ____________ _ Cell No.: ___________ _
EMAIL Address: _____________ _ Fax No.: ___________ _
E. PROJECT CONT ACT (Typically Environmental Engineering Finn)
Name and Title: __ ___,J::..::a:_.y....,La=w.!.'s=o=n,,__. =D=w=e=ct=o=r -=o=f -=E=c=ol=o'-"gi=·c=a::..1-=S=ervi:..:.:.· c=es=----------------
Company Name ___ P=---r=oc..::irr:,c_es=s---"'E=n=Vll'-=·=o=nm=en=ta=l ...,In=c"-. ___________________ _
Mailing Address: ----=-P=.O=•..=B=o=x=5=8"""8'""'4 _______________________ _
City: Winston-Salem State: NC Zip Code:27113 County:---"'F---"'o=rs ...... vth~---
Day Tele No.: 336-722-9999 Cell No.: 336-782-9726
EMAIL Address: jlawson@ progressenv.com Fax No.: 336-722-9998
F. PHYSICAL LOCATION OF WELL SITE
(1) Facility Name & Address: ---=G"""o"""G=a=s -'-'-#2=------=1=1~16~So=u=th~C=o=ll=e g=e~R=o=a=d-----_________ _
City: ___ W~ilm=in=lrt=o=n~ _______ County; New Hanover Zip Code: 2=8~4~0~3 __ _
(2) Geographic Coordinates: Latitude**: ---~34~0 ------11' 52 .45" or ___ 0 _____ _
Longitude**: 77° 53 ' 10.92 " or 0
Reference Datum: ________ .Accuracy: _______ _
Method of Collection:_G=o=o=g=le==E=arth=----------
**FOR AIR INJECTION AND AQUIFER TEST WELLS ONLY: A FACILITY SITE MAP WITH PROPERTY
BOUNDARIES MAY BE SUBMITTED IN LIEU OF GEOGRAPHIC COORDINATES.
G. TREATMENT AREA
Land surface area of contaminant plume:~l=l ~.4~1=2~ ___ square feet
Land surface area of inj. well network:_~3_1_4~.2~ ___ square feet~ 10,000 ft 2 for small-scale injections)
Percent of contaminant plume area to be treated: 2. 7 5 (must be ~ 5% of plume for pilot test injections)
H. INJECTION ZONE MAPS -Attach the following to the notification.
(1) Contaminant plume map(s) with isoconcentration lines that show the horizontal extent of the
contaminant plume in soil and groundwater, existing and proposed monitoring wells, and existing and
proposed injection wells; and
(2) Cross-section(s) to the known or projected depth of contamination that show the horizontal and
vertical extent of the contaminant plume in soil and groundwater, changes in lithology, existing and
proposed monitoring wells, and existing and proposed injection wells.
(3) Potentiometric surface map(s) indicating the rate and direction of groundwater movement, plus
existing and proposed wells.
Deemed Pennitted GW Remediation NOi Rev . 3-1-2016 Page2
I. DESCRIPTION OF PROPOSED INJECTION ACTIVITIES -Provide a brief narrative regarding the
putpose, scope, and goals of the proposed injection activity. This should include the rate, volume, and
duration of injection over time.
In an effort to facilitate de2Tadation of ersistent dissolved-hase etroleum constituents at the site.
Progress pro poses to in ject a bioremedial product consisting of several non-pathogenic, naturall y
occurring. non-genetically modified strains of Bacillus spp. The bioremedial product will be introduced
into existing monitorine well MW-6 via sus pension within a five-foot , one inch Schedule 40 PVC pi pe
with 0.10 slotted screen. A small amount of sugar (a pproximately 1-2% of pip e volume ) will be added to
the PVC i e in order to stimulate microbial activi . Monitorina well MW-6 has exhibited ersistent
dissolved-phase p etroleum groundwater contamination. The goal is to introduce microbes into the
contaminant plume to facilitate petroleum de{!radation. Progress will collect a 2roundwater samp le from
monitorinl! well MW-6 rior to and a roximatel 30 davs following_ in"ection activities in order to
evaluate the effectiveness of the remedial product on dissolved-phase petroleum constituents and
influence on bacteriologi cal p o pulations under field conditions. The groundwater samples will be
analyzed for volatile orn:anic comp ounds (V OCs) b y Standard Method 6200B including ethanol, methvl
tert-bu 1 ether MTBE and iso ro 1 ether IPE . In addition. Pro ess will collect !ITOundwater
s for sis via heterotr e is used for estimatinu the
number of live culturable heterotrophic bacteria in water. Colonies may arise from airs , chains . clusters.
or sinel e cells all of w · included in the term "colon -formin unit Procress will collect
oeochemical data durin sam line: activities includinf! measurements of H tern erature conductivit
and dissolved oxy gen. Progress will conduct additional samplin g events (p er above) at a pproximately 30-
day intervals. Sample results will be comp ared to existin2 groundwater standards. as a ppro priate.
J. APPROVED INJECTANTS -Provide a MSDS for each injectant. Attach additional sheets if necessary.
NOTE: Only injectants approved by the NC Division of Public Health, Department of Health and Human
Sen1ices can be injected. Approved injectants can be found online at http ://de g.nc.!wv/about/divisions/water-
resources/water-resources-permits/wastewater-branch/ ground-water-protection/ gr ound-water-a pproved-in jectants.
All other substances must be reviewed by the DHHS prior to use. Contact the UIC Program for more info
(919-807-6496).
lnjectant: Bio-Cat Microbials Pro gre ss Blend -11 Strain
Volume of injectant: __ 0=·=2'-'g=a=ll=o=ns~------------------------
Concentration at point of injection: ~1 =IB~C=FU~/=g __________________ _
Percent if in a mixture with other injectants: ____________________ _
Injectant: Granulated Su gar
Volume of injectant: See above
Concentration at point of injection: _______________________ _
Percent if in a mixture with otlier injectants: ___ 1_-_2~%~----------------
Injectant: ----------------------------------
Volume of injectant:
Concentration at point of injection:
Percent if in a mixture with other injectants:
K WELL CONSTRUCTION DATA
(1) Number of injection wells: --~N~/=A~~Proposed 1 (MW-6 ) Existing (provide GW-1s)
Deemed Permitted GW Remediation NOi Rev. 3-1-2016 Page3
(2) For Proposed wells or Existing wells not having GW-1 s, provide well construction details for each
injection well in a diagram or table format. A single diagram or line in a table can be used for
multiple wells with the same construction details. Well construction details shall include the
following (indicate if construction is proposed or as-built):
(a) Well type as permanent, Geoprobe/DPT, or subsurface distribution infiltration gallery
(b) Depth below land surface of casing, each grout type and depth, screen, and sand pack
( c) Well contractor name and certification number
Deemed Permitted GW Remediation NOi Rev. 3-1-2016 Page4
L. SCHEDULES — Briefly describe the schedule for well construction and injection activities.
Monitoring well MW-6 was previoush installed Progress will conduct infection activities within
approximately two weeks following approval.
M. MONITORING PLAN — Describe below or in separate attachment a monitoring plan to be used to determine
if violations of groundwater quality standards specified in Subchapter 02L result from the injection activity.
Plan attached
N. SIGNATURE OF APPLICANT AND PROPERTY OWNER
APPLICANT: "I hereby certfy, under penalty of law, that I am familiar with the information submitted in
this document and all attachments thereto and that, based on my inquiry of those individuals immediately
responsible for obtaining said information, I believe that the information is true, accurate and complete. 1 am
aware that there are significant penalties, including the possibility of fines and imprisonment, for submitting
false information- I agree to construct, operate, maintain, repair. and if applicable, abandon the injection well
and sll related appurtenances in accordance with the 15A NCAC 02C 0200 Rules."
I\ ` r O ti�t1 5 �;r n Se(v 5
i attire o .a licant Print or a Full Name Title
� Pp Type
PROPERTY OWNER Of the property is not owned by the permit applicant):
"As owner of the property on which the injection well(s) are to be constructed and operated, 1 hereby consent
to allow the applicant to construct each infection well as outlined in this application and agree that it shall be
the responsibility of the applicant to ensure that the injection well(s) conform to the Well Construction
Standards (15A NCAC 02C .0200). "
"Owner" means any person who holds the fee or other property rights in the well being constructed. A
well is real property and its construction on land shall be deemed to vest ownership in the land owner, in
he absence of con' . agreement in writing.
Signature* of Property Owner (if different from applicant) Print or Type Full Name and Title
*An access agreement between the applicant and property owner may be submitted in lieu of a signature on this form.
Submit the completed notification package to:
DWR — UIC Program
1636 Mail Service Ccntcr
Raleigh, NC 27699-1636
Telephone: [phone]
Deemed Permitted GW Remediation NO1 Rev. 3-1-2016
Page 5

Figure 1
Topographic Site Map
Go Gas #2
1116 South College Road
Wilmington, New Hanover County, North Carolina
P.O. Box 5884
Winston-Salem, NC 27113
Telephone: [phone]
Fax: [phone]
USGS
United States Department of the Interior
USGS 7.5 Minute Series Topographic Map
Contour Interval: 10 feet
Scale: P- 2000'
Wilmington, North Carolina
Date: 2016
Project: Go Gas #2
Client:
Progress Joh #:
Date: January 2017

{
Il
LEGEND
n 15 5O
H mowirote Ku. L{JCATIUN
�•.�.•� PROPERTY 1.NE
MW-2
0
CHWI01 NAAIM 1 LOT
CURS JGINP- wmar
PPCAERP GIRO
WNOIP:Ikti GAS PGNI AIlCA 1 Q fa NrIAE7T CURBING 1' PIG+..)
RSPIIALj PRVEMCNi`
d.
u
3
CaNCPICDt
111lAMM T
JAB JAN 201T
JEL JAM 2017
SCALE
I AS $rter,A 1
MAME
MW-G
Ili 11 m rn o 9 m
MW-1O
AS-1
MW-y
14
[ —13—MW. 9
SOUTH COLLEGE RC
Progress
ENVIRONMENTAL, 1 NC.
�� -'---• 9GI.�S61A55 -...
A9P14R4-1 PAwySN1
cWJ R[ IE n)R T inPIC4.t
P.O. Bex saes
1MNSTON-SALEM. NORTH CAROLINA 27113 I
PHONE 33E1.722.9999 FA14 330.722.9998
FIGURE 2
SITE MAP
111 B SOUTH COLLEGE ROAD
WILMINGTON, NORTH CAROLINA

11
LJ
LEGEND
SCALE
$ MONI T'OR Wr:l.l. I.00A71ON
GROUNUWArE.R FLOW
171RranoN
---95 PO1LN 1'IOK'NIL'
CON FOUR um, {li.)
(Tb.o,t) G OIJNOWArCii
F; FVAFION ((t.)
MACH AOH PM IO LOT
cumput SUBJECT
EtrRmpiiy wit'
(NIA) a rRA
IyijGIC�rRANE
ir
1,1.1 1_.......11....... uw_,_._._..._._._._._._._.._.. ,�-� �I�11��111I�11,,
N A T IW-1 MIN-3
L .�.�
is MW-� 49 CI4CAETL rurmwc (Trr1CAL
>F I {NIA} LSNolt1rb cm mot WA. tN/Aj
0.
H I .0a
eS
flkAir, \
FAMVP'r 1,11717 ........
12}
1.1
I Iz
II I
ill
rAo
A"i' Lt P►4tI ENT
•
I ,
AREA
�� � cuncvr R
Ottaa
97.85
JAB
J9L
SCALE
AS 6HOV N
P.E NAYE
JAN 21317
oar
MW-4
(97.85) a:
MW-10A 97.17)
EP
(97,B2)fig w—
AS•-I
97.8
mow-
(a 18)
97.85
97.5
97.75
SOUTH COLLEGE RD
Progress
ENVIRONMENTAL. INC.
ASPHAL1 FAVCMENT
CONCNCTC ward
P.O. Bon 58S4
LMNSTON-SALEIN. NORTH CAROLINA 27113
PHONE [phone] FAX 33E1,722.999S
FIGURE 3
GROUNDWATER FLOVV MAP
1116 SOUTH COLLEGE ROAD
1MLMINGTON, NORTH CAROLINA

LEGEND
n'15 30'
$ !ANION S L1 I_UGA I1ON
R 158)
ULNYLNL C0NCIN -
7RATION (ug/i.)
BENZENE N0
rEC I.:C
Oa_ FOR BENZENE
'ucsl1..;
ll
c W4l WOK 949.90
PATEN %Op
ri/01 ea Qile)
com: k y..l1 CA. Mold r HEA
MW•-.
(n /o
{ ASNIRL1 PAA1ALN7
........
•
■
AI� 3
•
•
I
■
r! - . MW--11
1 M1;te. �'r r
1 ▪ 9 p `'1ti14tAW,7+
�� r# 1 t "lyh[1 , 10C 1 1
MW-10C� g. r 1 1 9
� (17) . �/Q. r J 1
mew ▪ s
• . W-
Me ••
•
MORE ■ 1CC lOI!_ 0I$I kE1C
1
7AAHN
LcIac
AS SHONT1
NAME
JAB
J8L
JAN 2017
JAN 2017
L 8 cuRAwp f+yrlC MJ
3'
59L\CRASS --�
nyThi 41 Nw847.111
SOUTH COI.I_ECE RD
Progress
I ENVIRONMENTAL, INC,
91/11 {FIE CLI184112 rwrcAl7
P.a. sox 6@84
VµNSTON-SALEM, NORTH CAROLtNA 27113
PHONE [phone] FAX [phone]
FIGURE 4
BENZENE ISOPLETH MAP
1116 SOUTH COLLEGE ROAD
WI LMI NGTON, NORTH CAROLINA

L
27
LEGEND
E
Q -15 30'
MONITOR WELL. L.00A110N
3tSX)
F1'IEX CON CEN-
IRATION (g/t)
+317l.. k9TEX NOT
fll 1ECTF[7
Brix kSaCaNf:rN-
IRA [ION '» . L
M'h'-3
styo:N l[u CM NM! AKA T*
i-i0)
7 „...„...,,. ,..„„,..,,. , "..
rr! r w r rr S'�V rma
-r —--cuirmv�a ic • - •
•• of •t we
ifQ
II y 4 }•
I
1 e
'
• r
_ rxs
Asriu:f Pttiesr y I �qw r �I .44
M0R
La ••
r M & I•
•
(3B) • •'4q5----: ,/ 1,, I t
! \ LAN AREAIS; •■•`�� •• �.. I
•
zapamoc I
ixA
GRAM. , rn
JAB JAN 2017
• JSL JAN :01'
S G A�F 'Pfl '!VLT
AS SHOLhN
fiLE NAME
•w�►�).. r-�
~ ~~ r w r r r r •w
WW�s • .__:__
EGG ion
e+u.LH VAIuoNL Lvr
ctl.0 Mks Su CT
IW PC 1f C64J
•
•
•
•
a
Ar,i' cvPurW i Y CAL}
cf
1.'
r.;2
1I1/I1IfI/I
•
•Ir
1-1104T NAy(M! IlI
304 \CRASS ..,..
ri
Mgl'HALT PAN1.1•12NT
ca+r+rir. _._. ._._.�.......�._ _.� _._
7
SOUTH COLLEGE RD
Progress
ENVIRONMENTAL, INC.
111100---
cieiME1E CURINhG ffn:cAy
P.O. SOX 5864
WINSTON-SALEM. NORTH CAROLINA 27113
PHONE 330,722,8400 FAX 338,722.0998
FIGURE 5
TOTAL BTEX ISOPLETH MAP
1115 SOUTH COLLEGE ROAD
WILMINGTON, NORTH CAROLINA

W4MTER PARK
PRE SBYTERIAN
CHURO-I
YiR/Gm5AGE
JAB
I Glti- E3
JSL
SiCkE
AS SHOWN
nil mak<
1zv
JAN 2017
e,,,m,e11AN 2077
Progress
ENVIRONMENTAL. INC.
Mom—
i E C E N D
Pollan) C€SCRIPIVN
$ Yost o WONITgnMG IIEL4
• TYPE IN gOXLT *O WELL
P.O. 130X 6994
V1INETQN-5AL.EJN, NORTH CAROLINA 27113
❑HONE 3313.722.9999 FAX 333.722 sage
a
SCALE IN EET
FIGURE 6
CROSS—SECTION LOCATION MAP
1116 SOUTH COLLEGE ROAD
WILMINGTON. NORTH CAROLTNA

100
90
80
70
E0
NITACTS REPRESENT GRADATIONAL CHANGES
:TWEEN UNITS. AND ARE APPROI(IMATE
ID
IOW-2
EL+99, 77
Mt"-1
EL.1GO.I4
TirIW-1
E L•1 OE100
E71ISRNG
CONCRETE SLAB
ESPMATEO EXTENT
OF UST BASIN
iEIII STENCI ASPHALT
MW-4
EL.100,25
i
0+00
0+C10
HORIZONTAL DISTANCE IN FEET
SCALE: 1'020' NORIZ.
1'.10' VERT,
1+00
.77
•
A'
- 110
103
90
70
80
).; SW WELL GRADED SANDS OR GRAVELLY SANDS.
LITTLE OR NO FINES
3 Ili SILTY SANDS. SAND -SILT EII%1RIRE5
7-3 CH INORGANIC CLAYS OF HIGH PLASTICITY, FAT CLAYS
ENIZAED
JAR
JSL
REALE
AS BHOVVN
rRX NMIC
El. eLEVA1I0N NF nor
I I SA4PLE INTERVAL
JAN 2217
JAN 2317
A ENECt
SCREEN INTERVAL
WATER 1AELE ELEVA1IOi1
A9 cc 7 RAY 00
emar
wan MO 90.iMUM 110.
KG
me
CROSS SECTION A—A'INURNETCK
nut
4.2
....maw maw
"9917-70 r GCT 2000
._r- AS SWAP 1°•6w Ix* 1 as. n St
Progress
ENVIRONMENTAL, INC.
P.O. Sox 6864
WINSTON-SALEM. NORTH CAROLINA 27113
PHONE [phone] FAX [phone]
FIGURE 7
CROSS-SECTION A to A'
1116 SOUTH COLLEGE ROAD
1MLMINGTON, NORTH CAROLINA

.O11S:
. CONTACTS REPRESENT GRADATIONAL CHANGES
BETWEEN UNITS AND ARE APPROXIMATE
.CFNO
110
I00
8
90
4
90 -
70
60
EXISTING CONCRETE STAR EXISTING ASPHALT
MW-I
EL-I00.24
1AW-3
EL-T00.11 EL Y100.00
0+00
MW-5
EL-100.25
ie p ; MAY 00-
L
0+50.
HORIZONTAL DISTANCE IN FEET
SCALE 1"-20. H0RI2.
ART.
+DO
100
as
d0
70
» BB
4
4 �y WELL GRADED SANOS OR GRAVELLY SANDS,
E • LITTLE OR ti0 FINES
SA1 SILTY SANDS. SAND -SILT MIXTURES
CH INORGANIC CLAYS OF HICK PLASTICITY, FAT CLAYS
CL INORGANIC CLAYS OF LOW TO MEDIUM PLASTICITY,
GRAVELLY CLAYS, SANDY CLAYS, SILTY CLAYS, LEAN CLAYS
mom
JAB
JOL
AB sliCVWN
JAN 2077
ip .. JPJY 20I7
EL ELEVATOR IN PELT
I
SAMPLE INTERVAL
SCREEN 9ITERYAI.
WATER TABLE ELEVATION
43 OF 2 MAY 00
IWO
OZAS
¶1fl NOM mu1X OA
11ILI06104. XXL
m4991SI-20 40CT2003
Progress
ENVIRONMENTAL, INC.
P.O. Box 5884
VNNETION-SALM. NORTH CAROLINA 27113
PHONE [phone],790 FAX [phone]
Wit
CROSS SEC1 B#
ham AS E10Y I 1TFsw �use n !T 1
ROM
4.;
FIGURE 8
CROSS-SECTION B to 8'
1118 SOUTH COLLEGE ROAD
1MLMINGTON, NORTH CAROLINA

NONRESIDENTIAL WELL CONSTRUCTION RECORD
North Carolina Department al -Environment and Natural Resources- Division of Water Quality
WELL CONTRACTOR CERTIFICATION # 3426
1. WELL CONTRACTOR:
Henry Nemargut
Well Contractor (Individual) Name
Henry Nemargut Engineering Services
WeU Contractor Company Name
STREET ADDRESS 2211 Chestnut S1roet
Wilmington NC 28405
City or Town Slate
( 910 ). 762-5475
Zip Code
Area code- Phone number
Z WELL INFORMATION:
SITE WELL ID #{tf applicable) MAW-6
STATE WELL FERMITH If applicable)
DWQ or OTHER PERMIT Cif applicable)
WELL USE (Check Applicable Box) Monitoring NI Municipal/Public ❑
Industrial/Commercial til Agricu tural 0 Recovery ❑ Injection ❑
lrrigationp Other 0 (fist use)
DATE DRILLED 4/8/11
EI PM Ft
New Hanover
TIME COMPLETED I:40 W
3. WELL LOCATION:
CITY: Wilmington COUNTY
1116 S. College Road, Wilmington. NG 28403
(Sheet Name, Numbers, Community. Ssnbditiwon, Lot No., Parcel, Tip Code)
TOPOGRAPHIC I LAND SETTING:
❑ Slope (Valley to Flat °Ridge 0 Other
(check appropriate box)
LATITUDE 3 34.21543
May be is degrees,
minutes, seconds or
inn decimal format
LONGITUDE 77.8886E
Latitude/longitude source: RIGPS El Topographic
(tocat'vn of well must be shown on a USGS lope
attached Io this form d' not using GPS)
4. FACILITY- .s tr. name a the djsiness wr1C0e Inc well is
FACILITY ID #(If applicable) 0-022087
map
map and
bested.
NAME OF FACILITY GoGes #2
STREET ADDRESS 1116 S- College Road
Wibeington NC 28403
City or Tcrkvn State Zip Code
CONTACT PERSON Reggie Stanley
MAID NG ADDRESS 3301 Bumt Mill Drive
Wilmington NC 28403
City or Town State
L 910 I- 762.47013
Zip Coda
YES © NO Fo
FT.
Area code - Phone number
5. WELL DETAILS:
a. TOTAL DEPTH: 14'
b, DOES WELL REPLACE EXISTING WELL?
c. WATER LEVEL Be1ow Top of Casing: 6.19
(Use'4" if Above Top or Casing)
d. TOP OF CASING IS 0 FT. Above Land Surface'
"Top of casing terminated at/ar below land surface may require
a variance in accordance with 15A NCAC 2C .0118.
e. YIELD (gpm): METHOD OF TEST
f. DISINFECTION: Type Amount
g. WATER ZONES (depth):
From To From To
From To From To
From To From To
6. CASING: Thickness/
Depths Diameter Vg ghhi M-at8rial
From 0' To _ Fi- _ U PVC:
From To Ft
From To Ft.
7. GROUT: Depth Material Method
From 0 To 3 Ft Portland Pour
From To Ft.
From To F1-
6. SCREEN: Depth Diameter Slot Size Material
From 4' To 14' Ft 2 • in. 0.01 PVC
Fran To Ft. ht. in.
From To FL in. in.
9. SAND/GRAVEL PACK
Depth Size Material
From 3' To 14' Ft. #2 Filter Sand
From To Ft,
From To Ft,
10. DRILLING LOG
From To
D. 6'
Formatiion Description
qrak Sgr19 War fbavkfngi
6' 10' ti _pray. Sand wlsar
10' 14' it. gray, fine Sand wIsift
11. REMARKS:
1 OD HEREBY CERTIFY THAT THIS WELL WAS CONSTRUCTED IN AcoortoAraCE WITH
15A NCAC 2C, WELL CON 'TRUCTION STANDARDS, AND THAT A COPY OF THE
N qq0 .AS SEEN PI 1DEDTOTHE W -
5/3,2011
SIGfif TUir • CERTIFIED W 1 CO CTOR DATE
hr7 Ate t1Cf4
PRINTED N, k�E OF PERSON CONSTRUCTING THE WELL
Submit the original to the Division of Water quality within 30 days. Attn: Information Mgt.,
'1617 Mali Service Center — Raleigh, NC 27699-1617 Phone No. [phone] ext 566.
Form GW-lb
Rev- 7/05

Progress
ENVIRONMENTAL INC
January 24, 201 7
Dr. Ken Rudo
North Carolina Health and Human Services
101 Blair Drive
Raleigh, North Carolina 27603
Subject:
Dear Dr. Rudo:
Sampling Plan
GoGas #2 Facility
1116 South College Road
Wilmington, New Hanover County, North Carolina
Groundwater Incident No. 32703
RECEfVED/NCDEQ/DWR
JAN 2 6 2017
Water Q~ality Regional
Operations Section
In response to your request, Progress Environmental, Inc. (Progress) is pleased to submit the following
Sampling Plan for proposed environmental services at the above-referenced site. The objective of this
plan is to evaluate bioremedial injectant influence on dissolved-phase petroleum groundwater
contamination and microbiological populations at the site. The site is an active convenience store located
within an urban area of Wilmington that that stores petroleum products in underground storage tanks
(USTs) for retail sale. Soil and groundwater contamination were initially identified in 1999 following
discovery of a leaking UST. The incident was subsequently closed in May 2004 by the North Carolina
Department of Environmental Quality (NCDEQ). The closure included filing of a Notice of Residual
Petroleum (NRP) which provides restrictions for the site including prohibition of the use of site
groundwater. The site and surrounding area are serviced by the municipal water-supply system.
A new release from beneath the remote dispenser islands was identified in March 2011. The NCDEQ has
approved remedial activities at the site including Aggressive Fluid Vapor Recovery (AFVR) and ongoing
groundwater sampling. In an effort to facilitate degradation of persistent dissolved-phase petroleum
constituents at the site, Progress proposes to inject a bioremedial product consisting of several non-
pathogenic, non-genetically modified, naturally occurring strains of Bacillus spp. The product will be
introduced into existing monitoring well l\tIW-6 via suspension within a one inch Schedule 40 PVC pipe
with 0.10 slotted screen. Monitoring well MW-6 has exhibited persistent dissolved-phase petroleum
contamination. The goal is to introduce microbes into the contaminant plume to facilitate petroleum
degradation.
Preliminary laboratory tests have indicated that the microbes can significantly degrade (i.e., > 90%
decrease in contaminant levels) petroleum constituents under laboratory conditions. Microbial ·product
information is provided below. Please note that this information is proprietary.
Product manufacturer name, address, phone number, and contact person:
Bio-Cat Microbials
689 Canterbury Road
Shakopee, Minnesota 55379
952-445-4251
Mike Nash
F ~ Box " 84 • Winston-Salem, NC ? · 113 5884
-~?. -• t " )F I e • -:i1t 7 ,? ::;..,c, F
www.progressenvironmental.com
Sampling Plan
GoGas #2 Facility, Wilmington, North Carolina
Groundwater Incident No. 32703
January 2 4, 2017
Genus/species of microorganism( s) contained in product:
Bacillus subtilis (strain proprietary)
Bacillus licheniformis (strain proprietary)
Bacillus amyloliquefaciens (strain proprietary)
Bacillus pumilus (strain proprietary)
Bacillus mojavensis (strain proprietary)
Brevibacillus laterosporus (strain proprietary)
Progress will collect a groundwater sample from monitoring well MW-6 prior to, and approximately 30
days following, injection activities in order to evaluate the effectiveness of the remedial product on
dissolved-phase petroleum constituents and influence on bacteriological populations under field
conditions. The groundwater samples will be analyzed for volatile organic compounds (VOCs) by
Standard Method 6200B including ethanol, methyl tert-butyl ether (MTBE), and isopropyl ether (IPE). In
addition, Progress will collect groundwater samples for analysis via heterotrophic plate count (HPC).
This procedure is used for estimating the number of live culturable heterotrophic bacteria in water.
Colonies may arise from pairs, chains, clusters, or single cells, all of which are included in the term
"colony-forming units" (CFU). Progress will collect geochemical data during sampling activities
including measurements of pH, temperature, conductivity, and dissolved oxygen. Progress will conduct
additional sampling events (per above) at approximately 30-day intervals or until plume stabilization
occurs. Sample results will be compared to existing groundwater standards, as appropriate.
Note that, in terms of potential breakdown products from metabolism of petroleum hydrocarbons by
Bacillus spp., the ultimate breakdown products are CO2, water, and Bacillus cells. Intermediary products
are possible and are addressed in the attached reference materials (Patowary, K, et al, "Degradation of
Polyaromatic Hydrocarbons Employing Biosurfactant-producing Bacillus pumilus KS2", Ann Microbiol
(2015) 65: 225-234). Additional supporting information regarding the proposed injectant is attached to
this plan.
2
Sampling Plan
GoGas #2 Facility, Wilmington, North Carolina
Groundwater Incident No. 32703
January 24, 2017
We appreciate your consideration and cooperation in the submission of this infom1ation. If you have any
questions concerning this submittal please feel free to contact us at [phone].
Sincerely,
PROGRESS ENVIRONMENTAL, INC.
~~
Directo~ili:~:gi~al Services
Certified Microbial Consultant
J~~
Director of Environmental Services
Licensed NC 1653
Attachments: Progress Blend Material Safety Data Sheet (MSDS)
SugarMSDS
Excerpt -Progress Letter of Supporting Documentation (Progress Blend) dated August 22,
2014)
Patowary, K, et al, "Degradation of Polyaromatic Hydrocarbons Employing
Biosurfactant-producing Bacillus pumilus KS2", Ann Microbiol (2015) 65: 225-234.
Bacillus spp. Reference Information
3
M aterial Safety D ata Sheet
&..
BIO·CAT Microbials
Form: Powder Dated: October 2013
Section 1: Chemical Product and Company Information
Product Name: Experimental Blend-11 Strain 11B CFU/g
Supplier/ Further Information: Bio-Cat Microbials
689 Canterbury Road Shakopee, MN 55379
(p)434-589-4777, (1)434.589.3301
Email: iinfo(@bio-cat.com www.bcmicrobials.com
Section 2: Composition/ Information on Ingredients
Chemical Name: NIA
Chemical Family: Bacterial
Molecular Weight: Not available
Hazard Classification: N/A
Formula: N/A
Synonyms: N/A
Shipping Name: N/A
CAS Number: Proprietary
CAS Number: 7647-14-5
Chemical: Proprietary bacteria blend
Chemical: Sodium chloride
Section 3: Hazards Identification
Health Hazard Information: Potential aUergic reaction and/or breathing problem if powder inhaled.
Potential Health Effects:
Eye: Mild irritant
Skin: Mild irritant
Ingestion: Ingestion of material is not known to result in significant adverse health effects.
Inhalation: May cause sensitization by inhalation in hypersensitive individuals. A void dust
generation.
Signs and Symptoms of Exposure: The allergic symptoms are such as runny nose, cough, sneeze,
languor,.slight attack of fever. Contact with powder causes irritation to sensitive skin and eyes.
Section 4: First Aid Measures
Inhalation: If inhaled remove from contaminated area to fresh air. Report situation. Seek medical
attention if allergic response is exhibited.
Eye Contact: In case of contact with eyes, flush eyes with low pressure water for at least 15 minutes.
If irritation develops, seek medical attention.
Skin Contact: In case of contact with skin, wash skin with soap and water. Remove contaminated
clothing and wash.
Ingestion: If swallowed, rinse mouth and throat thoroughly with tap water. Drink water.
-I -
Section 5: Fire Fighting Measures
Flash Point/Ignition Temperature: NFP A Rating: Health= 1
Flammability= 1
Reactivity = 0
Protection Against Fire & Explosions: Under normal use no special requirements. If extremely high
levels generated in ambient air, material can support combustion.
Explosion Characteristics: N/A
Hazardous Decomposition Products: None
Suitable Fire Extinguishing Media: Water, Foam, Halon
Section 6: Accidental Release Measures
If a spill occurs: S pilled product should be removed immediately to avoid formation of dust.
Vacuum or moisten with
water and collect into a sealable container for disposal Flush spill area with plenty of water (low
pressure) into approved sewer. Avoid formation of aerosols and dusts. Ensure sufficient ventilation.
Wash contaminated clothing.
Section 7: Handling and Storage
Handling: Never handle powder without appropriate personal protective equipments in accordance
with Section 8. Avoid formation of dust. Avoid splashing and high pressure washing. Ensure good
ventilation of the room when handling this product.
Storage: Store container in a dry, cool place.
Section 8: Exposure Controls/ Personal Protection
Respiratory Protection: None required under usual condition of use. However, if exposure potential
exists, refer to NIOSH Criteria Guides to determine appropriate unit.
Hand Protection: Impermeable gloves recommended.
Eye Protection: Protective glasses or eye shield.
Industrial Hygiene: Maintain good conditions of industrial hygiene.
Section 9: Physical and Chemical Properties
Boiling Point, 760 mm Hg: NIA Specific Gravity: N/A
Evaporation rate (butyl acetate = 1 ): N/ A % Volatile by Volume: N/ A
Appearance & Odor: Off white to Tan powder, typical fermentation odor. Pour Point: N/ A
Vapor Pressure at 20 degrees: N/A Vapor Density: (Air =1) N/A
Solubility in water, % by weight: Readily Soluble pH Value (1 % sol.): 3.8-8.0
Section 10: Stability and Reactivity
Stability: Stable
Hazardous Decomposition or Byproducts:N/A
Section 11: Toxicological Information
Inhalation of dust may cause respiratory allergy in susceptible individuals. Oral rat LD-50 >2g/kg-
classifies product as "non-toxic".
Not classified as a carcinogen by IARC, OSHA, or NTP.
-2-
Section 12: Ecological Information
Product is readily biodegradable.
Section 13: Disposal Considerations
No special disposal method required, except that in accordance with current local authority
regulations.
Section 14: Transport Information
Road/Rail: Not classified Sea: Not classified Air: Not classified
Section 15: Regulatory Information
The active ingredient and all components of the enzyme preparation are listed on the TSCA
Inventory.
Section 16: Other Information
Any person who experiences any allergic or sensitive reactions to this powder should refrain from
handling it again.
The information contained in this Safety Data Sheet, as of the issue date, is believed to be true and
correct. However, the accuracy or completeness of this information and any recommendations or
suggestions are made without warranty or guarantee. Since the conditions of use are beyond the
control of the company, it is the responsibility of the user to determine the conditions of safe use of
this product. The information does not represent analytical specifications.
-3-
Science LdIJ .com
Chemicals & Laboratory Equipment
Material Safety Data Sheet
Sucrose MSDS
Health R
Fire
Reactivity 0
Personal
Protection
Section 1: Chemical Product and Company identification
Product Name: Sucrose
Catalog Codes: SLS4048, SLS3253, SLS1036
CAS#: 57-50-1
RTECS: WN6500000
TSCA: TSCA 8(h) inventory: Sucrose
CI#: Not available.
Synonym: beta-D-Fructofuranosyl-alpha-D-
glucopyranoside
Chemical Name: Sucrose
Chemical Formula: C121122011
Contact Information:
5ciencelab.com, inc.
14025 Smith Rd.
Houston, Texas 77396
US Sales: 1-800-901-7247
International Sales: 1-281-441-4400
Order Online: ScienceLab.com
CHEMTREC (24HR Emergency Telephone), call:
1-800-424-9300
International CHEMTREC, call: 1-703-527-3887
For non -emergency assistance, call: 1-281-441-4400
Section 2: Composition and Information on Ingredients
Composition:
Name
Sucrose
CAS #
57-50-1
Toxicological Data on ingredients: Not applicable.
% by Weight
100
Section 3: Hazards Identification
Potential Acute Health Effects: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of
inhalation.
Potential Chronic Health Effects:
CARCINOGENIC EFFECTS: A4 (Not classifiable for human or animal_) by ACGII•i. MUTAGENIC EFFECTS: Mutagenic for
bacteria and/or yeast. TERATOGENIC EFFECTS: Not available. DEVELOPMENTAL TOXICITY: Not available. Repeated or
prolonged exposure is not known to aggravate medical condition.
Section 4: First Aid Measures
Eye Contact:
Check for and remove any contact lenses. In case of contact, immediately flush eyes with plenty of water for at least 15
minutes. Cold water may be used_ Get medical attention if irritation occurs.
P. 1

Skin Contact:
Wash with soap and water. Cover the irritated skin with an emollient. Get medical attention if irritation develops. Cold water
may be used.
Serious Skin Contact: Not available.
Inhalation:
If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Get medical
attention.
Serious Inhalation: Not available.
Ingestion:
Do NOT induce vomiting unless directed to do so by medical personnel. Never give anything by mouth to an unconscious
person. Loosen tight clothing such as a collar, tie, belt or waistband. Get medical attention if symptoms appear.
Serious Ingestion: Not available.
Section 5: Fire and Explosion Data
Flammability of the Product: May be combustible at high temperature.
Auto-Ignition Temperature: Not available.
Flash Points: CLOSED CUP: Higher than 93.3°C (200°F).
Flammable Limits: Not available.
Products of Combustion: These products are carbon oxides (CO, CO2).
Fire Hazards in Presence of Various Substances: Slightly flammable to flammable in presence of heat.
Explosion Hazards in Presence of Various Substances:
Risks of explosion of the product in presence of mechanical impact: Not available. Risks of explosion of the product in
presence of static discharge: Not available.
Fire Fighting Media and Instructions:
SMALL FIRE: Use DRY chemical powder. LARGE FIRE: Use water spray, fog or foam. Do not use water jet.
Special Remarks on Fire Hazards: Not available.
Special Remarks on Explosion Hazards: Not available.
Section 6: Accidental Release Measures
Small Spill:
Use appropriate tools to put the spilled solid in a convenient waste disposal container. Finish cleaning by spreading water on
the contaminated surface and dispose of according to local and regional authority requirements.
Large Spill:
Use a shovel to put the material into a convenient waste disposal container. Finish cleaning by spreading water on the
contaminated surface and allow to evacuate through the sanitary system. Be careful that the product is not present at a
concentration level above TLV. Check TLV on the MSDS and with local authorities.
Section 7: Handling and Storage
Precautions:
Keep locked up .. Keep away from heat. Keep away from sources of ignition. Empty containers pose a fire risk, evaporate
the residue under a fume hood. Ground all equipment containing material. Do not ingest. Do not breathe dust. Wear suitable
protective clothing. If ingested, seek medical advice immediately and show the container or the label. Keep away from
incompatibles such as oxidizing agents, acids.
p.2
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area. Do not store above 23°C (73.4 °F).
Section 8: Exposure Controls/Personal Protection
Engineering Controls:
Use process enclosures, local exhaust ventilation, or other engineering controls to keep airborne levels below recommended
exposure limits. If user operations generate dust, fume or mist, use ventilation to keep exposure to airborne contaminants
below the exposure limit.
Personal Protection:
Safety glasses. Lab coat. Vapor and dust respirator. Be sure to use an approved/certified respirator or equivalent. Gloves.
Suggested protective clothing might not be sufficient; consult a specialist BEFORE handling this product.
Personal Protection in Case of a Large Spill:
Splash goggles. Full suit. Vapor and dust respirator. Boots. Gloves. A self contained breathing apparatus should be used to
avoid inhalation of the product. Suggested protective clothing might not be sufficient; consult a specialist BEFORE handling
this product.
Exposure Limits:
TWA: 15 (mg/rn3) from OSHA (PEL) [United States] Inhalation Total. TWA: 10 (mg/m3) from ACGIH (TLV) [United States]
[1999] Inhalation Total. TWA: 10 (mg/m3) from NIOSH Inhalation Total. TWA: 5 (mg/m3) from NIOSH Inhalation Respirable.
TWA: 5 (mg/m3) from OSHA (PEL) [United States] Inhalation Respirable.3 Consult local authorities for acceptable exposure
limits.
Section 9: Physical and Chemical Properties
Physical state and appearance: Solid. (Crystalline granules solid.)
Odor: Characteristic Carmel to Odorless.
Taste: Sweet.
Molecular Weight: 342.3 g/mole
Color: White.
pH (1% soln/water): Not available.
Boiling Point: Not available.
Melting Point: 186°C (366.8°F)
Critical Temperature: Not available.
Specific Gravity: 1.587 (Water = 1)
Vapor Pressure: Not applicable.
Vapor Density: Not available.
Volatility: Not available.
Odor Threshold: Not available.
Water/Oil Dist. Coeff.: The product is more soluble in water; log(oil/water) = -3. 7
lonicity (in Water): Not available.
Dispersion Properties: See solubility in water, methanol.
Solubility:
Easily soluble in cold water. Partially soluble in methanol. Insoluble in diethyl ether.
Section 10: Stability and Reactivity Data
p.3
Stability: The product is stable.
Instability Temperature: Not available.
Conditions of Instability: Excess heat, incompatible materials
Incompatibility with various substances: Reactive with oxidizing agents, acids.
Corrosivity: Not available.
Special Remarks on Reactivity: Reactive with sulfuric acid, nitric acid, and oxidizers.
Special Remarks on Corrosivity: Not available.
Polymerization: Will not occur.
Section 11; Toxicological Information
Routes of Entry: Inhalation. Ingestion.
Toxicity to Animals: Acute oral toxicity (LD50): 29700 mg/kg [Rat].
Chronic Effects on Humans:
CARCINOGENIC EFFECTS: A4 (Not classifiable for human or animal.) by ACGIH. MUTAGENIC EFFECTS: Mutagenic for
bacteria and/or yeast.
Other Toxic Effects on Humans: Slightly hazardous in case of skin contact (irritant), of ingestion, of inhalation.
Special Remarks on Toxicity to Animals: Not available.
Special Remarks on Chronic Effects on Humans:
No adverse reproductive affects have been found in humans. However at extremely high oral doses of 683,000 mg/kg given
to rats during pregnancy showed some effects on newborn (growth, developmental anomalies of central nervous system).
Passes through the placental barrier in human.
Special Remarks on other Toxic Effects on Humans:
Acute Potential Health Effects: Skin : May cause skin irritation. Low hazard for usual industrial handling. Eyes: Dust may cause
mechanical irritation. Inhalation: Excessive inhalation may cause minor respiratory irritation. Ingestion: Ingestion of large
amounts may cause gastrointestinal (digestive) tract irritation. Expected to be a low ingestion hazard. Chronic Potential Health
Effects: no information.
Ecotoxicity: Not available.
BODS and COD: Not available.
Products of Biodegradation:
Section 12: Ecological Information
Possibly hazardous short term degradation products are not likely. However, long term degradation products may arise.
Toxicity of the Products of Biodegradation: The product itself and its products of degradation are not toxic.
Special Remarks on the Products of Biodegradation: Not available.
Section 13: Disposal Considerations
Waste Disposal:
Waste must be disposed of in accordance with federal, state and local environmental control regulations.
Section 14: Transport Information
p .4
DOT Classification: Not a DOT controlled material (United States).
Identification: Not applicable.
Special Provisions for Transport: Not applicable.
Section 15: Other Regulatory Information
Federal and State Regulations:
Rhode Island RTK hazardous substances: Sucrose Pennsylvania RTK: Sucrose Minnesota: Sucrose Massachusetts RTK:
Sucrose Tennessee: Sucrose TSCA 8(b) inventory: Sucrose
Other Regulations: EINECS: This product is on the European Inventory of Existing Commercial Chemical Substances.
Other Classifications:
WHMIS (Canada): Not controlled under WHMIS (Canada).
DSCL (EEC):
This product is not classified according to the EU regulations. S24/25-Avoid contact with skin and eyes.
HMIS (U.S.A.):
Health Hazard: 1
Fire Hazard: 1
Reactivity: 0
Personal Protection: X
National Fire Protection Association (U.S.A.):
Health: 1
Flammability: 1
Reactivity: 0
Specific hazard:
Protective Equipment:
Gloves. Lab coat. Vapor and dust respirator. Be sure to use an approved/certified respirator or equivalent. Safety glasses.
Section 16: Other Information
References: Not available.
Other Special Considerations: Not available.
Created: 10/10/2005 08:28 PM
Last Updated: 05/21/2013 12:00 PM
The information above is believed to be accurate and represents the best information cu"ently available to us. However, we
make no wa"anty of merchantability or any other warranty, express or implied, with respect to such information, and we assume
no liability resulting from its use. Users should make their own investigations to determine the suitability of the information for
their particular purposes. In no event shall ScienceLab.com be liable for any claims, losses, or damages of any third party or for
lost profits or any special, indirect, incidental, consequential or exemplary damages, howsoever arising, even if ScienceLab. com
has been advised of the possibility of such damages.
p.5
Supporting Documentation
Bio-Cat Microbials Experimental Blend -11 Strain (Progress Blend)
August 22, 2014
How long will product remain in subsurface? How will it travel over time?
The Bacillus species included in the Progress Blend are all non-genetically modified and native to soils
and groundwater in the United States. Since these species are spore formers they can survive in the soil at
low levels for an extended period. The Bacillus species can be motile but will travel over time in the
same way that the native soil Bacillus travel. Since these Bacillus do not contain genetic markers they
would be impossible to distinguish from native soil Bacillus after soil treatment.
Bacillus includes the foodbome pathogenic species Bacillus cereus. There is no evidence at this time of
waterborne transmission. Bacillus cereus is not one of the species included in the Progress Blend, but
even this food-borne pathogen is considered safe.
See discussion and references from the World Health Organization below on the safety of Bacillus in soil
and water.
h ttp ://www.who.int/water sanitation health/dwq/GDWI lrevland2.pdf
11.1.3 Bacillus
General descri ption
Bacillus spp. are large ( 4-10mm), Gram-positive, strictly aerobic or facultatively anaerobic encapsulated
bacilli. They have the important feature of producing spores that are exceptionally resistant to
unfavourable conditions. Bacillus spp. are classified into the subgroups B. polymyxa, B. subtilis (which
includes B. cereus and B . lichenifonnis), B. brevis andB. anthracis.
Human health effects
Although most Bacillus spp. are harmless, a few are pathogenic to humans and animals. Bacillus cereus
causes food poisoning similar to staphylococcal food poisoning. Some strains produce heat-stable toxin
in food that is associated with spore germination and gives rise to a syndrome of vomiting within 1-5
hours of ingestion. Other strains produce a heat-labile enterotoxin after ingestion that ca~ diarrhoea
within 10-15 hours. Bacillus cereus is known to cause bacteraemia in immunocompromised patients as
well as symptoms such as vomiting and diarrhoea. Bacillus anthracis causes anthrax in humans and
animals.
Source and occurrence
Bacillus spp. commonly occur in a wide range of natural environments, such as soil and water. They form
part of the HPC bacteria, which are readily detected in most drinking-water supplies.
Routes of ex posure
Infection with Bacillus spp. is associated with the consumption of a variety of foods, especially rice,
pastas and vegetables, as well as raw milk and meat products. Disease may result from the ingestion of
the organisms or toxins produced by the organisms. Drinking-water has not been identified as a source of
infection of pathogenic Bacillus spp., including Bacillus cereus. Waterborne transmission of Bacillus
gastroenteritis has not been confirmed.
Si gni ficance in drinking-water
Bacillus spp. are often detected in drinking-water supplies, even supplies treated and disinfected by
acceptable procedures. This is largely due to the resistance of spores to disinfection processes. Owing to a
lack of evidence that waterborne Bacillus spp. are clinically significant, specific management strategies
are not required.
2
Supporting Documentation
Bio-Cat Microbials Experimental Blend -11 Strain (Progress Blend)
August 22, 2014
Selected bibliography
Bartram J et al., eds. (2003) Heterotrophic plate counts and drinking-water safety: the significance of
HPCs for water quality and human health. WHO Emerging Issues in Water and Infectious Disease
Series. London, IW A Publishing.
What will contaminants metabolize into?
The Bacillus in the Progress Blend will break down the petroleum hydrocarbons according to known
microbial pathways (see Attachment). At times the petroleum hydrocarbons will be broken down into
metabolites that can be further metabolized by other native soil bacteria.
See discussion and references below:
h ttp ://www.ncbi.nlm.nih.'Z.ov/pubmed/7783002
J Basic Microbial. 1995;35(2):83-92.
Phenol and cresol metabolism in Bacillus pumilus isolated from contaminated groundwater.
Gunther K 1
, Schlosser D, Fritsche W.
Abstract
From an aquifier contaminated with phenolic compounds seven bacterial strains able to grow on phenol
and several mono-and disubstituted alkylphenols as sole source of carbon and energy were isolated. Five
isolates belong to the genus Pseudomonas, two to the genus Bacillus. The isolate most active in
utilization of the applied xenobiotics was identified as Bacillus pumilus and used for the investigation of
the degradation pathways in liquid cultures. Cells of this strain precultured on phenol were able to utilize
para-cresol as sole carbon source via the oxidation of the methylsubstituent and intradiol ring cleavage of
the resulting protocatechuic acid, whereas an intradiol ring fission of the intermediate 4-methylcatechol
led to 4-methylmuconolactone as dead end-product. Cells precultured on meta-and ortho-cresol were able
to utilize the respective compounds as sole carbon sources via 3-methylcatechol, which induced the
following extradiol ring fission pathway. Cells precultured on phenol were able to cooxidize meta-as well
as ortho-cresol to 3-methylcatechol, which was cleaved via an intradiol ring fission, finally leading to the
dead end-product 2-methylmuconolactone.
h ttp ://w,v,v.2:enome.jp/keQ:g-bin!show pathwav?bcl00624
See Attachment.
Will product act as a spore/surfactant?
The Bacillus species in the Progress Blend are all spore formers and, thus, they will certainly "act as a
spore". In terms of acting as a surfactant-Bacillus, especially petroleum hydrocarbon utilizing Bacillus,
are known to produce a number of naturally occurring bio-surfactants which emulsify the petroleum
hydrocarbons and increase degradation.
See discussion and references below:
http ://w'V'.'\V.znaturforsch.com/ac/v59c/s59c0205.pdf
Enhanced Hydrocarbon Biodegradation by a Newly Isolated Bacillus subtilis Strain
Nelly Christovaa, *, Borjana Tulevaa, and Boryana Nikolova-Damyanovab
3
Supporrin;g Documentation
Bio-Cat Microbials Experimental Blend —11 Strain (Progress Blend)
August22 2014
The relation between hydrocarbon degradation and biosurfactarrt (rhamnolipid) production by a new
Bacillus subtilis 22BN strain was investigated_ The strain was isolated for its capacity to utilize n-
hexadecane and naphthalene and at the same time to produce surfaceactive compound at high
concentrations (1.5 D 2.0 g ID1). Biosurfactant production was detected by surface tension lowering and
emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 d
1% and 75 d 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity
showed that the combination of slightly soluble substrate and rhamnolipid developed higher
hydrophobicity correlated with increased utilization of both hydrocarbon substrates_ To our knowledge,
this is the first report of Bacillus sublilis strain that degrades hydrophobic compounds and at the same
time produces rhamnolipid biosurfactant
hup://www.clenomedp/ke..t!-binishow pathwav?bc100624
See Attachment.
We appreciate your consideration and cooperation in the submission of this information. If you have any
questions concerning this submittal please feel fee to contact us at [phone].
Sincerely,
PROGRESS ENVIRONMENTAL, INC.
944-41. Ltrtil acitaike
Joseph S. Lawson III
Director of Ecological Services
Jeffrey A_ Balisieper, L.G.
Director of Environmental Services
Licensed NC I653
Attachments: Polycyclic Aromatic Hydrocarbon Degradation
Progress Blend Material Safety Data Sheet
4

IPOLYCYCLIC AROMATIC HYDROCARBON DEGRADATION 1
3,4 d V i scoau e
r 9' W1 L1412- '��
1J 13%f f
Rm.= i — Illdir, ' a-O - -1 adz }— _-►c% FyiES I{� i FLi1 - —.}— i7i�
oaro
ate
I
6esa;▪ 4dieena
1,1_ 1- 3fvsi —
r-1 112.1I, -- 3 11417.1---
r q��
I Y 3 4 lAl nsyF l.�mr]6d 2�nk and osossme
. --1 U4i2 } ••?:: ni 3-R-H9�SP Aii
[, 6Om.ae a.Formyll-ird®eae ]fie
1 141 -- ice 13.I. 1i111.1 sr— --- O----- i c.--; 1 4 1-"0-13"1.133F-e<
1 iArb5e4in- 4 '-Iedwm 3.�
aybd° ei2 xi3
Andineens-
91A.be =dace= 9,10.6a8oqasia
a1'yafeethX401)-
alit ci t[ _H. 3-Pl. 7 HR g
—P—+0ue0372• 2 J-1.0 'Ifln, 7 rJ .
2.3 aay rle.e
06,7-6raraeanrm i
f-
-jIl41L. ` PL 831, � � L 4P1raa$in1
jizeimAxert
--1112.1#. sae- 1- i ��__
Phersehm
1,2ozde 3,4-17hydmry.
11911,
cirr 4A
12$,2.21oboF3trin.l
45i 4S1:17dm
2522e 114.-. > 1.0 [i i L9a
1
DO,j
•1
—••D--I 337. 1—►O�
1-H1 � 17�,nae ihnaymo: i i.6
114,, ar!7
1 1-51 izio as-
1.14-r 1 .�
; ?.s�ma<
1-14fincr 16-e13
.4.it
222-- --- ��' 7p1 1.6-Dare my
a
4�ee" 1.cs 4� F'Se�
Ppraal]e
i•o-1 114131 1—rp- ---- : • e.emee degradation
54&-51411 Cedr.b03 C' ghe�3s.�em
1.-de
3
2 " 11- Paar
U 1hkldoccF
04-1
dicipaithan
mirameat
1�A �Fio-lLyb}
two—, 9. i0.171h9densg r-----ia irabosy�" co9_1 I • - _ [$iyreae
Ufa n�-■O—{17311. - {4413,. 7- 1Q{notr�CdeAcLryna,.iax
YiL.1- 1 :�.'An-.r ewc.xal.e
1J41� �F=--{ 13 1---0-0^
Senms1,flira► 4111.71�rn ntar +arr
llosole7
✓ beat] 1.
2C1524111542
(c)Kaa.ldr. lairanalaras
11. S 2"173nry.
gIrmewideledi
.1.169
3-Hyrdox}
bramtlr
3..1P
I mail
Pis
bewail
f 113.1a1
sesorkehyer
H®meb Ciegleaball

Ann Microbiol (2015) 65:225-234
DOI 10.1007/s13213-014-0854-7
ORIGINAL ARTICLE
Degradation of polyaromatic hydrocarbons employing
biosurfactant-producing Bacillus pumilus KS2
Kaustuvmani Patowary • Rasbmi Rekha Saikia •
Mohan Chandra Kalita • Suresh Deka
Received: 29 August 2013 I Accepted: 28 February 2014/Published online: 21 March 2014
© Springer-Verlag Berlin Heidelberg and the University of Milan 2014
Abstract An efficient hydrocarbon-degrading native bacteri-
al strain Bacillus pumilus KS2 (identified by partial l 6S
rDNA gene sequencing) was isolated from crude oil-
contaminated soil collected from oil fields of Lakowa,
Sivasagar district of Assam, India. Experiments were conduct-
ed under laboratory conditions to determine the efficiency of
this biosurfactant-producing strain to degrade
polycyclicaromatic hydrocarbons (PAHs). Quantification of
the capacity of the biosurfactant to reduce the surface tension
(ST) of the culture medium was used as a measure of
biosurfactant production. In terms of total petroleum hydro-
carbon (TPH) degradation, strain KS2 was able to degrade
80.44 % of the TPH by 4 weeks of incubation. It also dem-
onstrated efficient degradation of PAHs, completely
degrading nine of the 16 major PAHs present in the crude
oil sample. Strain KS2 also produced biosurfactant which,
based on biochemical and FTIR analyses, was glycolipid in
nature. To our knowledge, this is the first report showing the
potential of a native strain of the North-East region ofindia for
efficient degradation ofTPH and PAHs and, consequently, in
the remediation of hydrocarbons from contaminated sites.
Keywords Bacillus pumilus KS2 • PAH degradation •
Biosurfactant • Crude oil • Native strain
K. Patowary • S. Deka (E)
Environmental Biotechnology Laboratory, Life Sciences Division,
Institute of Advanced Study in Science and Technology, Guwahati,
Assam, India
e-mail: [e-mail]
R.R. Saikia
Department of Zoology, Jagannath Barooah College, Jorhat. Assam,
India
M. C. Kalita
Department of Bioengineering and Technology, Gauhati University.
Guwahati, Assam. India
Introduction
Oil pollution is a serious environmental problem worldwide.
Various activities associated with oil exploration cause envi-
ronmental pollution, including geophysical exploration, dril-
ling of wells, pressure control and management of oil and
natural gas gushing from the well, transportation and refining
of crude oil. Crude oil is a homogeneous but complex mixture
of hundreds of different hydrocarbons which vary widely in
their characteristics. These compounds are toxic, persistent
and have negative influence on living organisms, making it
imperative to develop a technology for cleaning up polluted
sites. Microorganisms capable of degrading hydrocarbons in
various forms (Klug and Markovetz 1971) can be found in
many environments depending on the specific compounds
present in the environment (Atlas 1981). However, the rate
of degradation of hydrocarbonaceous compounds in nature is
limited due to their hydrophobic property which leads to their
limited solubility in ground water and tendency to partition to
the soil matrix. This partitioning can account for as much as
90-95 % or more of the total contaminant mass. As a conse-
quence, hydrocarbon contaminants exhibit moderate to poor
recovery by physicochemical treatments, limited bioavailabil-
ity to microorganisms and limited availability to oxidative and
reductive chemicals when subjected to in situ and/or ex situ
applications (Pacwa-Plociniczak et al. 2011 ). A promising
method that can improve the effectiveness of bioremediation
of hydrocarbon-contaminated environment is the use of
biosurfactants. Biosurfactants are surface-active amphipathic
molecules produced by certain microorganisms. They have a
wide structural diversity, ranging from glycolipids,
lipopeptides and lipoproteins to fatty acids, neutral lipids,
phospholipids and polymeric and particulate biosurfactants
(Das et al. 2008). Biosurfactants reduce surface tension (ST)
and critical micelle dilution in both aqueous solution and
hydrocarbon mixtures, thereby facilitating the creation of
~ Springer
226
micro-emulsions with the formation of micelle in which
hydrocarbons can solubilize in water or water in hydro-
carbons (Banat 1995). Biosurfactants are often less toxic
and more biodegradable than synthetic surfactants and
thus are particularly suited for environmental applica-
tions such as hydrocarbon remediation (Oberbremer
et al. 1990). Most biosurfactants, in comparison to
chemical surfactants, have shorter persistence in the environ-
ment (Georgiou et al. 1992) and are more effective in enhanc-
ing the solubility and biodegradation of petroleum hydrocar-
bons, including polycyclic aromatic hydrocamons (PAHs)
(Wong et al. 2004; Hickey et al. 2007).
Crude oil contains more than 30 % PAHs. PAHs are im-
portant environmental contaminants because of their hydro-
phobic and recalcitrant nature {Toledo et al. 2006) and addi-
tionally are important health threats to human and animal life
(Samanta et al. 2002). They occur in nature as a result of
incomplete combustion of organic matter, as well as from
many anthropogenic sources, including cigarette smoke and
automobile exhaust (Jacques et al. 2007). A number of studies
on degradation of multiple PAHs by biosurfactant-producing
bacteria have been published. Ganeshalingam et al. (I 994)
suggested that applying surfactants as immobilizing agents
might be one approach to enhance the solubility of PAHs.
Biosurfactants are capable of increasing the bioavailability of
poorly soluble PAHs (Gilewicz et al. 1997; Olivera et al.
2003). Nie et al. (2010) studied the biosurfactant-producing
Pseudomonas aeroginosa NY3 as one of the components of
PAH-degrading bacterial consortia tested for their efficacy in
degrading a mixture containing equal amounts of fluorene,
anthracene, phenanthrene, pyrene and fluoranthene. These
authors reported that this bacterium could degrade all
five of these PAHs at different degradation rates. The
ability of members of the genus Bacillus to degrade
PAHs has also been reported. However, experimental
data on hydrocarbon degradation by biosurfactant-
producing Bacillus pumilus are relatively scarce. Several
bacterial strains, including B . pumilus JL, have demon-
strated the capacity to degrade diesel and used engine
oil effectively in liquid media (Mandri and Lin 2007;
Singh and Lin 2008). In a PAH degradation study by
bacterial consortia, Ma et al. (2010) observed that
B . pumilus was one of the members of the consortia,
but these authors did not determine whether the bacte-
rium was responsible for degradation of any specific
PAH. Khanna et al. (2011) reported that B. pumilus
(PK-12, MTCC 1002) could metabolically take up
64 % of pyrene from its growth medium. Yuliani
et al. (2012) recently showed that B. pumilus C 15
has the capability to degrade the PAHs pyrene and
phenanthrene because the strain possesses the
dioxyrenese nidA and nahAc gene which are responsible
for PAH uptake.
~ Springer
Ann Microbiol (2015) 65:225-234
The aim of this study was to isolate an efficient
biosurfactant-producing/hydrocarbon-degrading native bacte-
rial strain that can be used for the decontamination of the sites
contaminated with petroleum hydrocarbons.
Materials and methods
Isolation of microorganism
Soil samples were collected from crude oil-contaminated sites
of the Lakowa oil fields, situated in upper Assam, India, for
isolation of biosurfactant-producing bacterial strains with
hydrocarbon-degrading property. To this end, I g of collected
soil sample was added to a 500-mL Erlenmeyer flask contain-
ing 100 mL of sterilized nutrient broth and mineral salt solu-
tion at 1: 1 ratio (Francy et al. 1991) to which 2 % v/v crude oil
was added as the sole carbon source. The flasks were incu-
bated at 35 °C in a rotary shaker (model Orbitek LJEIL;
Scigenics Biotech, Bangalore, India) at 150 rpm. After 3 days
of incubation, 5 mL of culture broth was sampled from each
flask and transferred into a second batch of flasks containing
fresh medium; these flasks were then incubated under the
same conditions to decrease the unwanted microbial load.
This process was repeated three times, and each time 5 mL
of culture broth was withdrawn from the "older" flasks and
transfered into new ones. Serial dilutions of the culture broth
from the last batch of flasks were inoculated onto diesel-
containing (2 % v/v) mineral agar plates and the plates incu-
bated at 35 °C for the development of bacterial colo-
nies. The morphologically different bacterial colonies
which developed on the plates were streaked on nutrient
agar plates to obtain pure cultures of the isolates , and
these pure cultures were maintained on nutrient agar
slants and kept at 4 °C in the refrigerator.
Screening for biosurfactant-producing bacteria
The isolated bacterial strains grown in nutrient broth for 24 h
at 35 °C with shaking at 150 rpm were used as mother
inoculum. A 5-mL sample of each mother inoculum of each
isolate was transferred to a 500-mL Erlenmeyer flask contain-
ing I 00 mL sterilized mineral medium with 2 %w/v glucose
as the carbon source and incubated at 35 °C with shaking at
150 rpm. The composition of the mineral medium used was
{g/L): NH4NO3 (4.0), KCI (0.1), KH 2PO4 (0.5), K2HPO4
(1.0), CaCb (0.01), MgSO4•7H2 O (0.5), FeSO4-7H2 O
(0.0l ), yeast extract (0.1) and IO mL of trace element solution
containing (g/L) 0.26 g H3BO3, 0.5 g CuSO4 ·5H2O, 0.5 g
MnSO4-7H2 O, 0.06 g (N}4)6Mo 7Oz-4H 2O and 0.7 g ZnSO4 •
7H 2O (Saikia et al. 2012). The pH of the medium was adjusted
to 7.0±0.2. The production of biosurfactant by the bacterial
Ann Microbial (2015) 65:225-234
isolates was assayed in terms of drop collapse assay and ST
reduction of the culture medium.
Drop collapse assay
A single drop of crude oil was placed on a glass slide,
following which a single drop of 48-h-grown culture broth
was dropped onto the crude oil drop (Bodour and Miller-
Maier 1998) and drop collapse activity was observed.
ST measurement
Surface tension reduction was measured at 24-h intervals up to
the fifth day using a tensiometer (model Kl l; Kruss Optronic,
Hamburg, Germany). The values reported are the mean of five
measurements. The isolates which reduced the ST of the
medium to <45 mN/m were selected as efficient isolates for
further experiments.
Screening of the most efficient hydrocarbon-degrading
bacterial isolate
The method of Rahman et al. (2002), with minor modifica-
tions, was used to screen the bacterial isolates for the most
efficient hydrocarbon-degrading bacterium. As a first step,
mother cultures were prepared from all of the biosurfactant-
producing bacterial isolates in nutrient broth as per the proce-
dure mentioned above. For the screening, a 5-mL sample of
the mother culture of each bacterium was inoculated into a
500-mL Erlenmeyer flask containing 100 mL of sterilized
mineral medium with crude oil 2 % (v/v) collected from the
Noonmati refinery, Guwahati, India, and cultured in a shaking
incubator at 35 °C and 150 rpm for 7 days. The bacte-
rial growth in the medium of each flask was measured
by determining the optical density (OD) at 600 nm
using a UV-Vis spectrophotometer (model UV-1800;
Shimadzu, Kyoto, Japan). The bacterial strain showing
the maximum growth in the crude oil-containing medi-
um was selected for further study.
Identification of the bacterial strain
The most efficient hydrocarbon-degrading/biosurfactant-pro-
ducing bacterial isolate was identified by l 6S rDNA sequenc-
ing and subsequent alignment of the sequence in the NCBI
GenBank (performed by National Center for Cell Science,
Pune, India). Following standard protocols, we extracted ge-
nomic DNA from a pure culture and amplified a l 6S rDNA
:fragment of approximately 1.5 kb using Taq DNA polymer-
ase. The :fragment was bi-directionally sequenced using uni-
versal bacterial primers UFUL (GCCTAACACATGCAAG
TCGA) and URUL (CGTATTACCGCGGCTGCTG)
(Nilsson and Strom 2002). Sequence data were aligned and
227
analyzed to identify the closest homolog of the isolated bac-
terial strain. A neighbor-joining phylogenetic tree
(Fig. 1) was constructed with the aligned l 6S rDNA
gene sequences using 1,000 bootstrap replication with
MEGA5 software (Tamura et al. 2011 ).
Study of total petroleum hydrocarbon degradation
Crude oil (2 g) was added to each of twenty 500-mL Erlen-
meyer flasks, following which 100 mL of mineral media was
added and the mixture mixed thoroughly. The mixture was
then sterilized to kill the unwanted microorganisms present in
the crude oil. Microbial growth was initiated in 15 of the 20
flasks by inoculating 5 mL of the strain KS2 mother culture
into each flask (OD600 1.8). The remaining five flasks were
kept as abiotic controls and were not inoculated with the
bacterial culture. The flasks were incubated in a rotary shaker
at 3 5 °C to study the degradation of crude oil for a period of 1,
2, 3, 4 and 5 weeks (Kumari et al. 2012). Crude oil was
extracted from three flasks containing culture media and from
one from control flask at weekly intervals using a solvent
extraction method with petroleum benzene (boiling range
40-60 °C). The solvent was evaporated in a rotary evaporator,
and the hydrocarbon solution was captured in a clean, previ-
ously weighed beaker and kept for few days to obtain the
constant weight. The amount of crude oil degraded was then
determined gravimetrically (Mittal and Singh 2009).
Study of degradation of PAHs
The degradation of PAHs was analyzed in the crude oil
samples extracted from the flasks containing mineral media
and crude oil inoculated with B. pumilus strain KS2 mother
culture after 4 weeks of culture. Crude oil from the abiotic
control was also extracted for analysis. Samples were ana-
lyzed by gas chromatography (GC) on a Master GC chro-
matograph (DANI, Cologno Monzese, Italy) equipped with a
flame ionization detector and DN5 capillary column with
Supelco (Sigma-Aldrich, St. Louis, MO) standards. The
flow rate of carrier gas was 25 mL/min, and the fuel
source was supplied at 40 mL/min. Air was supplied as
oxidant at a rate of 280 mL/min. The initial oven
temperature was 160 °C for 2 min.
Extraction ofbiosurfactant
To extract the biosurfactant from the culture media, we cen-
trifuged 48-h-old culture broth at I 0,000 rpm for 20 min at
4 °C to obtain the cell-free supernatant. The clear supernatant
served as the source of crude biosurfactant. The pH was
adjusted to pH 2 by adding 6 N HCl to the collected superna-
tant. The acidified supernatant was kept at 4 °C overnight,
following which the biosurfactant was extracted continuously
~ Springer
228 Ann Micmbiol (2415) 65^25-234
Fig. 1 Phylogenctic tree of
Bacillus pumilus strain KS2 based
on l6S rDNA sequencing and its
closest relatives. Bar 0.02
nucleotide substimdons, values in
parenthesis GenBank accession
number
0.02
with vigorous shaking in a'mixtuure of supernatant and ethyl
acetate (ratio i :I) at room temperature and then left static for
phase separation. The organic phase was then transfencd to a
rotary evaporator and a viscous solid product was recovered
(i.e. crude biosurfactant) after solvent evaporation at 40 °C
under reduced pressure (George and Jayachandran 2009). The
crude biosurfactant was subsequently dried and determined
gravimetrically.
Characterization ofbiosurfer tam
Biochemical
The presence of different bio-molecules in the extracted crude
biosurfactant was determined by different biochemical assays
according to standard procedures reported by Sawhney and
Singh (2000). The ninhydrin test was carried out to determine
the presence of amino acids and their polymer proteins, the
enthrone test was performed to determine the presence of a
carbohydrate moiety in the biosurfactent sample and the sa-
ponification test was done to estimate lipid content.
In the ninhydrin test, 5 drops ofninhydrin solution were added
to 5 nmL of cell -free supernatant and the mixture kept for 5 min in
a boiling water bath. Color formation indicated the presence of
amino acids and the polymer proteins. In the anthone test, equal
C} Springer
Bacillus stratoosphericus (kF016032)
Bacillus aftitudnis (KC833O47)
Bacillus safensis (KFO17359)
Bacillus pumilus (JN03?409)
• Bacillus purnilus (KF021245)
Bacillus pumilus (HQ003450)
Bacillus amyloliquefaciens (JX674030)
Bacillus sustilis (GU220584)
— Bacillus mofaV nsis (KF05.4921)
Bacillus circulans (f0409560)
Bacillus fiche► iformis (G11269542)
Bacillus megaterium (H0166111)
Bacillus census (EIJ513393)
Bacillus simplex (JF343181)
Lysinibacillus fusiformis (JQ900545)
- Bacillus bathos (H14440045)
Bacillus ►enrols (HM439778)
Lactobacillus rhamraosus (EF495247)
Brevibacillus brevis (EU6935161
amounts of culture supernatant and enthrone reagent (5 mL) were
mixed, and a color change indicated the presence of a carbohy-
drate moiety_ The saponification test was carried out by mixing
5 mL of cell -flee supernatant with 2 mL of 2 % NaOI l solution
followed by vigorous shaking (i.e. saponification).
Spectral analysis
Spectral analyses were caned out to examine the type of
biosurfactant produced by determining the functional groups
present in the biosurhactant. For the analysis of functional
groups, the extracted biosurfactant was analyzed in a Vector-
22 Fourier transform infrared (F ill() spectrometer (Broker
Corp., Fremont, CA). The spectral region used was 4,000-
400 cmft at a resolution 4 cm -t using a KBr palate of 0.26-
mrn thickness.
Statistical analysis
AU of the experiments were carried out three times and studied
in triplicate. Results represent the mean ± standard deviation.
One-way analysis of variance (ANOVA) with the least signif-
icant difference (LSD) test was conducted to determine the
significant differences in hydrocarbon degradation efficacy of
the bacterial strain at different time periods_ SPSS vet 17

Ann Microbiol (2015) 65:225-234
229
Table 1 Surface tension measurements' of the bacterial isolate on glucose containing mineral medium at different time intervals
Bacterial isolates ST at 0 h ST at 24 h of culture ST at 48 h of culture ST at 72 h of culture ST at 96 h of culture ST at ] 20 h of culurre
Ct'
KS1
KS2`
KS3`
KS4
K55
KS6
KS7`
KS8
KS9`
KS10
KS1]
KS12`
71.1 ±030
67.9±0.40
59_1 *0.30
60.8+0.30
69.8+0.12
60.8+0.15
69.4+0.16
62.9+0,40
68.8*0.12
59.4+0.30
63.8+0.15
68.3+0.14
60.9+0.41
71.0+07A
69.410.44
48.5+0.0.25
45.8+0.44
68.510_17
57.7+0_21
59.21--024
47.4+0.21
60.5+0.17
44.5+0.25
58.4+0,21
622+024
45.4+0.23
71.0+0.40
66.5+0,43
38.5+0.30
31.4+0.22
60.1 +024
54.4+0.16
58.6+0.22
33.4+0.41
54.1+0.24
32.5+0.30
50.2*0.16
58.6+0.22
3.3*0.27
69.9+0.30
65.0+0.31
40.2+0.25
34.5+0.23
60.0t0.43
57.8+023
57.1 3035
37.3+0.12
57,8+0.43
38.3+0.25
52.7+0.23
57.I+0.35
38.4+032
69.9+0,23
65.0+033
41.7+0.15
36.9+0.24
62.7+0_41
57.0+0.16
56.2+0.40
41_210.31
60.7*0.41
42.8+0.15
56.1*0,16
59.2+0.40
42.4+0.41
69.8+0.27
65.2+0.23
43.7+015
41.8+0.28
62.8+031
573+0.26
56.1 +0.30
443+0.22
62.8+031
44_3+0.25
57.31026
61.7+0.30
47_1 +0.12
`Surface tension (ST) measurements (in mNlrn) are
Abiotic control
`Denotes the biossufaclant-producing strains
given as the mean * standard deviation (SD) of five measurements
software (SPSS, Chicago, IL) was used to carry out the Screening for the most elflcieat hydrocarbon -degrading
statistical analysis. bacterial strain
Results and discussion
Screening for biosurfactant-producing bacteria
A total of I morphologically different bacterial colonies were
isolated from the collected soil samples. These 12 isolates
were screened for biosurfactant production.
Drop collapse assay
The culture broths of five of the 12 bacteria! isolates were able
to collapse the drop of crude oil, indicating the presence of
biosurfactant in the respective culture broth medium. Drop
collapse, when observed, occurred almost immediately but
always within 1 min of the addition of culture broth. The
remaining bacterial culture broths were unable to collapse
the drop of crude oil even after 1 min.
ST measurement
All five bacterial isolates able to collapse the drop of crude oil
(positive drop collapse assay result) were able to reduce the
ST to <45 rrnN/rn (Table 1). According to Vitarrtontes-Ramos
et al_ (2010), , isolates able to reduce the ST of the medium to
-545 mNIM can be considered to be biosurfactant-producing
microbes. Based on these results, we studied these five bacte-
rial isolates for degradation of hydrocarbon.
Among the five biosurfactant-producing bacteria! isolates,
bacterial strain KS2 showed the highest growth (DDT)
after 1 week of incubation in mineral media containing
2 % (wlv) crude oil (Fig. 2). In contrast, of all five
biosurfactant-producing bacterial isolates, strain KS2 pro-
duced the lowest amount of biosurfactant when cultured in
medium containing 2 % (w/v) glucose (Fig. 3). Strain KS2
showed maximum growth on culture day 7. This result
indicates that the petrolcurn hydrocarbon was best utilized
by the KS2 strain through degradation and establishes that
not all efficient biosurfactant producers arc good degraders
O ticsl density SOD 800)
0_9
0.8
0.7
0.6
0.5
OA
0.3
0,2
0.1
0
0.72
Biosurlactant producing isolates
0.534
+00ai[In day -a- opal lhday
Fig. 2 Growth characterization of biosurfactant-producing isolates in
mineral medium containing crude oil as the carbon source. Bars Standard
error (SE) of three determinations
Sprenger

230
Ann Microbial (2015) 65:225-234
Surface Tension (mWm) at 48th h
45 -
40
35
30
25
20
15
10
5
0
r7�
4
�k
3
2.5
2
1.5
0_5
0
Bloeurfactant extracted (g/L)
Biosurlactant producing isolates
--a— ST at 48th h t Biosurfactant extracted ( gl L)
Fig. 3 Surface tension (S7; mN/m) reduction and biosurfactant extrac-
tion (g/L) at 4$ h of culture ofhiosurfactant-producing isolates is mineral
medium with 2 % (wlv) ghroose. Bars SE of three determinations
of petroleum hydrocarbons—i.e. biosurfactant production/
hydrocarbon degradation is dependent upon the nature and
pope —dies of the respective strain. At 48 h of culture, bacte-
rial strain KS3 produced the highest amount of biosurfactant
(2.37 g/L), but bacterial growth in crude oil -containing
media on culture day 7 was 0.474 (DDT). In comparison,
bacterial strain KS2 produced the lowest amount of
biosurfactant (0.3 g/L) at 48 h of incubation, but showed
its maximum growth in crude oil -containing media at culture
day 7 was 0.720 (OD) (Figs. 2, 3). Based on these results,
we selected strain KS2 for further degradation studies.
Identification of the most efficient isolate
The sequence of bacterial strain KS2 was submitted to the
NCBI GenBank database under accession no. gbIKF0212451
Fig. 4 Peiceutage degradation of
total petroleum hydrocarbons by
bacterial strain KS2 at different
time points during the incubation
period. Bars Standand deviation
(SD) of three determinations
Springer
90
80
0
70
a 60
L
ce
.a50
n • 40
0 • 30 -
20
10
51.95
Week 1
and a BLAST search was conducted to compare this sequence
with existing sequences. The results revealed that strain KS2
has a maximum similarity with Bacillus pumilus strain
NBGD45 (accession no. gb1HQ0034501) and Bacillus pumilus
strain DSX6 (accession no. gbi JN0374091).
.[Degradation of total petroleum hydrocarbons
The degradation of total petroleum hydrocarbon (TPH) by
bacterial strain KS2 at different time intervals is presented
graphically in Fig. 4. There was a trend towards increasing
TPH degradation with increasing length of culture up to the
fourth week of culture, with maximum TPH degradation
(80.45 %) at the fourth week of incubation and minimum
TPH degradation (51.95 %) at first week of incubation. The
differences in TPH degradation values at different time period
was found to be statistically significant, although there was no
significant increase in the value after fourth week of incuba-
tion (ANOVA LSD test,p<0.05).
Biosurfactants can enhance the degradation of hydrocar-
bons by two mechanisms, namely by increasing substrate
bioavailability for the microorganisms and by interacting with
tie surface of the bacterial cell to increase the hydrophobicity
of the surface, thereby allowing hydrophobic substrates to
associate more easily with bacterial cells (Mulligan and
Gibbs 2004). 13y reducing surface and interfacial tensions,
biosurfactants increase the surface areas of insoluble com-
pounds, leading to an increased mobility and bioavailability
of hydrocarbons. Taken together, biosurfactants ultimately
enhance the biodegradation and removal of hydrocarbons.
Therefore, the addition of biosurfactant producing bacteria
to a culture system can be expected to enhance hydrocarbon
biodegradation by mobilization, solubilization or emulsifica-
tion (Nguyen et al. 2008; Deziel et al. 1996; Nievas et al.
74.12
80A5
77A4
Week 2 Week 3 Week 4
Treatment (in weeks)
79.21
Week 5

Attu Micmbiol (2015) 65.775-234
231
1
a
1_
20
Fig. 5 Gas chnmmatograph of abiotic control at culture week 4
— crude oil _30_05_2043 410 - Detector B
2008). Kumari et al. (2012) reported that under their respec-
tive optimal culture conditions biosurfactant-producing strains
Pseudamonus sp. BP10 and Rhodacnccus sp. NJ2 degraded
60.6 and 49.5 % of TPH, respectively, after 30 days of
incubation in minimal salt media captaining 2 % of crude
nil. Based on the results of their comparative study on the
biosurfactant activity of crude oil -degrading bacteria and its
correlation to TPH degradation, Phan et al. (2013) reported
that I{hodococcus sp. UKMP-7T, Rhodococcus sp_ [IKMP-
5T, Pseudamonas aeruginosa UKMP-14T and Acinetobacter
baumanii WU41P-12T degraded 93.3+1.0, 62.4+2.6, 75.2±
0.6 and 62.8±0.5 % of TPH, respectively -
a
m 1.
—r
S
is
1
30 40
Degradation of PAHs
50
[Z
All 16 PAHs tested were detected in the chromatogram of the
contol crude oil sample (Fig. 5) which was compared with the
chromatogram of the bacterial treated experimental sample.
Following treatment with the KS2 strain, nine of the PAHs
present in the control had been degraded, including naphtha-
lene, acenaphthene, acenaphthylene, 2-bromonaphthalene,
fluorenc, bcnzo[a]pyrene, indeno[l, 2, 3-c, d]pyrene,
dibenx a, h]anthracene and benzo[g, h, i]perylene (Fig. 6).
A large number of microorganisms are reported to use
two- and three -ringed PAHs as the source of carbon and
10 20
Term
Fig. 6 Gas chmmatograph of crude oil treated with strain KS2 at culture week 4
— Y.s•2-d _30_05_2013_9 - DeIecror 8
30
Springer

232
Ann Microbic] (2015) 65:225--234
Fig. 7 Fourier transform infrared
spectroscopy spectra of the crude
hiosurfaclant obtained from strain
KS2
°% Transmittance
to
0.9
0.8
0.7
0.6
0.5
O.d
0.3
4000 3500 300E 2500 2000 1500 1000
Waventtmber (cm'')
2926
CH Stretching
3388
ON Stretching
1735
C=0 ester.
1645
00-0
1085
1Q98
C W Mimic/Arm
energy (Bamforth and Singleton 2005). Only limited num-
bers of microorganisms are capable of degrading PAHs
with four or more fused aromatic rings (Harayarna 1997).
Kelley and Cernigilia (1995) reported the degradation of
benzo[ajpyrene in a mixture of PAHs by Mycobacterium
sp., and Ye et al. (1996) reported that a Sphingomonas
paucimubilis strain can degrade the five -ring PAH
dibenz[a,b]anthracene and benzo[b)fluoranthene. In our
study, the experimental strain Bacillus purnilus KS2
showed a wide range of PAH degrading ability, ranging
from two -ringed structures to six -ringed fused structures,
i.e., it was able to degrade four PAHs with a two -ringed
structure (naphthalene, acenaphthene, acenaphthylene, 2-
bromonaphthalene), one PAH with a three -ringed structure
(fluorine), two PAI1s with a five -ringed structure
(benzo[a]pyrene and dibenz[a,h)anthracene) and two PAHs
with a six -ringed structure (indeno[1,2,3-c,d]pyrene and
benzo [g,h,ilperyiene).
Extraction of crude biosurfactant
The yield of biosurfactant from strain KS2 was 0.3 gfL. The
color of the crude biosurfactant was light honey.
Characterization of biosurfactant
Biochemical
Ruhcmann's purple complex formation was absent in the
ninbydrin test, indicating the absence of amino acids or pro-
teins in the biosurfactant. In the anthrone test for carbohy-
drates, however, the formation of a blue-green color
was observed, indicating the presence of carbohydrates
'r Springer
in the sample. In the saponification test, NaOH sapon-
ified the lipids present in the biosurfactants, indicating
the presence of lipids. These results indicate that the
crude biosurfactant produced by bacterial strain KS2
contains sugar and lipid molecules but not protein.
molecules.
FTTR analysis
In the spectrum for crude biosurfactant from strain KS2
(Fig. 7), strong absorption band was observed at
3,386 crri t and at 2,926 cm - I. Carbonyl stretching band
was found at 1,735 cm. Absorption bands at 1,645 crn-1
and 1,036 cm-1 were also observed in the spectrum. Com-
parison of FT1R spectrum with Bordoloi and Konwar (2009)
and Thenmozhi et al. (2011) revealed the presence of differ-
ent functional groups in the biosurfactant sample. Strong and
broad band of the hydroxyl group (--OH) free stretch was
observed at 3,386 cm t which was due to the presence of
hydrogen bonding. The absorption band observed at
2,926 cm-1 confirmed the presence of C-I-1 stretching vibra-
tions of hydrocarbon chain of alkyl (CHz- CH3) groups.
Carbonyl stretching band found at 1,735 cm-1 was charac-
teristic for ester compounds. The absorption at 1,645 cm '
was because of stretching of COO) group. The spectrum
also showed absorption band at 1,036 cm 1 which
corresponded to stretching vibration of -C-0-. The pattern
of absorption bands observed in FM analysis indicates that
some polysaccharide or polysaccharide -like substances are
present in the biosurfactant. So, from the above discussion it
can be concluded that the biosurfactant is of glycolipid in
nature.

Ann Microbial (2015) 65:225-234
Conclusion
We report here the isolation of a new biosurfactant-producing/
hydrocarbon-degrading bacterial strain (Bacillus pumilus
KS2) from crude oil-contaminated soil of the Lakowa oil field,
Upper Assam, India. The biosurfactant produced was glyco-
lipid in nature. Although the KS2 strain produced relatively
less biosurfuctant than the other biosurfactant-producing iso-
lates, it showed maximum growth on crude oil, indicating
maximum degradation of crude oil and PAils. Therefore, we
conclude that the degradation capacity of this bacterium is
strain specific but does not depend on the biosurfactant pro-
duction. Strain KS2 showed excellent degradability against a
number of very complex PAHs present in crude oil, which has
not been reported previously. Therefore, this strain could be
used for the decontamination of the sites contaminated with
toxic pollutants of PAHs. Further studies are underway to
scale up growth conditions for better degradation. However,
field trials are necessaiy to ascertain the laboratory-scale find-
ings which indicate that this bacterial strain has a potential use
in cleaning up hydrocarbon-contaminated sites.
Acknowledgments The authors would like to thank the Director, In-
stitute of Advanced Study in Science and Technology (IASST),
Guwahati, India for providing laboratoi:y facilities and encouraging the
research. Kaustuvmani Patowaiy is also grateful to the Department of
Science and Technology, Govt of India for providing assistance as a
Junior Research Fellow (JRF) to carry out the research work. We also
thank Dr. N. Sen Sanna, Associate Professor, and DLA. Devi, Assistant
Professor, IASST, for their assistance with the FTIR and GC analysis,
respectively, at IASST, Guwahati. We expression our appreciation to Dr.
Hemen Deka, RA, !ASST, for the statistical analysis and Rajeev K.
Brahma, Tezpur University, for the phylogenetic analysis.
References
Atlas RM (1981) Microbial degradation of petroleum hydrocarbons: an
environmental perspective. Microbial Rev 45(1):180-209
Bamforth SM, Singleton I (2005) Bioremediation of polycyclic aromatic
hydrocaibons: current knowledge and future directions. J Chem
Technol Biotechnol 80:723--736
Banat IM (1995} Biosmfactant production and possible uses in microbial
enhanced oil recovery and oil pollution remediation: review.
Bioresour Technol 55: 1-12
Bodour AA, Miller-Maier RM (1998) Application of a modified drop-
collapse technique for surfactant quantitation and screening of
biosurfuctant-producing microorganisms. J Microbial Methods 32:
273--280
Bordoloi NK, Konwar BK (2009) Bacterial biosurfactant in enhancing
solubility and metabolism of petroleum hydrocarbons. J Hazard
Mater 170:495-505
Das P, Mukherjee S, Sen R (2008) Improved bioavailability and biodeg-
radation of a model polyaromatic hydrocarbon by a biosurfactant
producing bacterium of marine origin. Chemosphere 72: 1229---1234
Deziel E, Paquette G, Vtllemw-R, Lepine F, Bisaillon J (1996) Biosurfuctant
production by a soil Pseudomonas strain growing on polycyclic aro-
matic hydroc:amons. Appl Environ Microbiol 62:1908-1912
233
Francy DS, Thomas JM, Raymond RL, Ward CH (1991) Emulsification
of hydrocarbons by subsurface bacteria J Ind Microbial 8:237-246
Ganeshalingam S, Legge RL, Anderson WA (1994) Surfactant enhanced
leaching of poly aromatic hydrocarbons from soil. Trans Inst Chem
Eng 72:247-251
George S, Jayachandran K (2009) Analysis of Rhamnolipid
biosurfactants produced through submerged fermentation using or-
ange fruit peelings as sole carbon source. Appl Biochem Biotechnol
158:694-705
Georgiou G, Lin S, Shanna MM (1992) Surface-active compounds from
microorganisms. Biotechnology 10:60-65
Gilewicz M, Ni'matuzahrob T, Nadalig H, Budzinski H, Downenq P,
Michotey V, Bertrand JC (1997) Isolation and characteriz.ation ofa
marine bacterium capable of utilizing 2-methylphenanthrene. Appl
Microbial Biotechnol 48:528-533
Harayama S (1997) Polycyclic aromatic hydrocarbon bioremediation
design. Curr Opin Biotechnol 8:268-273
Hickey AM, Gordon L, Dobson AD, Kelly CT, Doyle EM (2007) Effect
of surfactants on fluorantbene degradation by Pseudomonas
alcaligenes PA-10. Appl Microbial Biotechnol 74(4):851---856
Jacques RJS, Okeke BC, Bento FM, Peralba MCR, Camargo FAO (2007)
Characteriz.ation of a polycyclic aromatic hydrocaibon-degrading
microbial consortium from a petrochemical sludge land farming
site. Biorem J 11:1-11
Kelley I, Cernigilia CE (1995) Degradation of a mixture of high-
molecular-weight polycyclic aromatic hydro-carbons by a
Mycobacterium strain PYR-1. J Soil Contam 4:77-91
Khanna P, Goyal D, Khanna S (2011) Pyrene degradation by Bacillus
pumilus isolated from crude oil contaminated soil. Polycycl Aromat
Compd 31(1):1-15
Klug MJ, Markovetz AJ (1971) Utilization of aliphatic hydrocarbons by
microorganism. Adv Microb Physiol 5:1-4
Kumari B, Singh SN, Singh DP (2012) Characterization of two
biosurfuctant producing strains in crude oil degradation. Process
Biochem 47:2463--2471
Ma B , Chen H, He Y, Xu JM (2010) Isolations and consortia of PAH-
degrading bacteria from the mizosphere of four crops in PAH contam-
inated field. In: I 9th World Congress of Soil Science, Soil Solutions for
a Changing World Brisbane, pp 63---66 (published on DVD)
Mandri T, Lin J (2007) Isolation and characterization of engine oil
degrading indigenous microorganisms in Kwazulu-Natal, South
Africa. Afr J Biotechnol 6(1):23-27
Mittal A, Singh P (2009) Isolation ofhydrocarlxm degrading bacteria from
soils contaminated with crude oil spills. Indian J Exp Biol 47:760-765
Mulligan CN, Gibbs BF (2004) Types, production and applications of
biosurfactants. Proc Indian Natl Sci Acad 70:31-55
Nguyen TT, Youssef NH, Mcinerney MJ, Sabatini DA (2008)
Rhamnolipid biosurfactant mixtures for environmental remediation.
Water Res 42:1735-1743
Nie M, Ym X. Ren C, Wang Y, Xu F, Sheu Q (2010) Novel rhamnolipid
biosurfactants produced by a polycyclic aromatic hydrocarbon
degrading bacterium Pseudomonas aeruginosa strain NY3 .
Biotechnol Adv 28(5):635--643
Nievas ML, Commendatore MG, E stevas JL, Bucala V (2008)
Biodegradation pattern of hydrocarbons from a fuel oil-type com-
plex residue by an emulsifier producing microbial consortium. J
Hazard Mater 154:96-104
Nilsson WB, Strom MS (2002) Detection and identification of bacterial
pathogens of fish in kidney tissue using terminal restriction length
polymmphism (T-RFLP) analysis of 16S rRNA genes. Dis Aquat
Org 48:175-185
Oberbremer A. Miiller-Hurtig R, Wagner F (1990) Effect of the addition of
microbial surfactants on hydrocarlxm degradation in a soil population
in a stirred reactor. Appl Microbial Biotechnol 32(4):485-489
Olivera NL , Commendatore MG, Delgado 0 , Esteves JL (2003)
Microbial characterization and hydrocarbon biodegradation
~ Springer
234
potential of natural bilge waste microflora. J Ind Microbiol
Biotechnol 30(9):542-548
Pacwa-Plociniczak M, Plaza GA, Piotrowska-Seget Z, Cameotra SS
(2011) Environmental applications of biosurfactants: recent ad-
vances. J Mol Sci 12:633--654
Phan CW, Bakar NFA, Ham7.ah A (2013) A comparative study on
biosurfactant activity of crude oil-degrading bacteria and its corre-
lation to total petroleum hydrocarbon degradation. Biorem J 17(4):
240-251
Rahman KSM, Banat IM, Thahira J, Thayumanavan T ,
Lakshmanaperumay P (2002) Bioremecliation of gasoline con-
taminated soil by a bacterial consortimn amended with poultty litta-,
coir pith and rhamnolipid biosmfuctant Bioresour Technol 81 :25-32
Saikia RR, Deka S, Deka M, Banat IM (2012) Isolation ofbiosurfactant-
producing Pseudomonas aeroginosa RS29 from oil-contaminated
soil and evaluation of different nitrogen sou= in biosurfactant
production. Ann Microbiol 62:753-763
Samanta SK, Singh OV, Jain RK (2002) Polycyclic aromatic hydrocar-
bons: environmental pollution and bioremediation. Trends
Biotechnol 20(6):243-248
Sawhney SK, Singh R (2000) Introductory practical biochemist!)'. Narosa
Publishing House, New Delhi, pp 16-17
Singh C, Lin J (2008) Isolation and characte:riz.ation of diesel oil
degrading indigenous microorganisms in Kwazulu-Natal, South
Africa. Afr J Biotechnol 7(12):1927-1932
~ Springer
Ann Microbiol (2015) 65:225-234
Tamura K, Peterson D, Peterson N, Stecher G , Nei M, Kumar S (2011)
MEGA5: molecular evolutioll31)' genetics analysis using maximum
likelihood, evolutionary distance, and maximum parsimony
methods. Mol Biol Evol 28(10):2731 -2739
Thenmozhi R, Somalaksmi AN, Nagasathya A, Praveenkumar D,
Thajuddin N (2011) Characterisation ofbiosurfactant produced by
bacterial isolates from engine oil contaminated soil. Adv Environ
Biol 5(8):2402-2408
Toledo FL, Calvo C, Rodelas B, Lez-Lopez GJ (2006) Selection and
identification of bacteria isolated from waste crude oil with polycy-
clic aromatic hydrocarbons removal capacities. Syst Appl Microbiol
29:244-252
Viramontes-Ramos S, Portillo-Ruiz MC, Ballinas-Casarrubias ML ,
Torres-Muiioz N, Rivera-Chavira BE, Nevarez-Moorillon GV
(20 l 0) Selection of biosurfitctant/bioemulsifier-producing bacteria
from hydrocarbon contaminated soil. Braz J Microbiol 41 :668-675
Wong JWC, Fang M, Zhao Z, Xing B (2004) Effect of surfactants on
solubilization and degradation of phenanthrene under thermophilic
conditions. J Environ Qual 33:2015-2025
Ye D, Siddiqi MA, Maccubbin AE, Kumar S, Sikka HC (1996)
Degradation of polynuclear aromatic hydro-carbons by
Sphingomonas paucimobilis. Environ Sci Technol 30:136-142
Yuliani H, Sablan M, Hermansyah H , Wtjanarko A (2012) Selection and
identification of polyaromatic hydrocarl>on degrading bacteria.
World Appl Sci J 20(8):1133-1138
4 REVISED ROAD MAP TO THE PHYLUM FIRM/CUTES
Genus Bacillus
The majority of the Bacillus species with \<-alicUy published names
are phylogenetically grouped into subclusters within this genus.
However, some "-alidly named species of Bacillus are not phylo-
genetically related to the type species, B. suhtilis, and are more
closely related to other genera. The phylogenetic subclusters
·within the genus Bacillus are:
a: Bacillus suhtilis, amylnliquef aciens, atrophaeus, mojauensis, licheni-
f urmis, W1wrensis, vallismortis, including the very likely misclassi-
fied Paenibacillus popi1liae.
b: Bacillus f arraginis, furdii, f ortis, lentus, galnctosidilyticus
c: Bacillus asahii, batauiensi5, beraoeil(Jfans, circulans, colmii, fir-
mus, fle=,s, famarioli, inf emus, jeotgaJi, luciferensis, megat,erium, meth-
anolicus, niacini, naualis, psychrosaccharolJticus, simplex, soli, ttimi
d: Bacillus anthracis, r.ereus, mycoides, thuringiensis, weihenstepha-
rumsis
e: Bacillus aquimaris, maruflaui
f Bacillus badius, coagulans, thermoamylovorans, acidicola,
oleronius, sporothermodurans
g: Baci/fus alcalophilus, arsenicoseJenat clausii, gibsmzii, ha!Ddumns,
lwrikoshi~ kndwichiae, okhensis, okuhidensis, psewloalmliphilus,
pseudofamus
h: Bacillus tmenicus, barnaricus, gelatini, decolomtionis,
i: Bacillus carixmiphilus, endaphyticus, smithii,
j: Bacillus pallid1ts,
k: Bacillus fanicul1ts, panaciterrae
The Bacillus cluster contains three additional groups of
related genera: Anoxybacillus, Geobacillus, and Saccharococcus.
In addition to these taxa, which compose the family Bacillaceae
sensu stricto, other phylogenetic groups have been assigned to this
family (Garrity et al., 2005). Although the largest group appears to
warrant elevation to a novel family, it is retained \\ithin the Bacil-
laceae in the present outline. lhis cluster comprises the genera
.4.lkalibacillus (new; Jeon et al., 2005), Amphibacillus, Cerasibacilh.LS
(ne\\~ Nakamura et el., 2004), Filohacillus, Gracilibacillus, Halobacil,-
lus (new; Spring etal., 1996), Halolactibacillus (new; lshika\<\.t et al.,
2005), Lentihacilltts, Ot:eanobacillus, Paraliobacillus, Paucisalihacillus
(new; Nunes et al., 2006); not described in the current volume),
Pantibacillus, Salibacillus (not described in the current volume),
Tenuibacillu.s, Thala.ssobacillus (ne\\~ Garcia et al., 2005), and Vngiba-
cillu.s. The type strains of other species are positioned phylogeneti-
cally among the members of this lineage and merit taxonomical
emendation: BacilbJ.s halnplulm and Bacillm theroux:loacae, Sinococ-
cus, and iUarinocoecus. For this reason, ,UariTWCoccuswas transferred
from the Sfxm>lactobacillaceaein the current outline.
In addition, the genera Umbacillus, Marinibacillus, Je.otgaliba-
cillus, and Exiguobacterium were previously assigned to the Bacil-
laceae (Garrity et al., 2005). Ureibacillus fulls within the clade
represented by PlaTWCoccat:eae, and it was reassigned to that family.
Marinihacillus and Jeotgalihacillu.s are closely related to each other
as well as to Bacillus aminu110rans. This group is distantly related to
the Planococcaceae, and they are also assigned to that family. Lastly,
E"<iguobactmum is not closely related to any of the described fami-
lies, and it is assigned to a Family XII lncettae Sedis in the current
roadmap.
Bacillus schlegrdii and Bacillus solfatamerepresent their own deeply
branching lineage of the "Bacilli'.and warrant reclassification.
Family UA/icyclobacillaceae"
Only the type genus Alicycwbacillus is retained in this family, and
two genera pre\.iously classified with the Alicyclobacillaceae have
been reclassified (Garrity et al., 2005). According to the new
16S rRNA sequence analyses, Sulfobacillus represents a deep
branch of the "Clostridia", and it is now placed ,\ithin Family
XVII Incertae Sedis of the Cl,ostridiales. Pasteuria, which was also
previously classified within this family, is an obligate parasite of
invenebrates. Vl7hile it can be cultivated "ithin the body of its
prey, it has not been cultured axenically. Because of the substan-
tial phenotypic differences and low l 6S rRNA sequence similarity
with Alicycwbacillus, it is now classified \<\ithin its own family, Pas-
teuriaceae (see below). Lastly, Alicycl,obacillus possesses a moder-
ate relationship to Bacillus tusciae, which could be reclassified
to this family.
Family "Listeriaceae"
The monophyletic family "Listeriaceae" combines tl1e genera
Listeria and Brochothrix as in tl1e previous outline.
Family uPaenibacil/aceae"
The members of the £nnily "Paenibacillaceae are distributed .
between two ph}iogenetic dusters. Paenibacillus, Breuihacilhts, OJ/1-
nella (ne,\~ Kampfer et al., 2006) and Themwbacillus share a com-
mon origin and represent the first group. Some ,aal.idly named
Bacillus species are found among the Paenibacillus species: Bacillus
chitinoryticus, edaphicus, ehimensis, and mucilagi1wsus. The second
group comprises the genera Aneurinibacilltts, A.m11Wniphilus, and
O:cak,phagus. Although not clearly monophyletic, these two clusters
are often associated together in several types of analyses. TI1us, in
the absence of clear evidence for a separation, the second cluster
is retained l\ithin the family. In contrast, Thennicanus, which was
classified \\ithin this family by Garrity et al . (2005), appears to
represent a novel lineage of the Bacilli. In recognition of its
ambiguous status, it was reclassified l\ithin Family X Incertae Set/is.
Family Pasteuriaceae
This family contains Pasteuria, an obligate parasite of inver-
tebrates which has not yet been cultivated outside of its host.
Although this genus ~.tS previously classified within the
"Alicyclobacillaceae~, the current analyses suggest that it is
more closely associated with the "Thermoactinomycetaceae. In
spite of the similarities in morphology and rR."IJA sequences
between Pasteuria and Thermoactinomycetes, these genera were
not combined into a single family for two reasons. First, in the
absence of an axenic culture of Pasteuria, additional pheno-
typic and genotypic evidence for combining these organisms
into a single family are not available. Second, the obligately
pathogenic nature of Pasteuria was judged to be distinctive
enough to warrant a unique classification in the absence of
evidence to the contrary.
Family Planococcaceae
The family Plan-0coccaceoe is a clearly monophyletic 1mit that con-
tains the genera Planococcu.s, Filihactei; Kmthia, Planomiavbium,
and Spomsarcina as well as three genera transferred from tl1e Bacil-
laceaR (]eotgalibacilltts, l\1arinibacillus, and Uwibacillu.s) and Caryo-
phanmz. Caryupha11on is the only genus of the Caryophanaceae in
42 FAMILY I. BACILLACEAE
along with one or more agents to which the organism is nor-
mally sensitive; given supportive sensitivity testing, a penicillin
may be used to complete treatment. The same approach
is recommended for cutaneous infections (Bell et al., 2002).
Doxycycline does not penetrate the central nervous system well,
and so is not appropriate for the treatment of meningitis.
Despite the well-established importance of Bacillus cereus as
an oppoITWlistic pathogen, there have been rather few studies
of its antibiotic sensitivity, and most information has to be
gleaned from the reports ofindividual cases or outbreaks. Bacil-
lus cereus and Bacillus thuringiensis produce a broad spectrum
!3-lactamase and are thus resistant to penicillin, ampicillin, and
cephalosporins; they are also resistant to trimethoprim. An
in vitro study of 54 isolates from blood cultures by disk diffusion
assay found that all strains were susceptible to imipenem and
\,'ancomycin and that most were sensitive to chloramphenicol,
ciprofloxacin, ery-rhromycin and gentamicin (with 2, 2, 6 and
7% strains, respectively, showing moderate or intermediate sen-
sitil'ities), while 22 and 3 7% of strains showed only moderate or
intermediate susceptibilities to clindamycin and tetraclycline,
respectively (Weber et al., 1988); in the same study, microdi-
lution tests showed susceptibility to imipenem, vancomycin,
chloramphenicol, gentamicin and ciprofloxacin with MICs of
0.25-4, 0.25---2, 2.0-4.0, 0.25---2 and 0.25---1.0mg/l, respectively.
A plasmid carrying resistance to tetracycline in Bacillus cereus
has been transferred to a strain of Bacillus subtilis and stably
maintained (Bernhard et al., 1978).
Although strains are almost always susceptible to clindamy-
cin, erythromycin, chloramphenicol, vancomycin, and the
arninoglycosides and are usually sensitive to tetracycline and
sulfonamides, there have been several reports of treatment fail-
ures ,~1th some of these drugs: a fulminant meningitis which
did not respond to chloramphenicol (Marshman et al., 2000);
a fulminant infection in a neonate which 'l\'aS refractory to
treatment that included vancomycin, gentamicin, imipenem,
clindamycin, and ciprofloxacin (Tuladhar et al., 2000); failure
of \'allcomycin to eliminate the organism from cerebrospinal
fluid in association with a fluid shunt infection (Bemer et al.,
1997); persistent bacteremias with strains sho\\ing resistance to
vancomycin in two hernodialysis patients (A von Gottberg and
,v. \'all Nierop, personal communication). Oral ciprofloxacin
has been used successfully in the treatment of Bacillus cereus
wound infections. Clindamycin \\1th gentamicin, given early,
appears to be the best treatment for ophthalmic infections
caused by Bacillus cereus, and e:iq>eriments with rabbits suggest
that intravitreal corticosteroids and antibiotics may be effective
in such cases (Liu et al., 2000).
Information is sparse on treatment of infections \\1th other
Bacillus species. Gentamicin \\'aS effective in treating a case
of Bacillus licheniformis ophthalmitis and cephalosporin \\'aS
effective against Bacillus lichenifrmnis bacteremia/septicemia.
Resistance to macrolides appears to occur naturally in Bacillus
lichenifunnis (Docherty et al., 1981). Bacillus subtilis endocarditis
in a drug abuser was successfully treated with cephalosporin,
and gentamicin \\'aS successful against a Bacillus suhtilis septi-
cemia. Penicillin, or its derivatives, or cephalosporins probably
form the best first choices for treatment of infections attributed
to other Bacillus species. In the study by Weber et al. (1988),
isolates of Bacillus megat.erium (13 strains), Bacillus pumilm (4),
BacilJ11.5 subtilis (4), Bacillus circulans (3), Bacillus amyloliquefaciens (2)
and Bacillus lichenifunnis (1), along \\ith five strains of Bacil-
lus (now Paenibacillus) polymyxa and three unidentified strains
from blood cultures, over 95% of isolates were susceptible to
imipenem, ciprofloxacin and vancomycin; while between 75%
and 90% were susceptible to penicillins, cephalosporins and
chloramphenicol. Isolates of" Bacillus polymyxa" and Bacillus cir-
culans were more likely to be resistant to the penicillins and
cephalosporins than strains of the other species -it is possi-
ble that some or all of the strains identified as Bacillus circu-
lans might now be accommodated in Paenihacillus, along with
"Bacillus polymyxa." An infection of a human bite wound with
an organism identified as Bacillus circulans did not respond to
treatment with amoxycillin and flucloxacillin, but was resolved
with clindamycin (Goucm,'aard et al., 1995). A recurrent septi-
cemia with Bacillus subtilis in an immunocompromised patient
yielded two isolates, both of which could be recovered from the
probiotic preparation that the patient had been taking; one
isolate \\'aS resistant to penicillin, erythromycin, rifumpin and
novobiocin, while the other'l\'aS sensitive to rifampin and novo-
biocin but resistant to chloramphenicol (Oggioni et al., 1998).
A strain of Bacillus circulnns showing 'v'allcomycin resistance has
been isolated from an Italian clinical specimen (Ligozzi et al.,
1998). Vancomycin resistance \\'aS reported for a strain of Bacillus
(now Paenihacillus) popilliaein 1965, and isolates of this species dat-
ing back to 1945 have been shmm to carry a vanA-and vanB-like
gene, that is to say a gene resembling those responsible for high-
level \,'allcomycin resistance in enterococci. Vancomycin-resistant
enterococci (\IRE) were first reported in 1986, and so it has been
suggested that the resistance genes in Bacillus popilliae and VRE
may share a common ancestor, or even that the gene in Bacillus
popillwe itself may have been the precursor of those in VRE; Bacil-
lus popilliae has been used for over 50 years as a biopesticide, and
no other potential source of van.4 and vanB has been identified
(Rippere et al., 1998). Of two South African vancomycin-resistant
clinical isolates, one ,~'aS identified as Poenibacillus thiamitwf)'ticus
and the other 'l\'aS unidentified but considered to be related to
Bacillus lentus (Forsyth and Logan, unpublished); the latter \\'aS
isolated from a case of neonatal sepsis, and has been shown to
have inducible resistance to \-'llllcomycin and teicoplanin; this is
in contrast to the Bacillus cimdans and Paenibacillus thiamirw~vticus
isolates mentioned above, in whicl1 expression of resistance \\'as
found to be constitutive (A. von Gottberg and W. ,'all Nierop,
personal communication).
Isolates of novel Bacillm species from pristine .'\ntarctic envi-
ronments showed sensiti,ity to: ampicillin, cl1loramphenicol,
colistin sulfate, kanamycin, nalidixic acid (Bacillm fwnarioli
resistant}, nitrofurantoin, streptomycin and tetracycline (L-ogan
et al., 2000, 2002b, and unpublished information).
Pathogenicity. The majority of Bacillus species apparently
have little or no pathogenic potential and are rarely associated
with disease in humans or other animals. The principal excep-
tions to this are Bacillus anthracis (anthrax), Bacillus cereus (food
poisoning and opportunistic infections), and Bacillm thuringi-
ensis (patl10genic to invertebrates), but a number of other spe-
cies, particularly Bacillus lichenifrmnis, have been implicated in
food poisoning and other human and animal infections. The
resistance of the spores to heat, radiation, disinfectants, and
desiccation also results in Bacillus species being troublesome
contaminants in the operating room, on surgical dressings, in
pharmaceutical products and in foods.
J Microbial Biotechnni. (2012), 22(12), 1597--1604
htep:/ldx.doi.org/] 0.40 I4/jmh.1204.04013
First published online October 4, 20 12
plSSN 1017-7B25 e1SSN i738-8872
RE1'11:►1
JOURNAL
MICROBIOLOGY
BIOTECHNOLOGY
Th. animr smcti 1v htvcvotgr ad iscr....K+wvv
Classification of Bacillus Beneficial Substances Related to Plants, Humans
and Animals
Moagkolt'.hanaruk, Wiyada*
Department of Microbiology. Faculty of Science, Khon Kaen University 123 Miiraparp Road, Khan Kaen 40002, Thailand
Received: April 9, 20121 Revised: July 28, 2012 / Accepted: July 30, 2012
Genus Bacillus is a spore -forming bacterium that has
unique properties in cell differentiation, allowing the
forming of spores in stress conditions and activated in the
vegetative cell, with suitable environments occurring
during the life cycle acting as a trigger. Their habitat is
mainly in soil; thus, many species of Bacillus are associated
with plants as well as rhiensphere bacteria and endopkytic
bacteria. Signal transduction is the principal mechanism
of interactions, hoth within the cell community and with
the external environment, which provides the subsequent
functions or properties for the cell. The antimicrobial
compounds of Bacillus sp. are potentially useful products,
which have been used in agriculture for the inhibition of
pbytopatbogens, for the stimulation of plant growth, and
in the food industry as probiotics. There are two systems
for the synthesis of these substances: nonribosomal
synthesis of cyclic lipopeptides (NRFS) and polyketides
(PKS). For each group, the structures, properties, and
genes of the main products are descried. The different
compounds described and the way in which they co -exist
exhibit the relationship of Bacillus substances to plants,
humans, and animals.
Keywords: Quorum sensing. quorum quenching, cyclic
lipopeptides, polyketides, bacteriocins
Spore -forming Bacillus species have been well known for
many years as plant biocontrol producers (fungicides,
bactericides, and fertilizers), probintics, and pathogens.
Their properties are diverse depending on the species or
subspecies. The Bacillus sp. is used as a genetic model of
Gram-positive bacteria that show a large number of swains
in the database for the whole genome. They are also
closely related to many species in the group Bacillaceae
*Corresponding author
Phone: [phone]; Fax: [phone]:
E-mail: [e-mail]
such as B. subnTis, B. anylaliquefaciens, and 8. Iicheniformis;
the distance of the number of nucleotide substitutions
between species is less than 031 [62]_ According to the
genetics, gene transfer occurs spontaneously among these
stains. The gene evolution is also caused by horizontal
gene transfer; for example, gene transfer from Archaea to
Bacillus species is about 1.7% [24]. B. subtilis has been
observed in the upper layers (1-3 cm) of a variety of soils.
Through sporulation, B. sut5tilis adapts to unfavorable
conditions with a highly resistant dormant endospore,
surviving for years before revitalization via spore
gemination and outgrowth. Endospore formation takes the
unusual form of asymmetric cell division, followed by the
engulfment of the forespore by the mother cell. Dormant
spores show properties that differ from those of growing
cells, with an increased resistance to the effects of
chemicals, heat, mechanical disruption, W irradiation,
and enzymes [56]. Each spore comprises a thick;
proteinaceous shell — known as the coat — and internally a
cortex, inner membrane, and core_ Analysis of these
components explains the high resistance of the dormant
spore, and further shows their changing roles when spore
germination is triggered by an external catalyst. A
germinant penetrates the coat and cortex to reach a
gemination receptor in the inner membrane. Depending
on the particular germinant and specific receptor of the
spure, a range of gene products and their coding genes (ger
genes) are exhibited and identified by mutations at various
stages of spore germination [9, 15, 22, 39, 47]. The various
mutants show differing responses to germinants as a result
of gene and operon duplication and divergence. In addition
to nutrients, spores are germinated by a variety of non -
nutrient stimuli [16, 55], which allows the non -nutrient
chemical-, enzyme-, or pressure -based germination of
spores in many species of endospore-forming bacteria.
Why have these spores remained of interest to many
fields in current study? Because they contain endospores
that can release to be free in environments. The spores

l598 Wiyada Mongkolthanaruk
have cortex peptidoglycan and a coat structure, which
combines obvious different structures and protects the
spore cell from stress conditions [36, 4-0, 41]. The spores
can become animated when they are triggered by some
nutrients or chemicals, and then go through outgrowth,
increasing the amount of vegetative cell. It is difficult to
kill the Bacillus strains; this is an advantage for beneficial
applications as they have a long life cycle, but is a
disadvantage for controlling spore-forming diseases.
Bacillus anthracis, animal and human pathogens known as
anthrax, is a biological weapon worldwide and remains a
serious problems for the US army [23] as it cannot be
killed completely from the spores. Knowledge of the
sporulation [20] and germination processes [37] is the
major key to understanding and controlling this organism.
To scientific advantage, Bacillus sp. is the friendliest
species for the human, animal, plant, and environment. It
also produces different kinds of enzymes used in industry,
such as amylase, lipase, protease, and laccase [50J. The
Bacillus species is found mainly in soil and in the
gastrointestinal tract in both animals and humans. As such,
it has been reported to be an antimicrobial producer and
probiotic bacteria.
In this article, the antimicrobial compounds produced
from Bacillus sp. are the effective products for beneficial
uses. There are two systems involved in antimicrobial
compounds synthesis: nonribosomal peptide synthetases
(NRPS) and polyketide synthases (PKS). The bacterial
community is also involved in the regulation of synthesiz.ed
metabolites and the efficiency of compound activities in
environments.
INTERACTION AND COMMUNITY OF BACILLUS SP. IN
HABITATS
The fundamental function of active compounds from
Bacillus species is the inhibition of plant pathogens. Many
species are plant-associated bacteria, such as B. subtilis, B.
amyloliquefaciens, B. licheniformis, B. pasteurii, B.
cereus, B. pumilus, B. mycoides, and B. sphaericus, which
have been reported to be elicitors of induced systemic
resistance (ISR) [29]. The bacteria associate with plant-
form sessile biofilm ( exopolysaccharide/ lipopolysaccharide)
to attach and colonize on the plant surface. The genes
involved in biofilm and fruiting body formation are epsA-0,
which is an operon consisting of 15 gene exopolysaccharides
in B. subtilis [25]. The swarming motility controlled by
gene swrA is also involved in the colonization of surfaces.
Furthermore, the genes RBAM00750, RBAM00751, and
RBAM00754 of B. amyloliquefaciens are involved in
surface adhesion or biofilm formation [6]. Endophytic
bacterium B. subtilis was isolated from plant stem and also
showed strong inhibition of phytopathogens [31 ]. The
mechanism of entry to the plant cell is not demonstrated;
colonization of bacteria in the cell may involve the biofilm
and fruiting body formation as described above. The
rhizobacteria can use root exudates as signal molecules to
penetrate into the root of a plant. The bacterial populations
behave as biofilm communities for attachment and
aggregation with plants. Then, bacterial colonies move
into the root system through the swanning process. It has
been reported that a Bacillus cyclic lipopeptide, surfactin,
has the role of stable biofilm formation and is involved in
surface motility [4, 26]. Cell-cell communication is an
important mechanism for a microbial community in habitats,
known as quorum sensing. The quorum-sensing system
has a function to coordinate gene expression and regulate
virulence production [14]. There are two types of signal
communication; intraspecies communication and interspecies
communication. The interspecies signaling, such as
antibiotics molecules, can transfer the signal to other
bacteria; thereby the bacteria can also "eavesdrop" and
lead to alteration in factors contributing to the virulence or
persistence of bacterial pathogens as well as influencing
the development of beneficial microbial communities. The
intraspecies signaling is involved in virulence factors,
biofilm, and acts as an autoinducer [53]. Therefore, if the
quorum sensing is interrupted, it will decrease the
virulence of pathogens. Gram-negative bacteria produce
N-acyl homoserine lactones (AHLs) as a signal molecule
showing virulence cell of pathogens, whereas Gram-
positive bacteria have modified peptides to signal
virulence regulation, called autoinducing peptides (AIP).
The AIP of Staphylococcus aureus are peptides consisting
of 7-9 amino acids that form a thiolactone ring at the C-
terminal (Fig. I). The ComX and CSF (Competence and
Sporulation Factor, also PhrC) peptides are extracellular
signaling of B. subtilis; these affect its differentiation. The
signal peptides are generated by cleavage from larger
precursor peptide, and subsequent modifications by substitution
with isoprenyl groups, resulting in the formation oflactone
and thiolactone rings and lanthionines [3, 35]; for example,
the c-butyrolactones of Streptomyces spp. and !antibiotic
(]anthionine-containing antibiotic) mersacidin of Bacillus
sp. [53, 57].
1n contrast, there are 3 types of quorum quenching,
which is enzymatic degradation of AHLs; known as · AHL
acylases (or AHL amidases), AHL lactonases, and AHL
oxidoreductase. The AHL acylases cleave the molecule
( quorum sensing) into a free homoserine lactone and a fatty
acid, whereas the AHL lactonases hydrolyze the lactone
ring, yielding a homoserine [2, 11]. The third enzyme also
cleaves the molecule to yield homoserine lactone [59].
Quorum-quenching bacteria in environments have been
reported upon; each sample obtained at least one quorum-
quenching bacterium [8]. For instance, B. thuringiensis is
able to break down the signal of quenching through
AIP 0 AIP-IV
000000
CLASSIFICATION of BACH1.11S BET:EFiL`[AL SUBSTANCES 1599
Cora
CSF
rig. I. The four AlP signals of S. aurora and the CosnX and CSF peptides signaling of B suhfilis.
The amino acid sequence of each signal is shown. The AN signals are boxed into three inhibitory classes and show the cross -inhibitory esoups. Any I End
AIP-IV differ by only one amino acid and function interchangeably [32, 34, 481.
degradation with MIL lactonasses (AiiA), resulting in the
silencing of virulence in pathogens without changing the
number or composition of cells [45, 63]. The AiiA,
quorum -quenching activity, was found in Bacillus spp. and
also found in Grarn-negative bacteria, such as Agrobacterium
lumefaciens, Pseudomonas aeruginosa, Arthrobacter sp.,
Rh&iococcus sp., Variovorar paradoxes, and Acinciabacrer
[46, 51]. The AiiA showed another role in the rhizosphere
competence of B. rhuringiensis on the plant mot system,
suggesting that AiiA is involved in the cell metabolism or
survival mechanism during cell growth [45]. Therefore,
the viability of B. thuringiencis is protected from AiiA to
form root colonization.
GROUPS OF BACILLUS SUBSTANCES AND THEIR
APPLICATIONS
Bioactive compounds of Bacillus sp. are divided into two
systems; (i) nonribosomal synthesis of cyclic iipopeptides
(NRPS) and (ii) polyketides (PKS), which are controlled
by many genes (Table 1). Most of the genes are clustered
Table 1. Major compounds from nonnibosomal peptide synthetases and polyketide syntheses.
Compounds Gene cluster involved Bacillus species
Functions
Nonribosomaal peptide synthelases (I RPS)
Iturin itu, !pa
Fengycin fen, Pps
5urfaetin srf, vex. nut, sfp
BacillomyciniY
Bacillibactin
Putative peptide
Baci l ysinientitaps in
ZwittermieinA
Polyketide syntheses (PKS)
Subtilin
SubtilosinA
TasA
Sublancin
Macrolactin
Bacillaene"
Difficidin
Mersacidin
bmy
dhh
nrs
bac. yw f
zwit
spec
sba, alb
las
sun, bdb
min
bae, pksX
cif
mrs
B. subrilis, B. amylaliquefaciens
B_ subtills. B. amyloliquefaciens
B. subrilrs, B. amylaliquefaciens
B. amyloliquefaciens
B. sublilis, B. amylaliquefaciens
B. amylaliquefaciens
B. subtilis, 1 amvloliquefaciens
B. cereus
B_ subrilis
B. subrilis, B. amylaliquefaciens
B. svbiilis
B_ subrilis
B. amylaliquefaciens
B. subrilis, B. amylaliquefaciens
B. amylaliquefaciens
B. amvloliquefaciens
Antifungal, hemolytic activities
Anti -filamentous fungi
Antiviral, aniimycoplasma activities
(Vollenbroich er al. [61 j)
Antifungal
Iron transport system; siderophores
Siderophores (Benner et al. MI)
Antimicrobial activity
Broad spectrum of antibacterial
(]ieela/. [19j)
Antimicrobial activity (lantibioric)
Antibacterial activity
Antibacterial activity
Antimicrobial activity (not 'antibiotic)
Anti -Gram-positive bacteria
Antibacterial activity
Antibacterial activity
Inhibit cell wall biosynthesis,
anti-Gram-positivc bacteria
(Bros er al. [5])
Refers to compounds produced from both NRPS and PKS_

1600 Wiyada Mongkolthanaruk
lturin structure
HO
Fig. 2. The structures ofiturin, surfactin, and fengycin (KEGG structure).
The cyclic lipopeptides contain fatty acid chain linked ·with amino acids (see text). The derivatives of compounds in each group come from different amino
acid components.
in the operon and are driven by multiple cascade pathways.
Different species of Bacillus offer different advantages in
biotechnology niches, due to divergent characteristics.
Lipopeptides (LPs) are active compounds showing
antimicrobial activity and act as immune stimulators by
reinforcing host resistance in terms of root colonization.
The cyclic LPs consist of surfactin, fengycin or plipastatin,
and iturin families; these are major groups containing base
structures related to other NRPS products. In the iturin
family, the main structure is heptapeptides linked to a 13-
amino fatty acid chain with a length of 14 to 17 carbons
(Fig. 2). The differences in heptapeptides show derivative
compounds of iturin (e.g., bacillomycin, mycosubtilin).
The members of surfactin are composed of a 13-hydroxy
fatty acid with 7 amino acids, whereas 10 amino acids
linked to a fl-hydroxy fatty acid chain show in fengycin
groups. The LPs have two roles in direct antagonism;
through the interruption of membrane permeabilization
properties of pathogens, and through root colonization
in the rhizosphere competence to induce host plant
immunization. However, the LPs cannot act with only one
function to cope with pathogenic organisms. They act in a
synergistic manner and also work with other rhizosphere
microbial populations. The control of plant diseases has
been reported with surfactin and iturin [34], surfactin and
fengycin [44], and iturin and fengycin [52]. Moreover, the
effect of these compounds on plant cells is specific to the
kind of plant because of differing composition in
phytosterols, mainly composed of sitosterol, stigmasterol,
campesterol, and in some species cholesterol [43]. These
affect the penetration of compowids or microbes in order
to protect host plants. The transcriptional mechanisms are
also important keys to synthesis compounds; these are
controlled by both nutritional conditions (carl>on, nitrogen,
iron) and physicochemical conditions (temperature, pH,
oxygen). The expression of surfactin is pH dependent,
whereas that of mycosubtilin is oxygen dependent [17].
Both temperature and pH influenced iturin D and
subtilosin A expressions [60]. Effective plant protection,
it is proposed, is to form a consortium that produces
various compowids in synchronicity, and independent to
environments. The consortium should be developed in
signal cell communication, transcriptional expression, and
the secretion system towards widespread use in host plants.
Macrolactin
Ba<;illaene
Difficidin
Fig. 3. Structmes of macrolactin (min, formerly pks2), bacillaene
(bac, formerly pksl), and difficidin (dif, formerly pks3),
polyketides of Bacillus spp. [54]
Polyketides are active compounds that exhibit major
antibacterial, immunosuppressive, or antitumor activities.
Their syntheses have nbosomal mechanisms using the
polyketide synthase gene cluster (PKS multienzyme
system). There are 3 operons in the PKS (pksI,pks2 and
pks3), which are identified in B. amyloliquefaciens [7].
The pks I, pks2, and pks3 genes use the same biosynthesis
pathway, type I PKS containing j3-ketoacyl synthase (KS),
trans-acyltransferase (AT), and acyl carrier proteins (ACP)
for basic domain, and subsequently the intermediate
compounds might combine with different groups in the
elongation step to generate individual compounds or a
novel bioactive compound. Bacillaene, encoded by the bae
(pks 1) gene, contains a linear structure conjugated to
hexaene (Fig. 3). The macrolactins consist of 24-
membered ring lactones with modifications, such as the
attachment of glucose ~-pyranoside, or they occur as linear
analogs [54]. This is controlled by the min (pks2) gene.
The dif(pks3) genes produce difficidin/oxydifficidin that is
a highly unsaturated 22-member macrolide with a rare
phosphate group. The modular PKS system starts at the C3
precursor and terminates at module 11 for macrolactin [54]
and difficidin [7], whereas the bacillaene synthesis
terminates at modules 16 and 17 [38]. Similar to B. subtilis,
the PKS system produces polyketide-like compounds
having antibacterial activity, which are encoded by the
pksX operon. The bae gene cluster showed similarity to
the pksX sequence region, and they contain two hybrid
NRPS-PKS at some parts of genes. The pks2 and pks3
genes may be transferred from other soil bacteria or
evolved from the ancestral pks operon by several gene
duplications [7]. However, these occurrences are not
CLASSIDCAllON OF BACTUUS BENERCIAL SUBSTANCES 160 I
Non~antibioticSUblancin
Fig. 4. Structures of Bacillus bacteriocins, subtilin, subtilosin A,
and sublancin.
These are single-peptide )antibiotics . Subtilin is a cationic pentacyclic
peptide. Subtilosin A and sublancin show thiol-disulfide bridge fonnation
at cysteines, resulting in inter-residual thioether bond forms[!]. However,
sublancin is not a !antibiotic, as shov.n by a new structure [42]. The non-
lantibiotic sublancin shows S-linked glycopeptides Oinked between a sugar
an!.I cysteine ).
involved in the modular PKS system. B. subtilis lacks the
sjjJ gene, which has the function of 4'-phosphopantetheiile
transferase; this results in an inability to synthesize
bacillaene, macrolactin, and difficidin. However, B. subtilis
can produce difficidin because of Sip-type PPTases (pptS
gene), which is a cognate 4'-phosphopantetheine transferase
(PPfase) for the posttranslational modification of fatty acid
synthases (FAS). The B . subtilis AI/3 contains pksM and
pksR genes for difficidin and bacillaenes biosynthesis [21].
Apart from the PKS system, !antibiotics are ribosomally
synthesized peptides having antimicrobial activity that
are found in Bacillus species; for example, subtilin and
nisin are from B. subtilis, and mersacidin is from B.
amyloliquefaciens [19]. These peptides are classified in
bacteriocins, which are antimicrobial peptides produced by
ribosomal synthesis.
Bacteriocins of genus Bacillus are divided into 3
classes that are independent from lactic acid bacteria
1602 Wiyada Mongkolthanaruk
(LAB) bacteriocins. Class I contains posttranslationally
modified peptides, such as subtilin, mersacidin, lichenicidin,
and subtilosin A; this class is similar to LAB bacteriocins
called !antibiotics. Class II is non-modified peptides (e.g.,
coagulin, thurincin, thuricin, lichenin), and class III features
large proteins (e.g., megacin) [I]. The !antibiotics are well
known, the most characteriz.ed, and used in 1he food,
agricultural, and pharmaceutical industries. They are small
molecules (3-10 kDa), containing precursors of unusual
amino acids (lanthionine and methyllanthionine ), which
are modified by the dehydration of serine and threonine
and the addition of cysteine residues. There are 2 groups of
)antibiotics; linearly shaped )antibiotics (e.g., subtilin,
nisin, epidermin) and globularly shaped lanttbiotics (e.g.,
mersacidin, subtilosin A, sublancin) (Fig. 4). Subtilin has
bactericidal activity synthesized by the spa operon [30]; it
is produced at high levels in starvation conditions. It is
likely that subtilin is released to inhibit oilier bacterial
growth, allowing Bacillus to then uptake greater nutrient
supply. Entianin is a novel compound, a subtilin-like
lantibiotic, of Bacillus subtilis subsp. spizizenii DSM15029;
it has autoinduction and antibiotic activities as subtilin
[13]. The cyclic, anionic peptide subtilosin A contains
head-to-tail amino acids with three disulfide bonds and a
linkage of a thiol to a-carbon of amino acids. It showed
bactericidal activity against human pa1hogens and hemolytic
activity. Sublancin is an S-linked glycopeptide containing
two disulfide bridges; it is not a lantibiotic, as shown in a
revised structure consisting of a sugar linked to cysteine-
22 residues (Fig. 4). It is a stable peptide and tolerant to
bo1h low and high pH; the loss of antimicrobial activity
results from a mistake of glycosylation with 1he correct
disulfide connectivity [42]. Quorum sensing or cell-cell
communication is associated wi1h the induction of
antimicrobial peptides or o1herpeptides as signal transducers.
The bacteriocins have a role as autoinducers in the
activation of gene clusters [27, 28]. Moreover, bacteriocin-
like inhibitory substances (BLIS) have been reported in
various Bacillus species. It is interesting, as it exhibits a
broad spectrum of antimicrobial activity, and is also stable
at a wide range of temperature and pH. The bacteriocins or
BLIS have the potential ability to prevent or control both
spoilage and pathogenic microorganisms. They are applied
in probiotics for human use (as a dietary supplement),
as animal feed, and are found in the food supply as
preservatives.
Within the three groups of antimicrobial compounds,
there are specific applications relating to 1he broad
functions of antimicrobial activity (Table 1 ). The cyclic
lipopeptides are used in crops, to protect plants from
phytopathogens. The compounds are tolerant to enzymes
(pronase, proteinase K) and organic solvents (butanol), and
are stable in low pH and high temperature [52]. These
properties render lipopeptides suitable for usage in agriculture
as a biocontrol and biofertilizing agent. Additionally, the
surfactins are important metabolites involved in quorum
sensing of cell-cell interactions in Bacillus sp. It has a
PPfase-dependent gene involved in NRPS for surfactin
and biofilm formation. Another lipopeptides producer,
Pseudomonas sp., exhibited different systems of pathway
synthesis and possessed good potential compounds for
biocontrol [49]. The polyketide and bacteriosin from PKS
systems are commonly used in pharmaceutical industries.
Moreover, the polyketides are also used as biocontrol
agents and 1he bacteriocins are commonly used in the food
industry as probiotics and preservatives. The Bacillus
species are classified to be generally recognized as safe
(GRAS) strains, and as such their usage is allowed in the
food industry. There are many commercial products
available as dietary supplements for human use (e.g., B.
cereus, B. clausii, B. pumilus [12]), and for veterinary use
(e.g., B. /icheniformis, B. coagulans, B. clausii, B. cereus
[10]). In these applications, the vegetative cell and spore of
Bacillus can stimulate immune systems in the gastrointestinal
tract. The secretion of compounds, such as coagulin,
amicoumacin, and subtilisin, effects the suppression of
pathogens, a characteristic that provides high potential as a
property of probiotics. Therefore, there are three mechanism
steps of Bacillus interaction with hosts; (1) stimulation of
immune systems, (2) suppression of gastrointestinal
pathogens, and (3) secretion of antimicrobial compounds.
Moreover, it has been reported that more than 10% of
inoculated Bacillus spore germination showed growth and
reforming sporulation [58]. However, recognition of
GRAS status, as safe for use in the food industry, is strain
specific; for example, B. subtilis var. Natto is a safe strain
for the production of proteolytic enzyme; B. subtilis
bacterium is not identified as a safe strain. This genus
produces the Bacillus enterotoxins, Nhe and Hbl, which
are unsafe for humans. There are many applications of
Bacillus substances apart from those previously mentioned,
including use as biosurfactant, biofuel, and with various
enzymes in various industries. Therefore, it is necessary to
closely consider the specific purposes in the analysis of
synthesis pathway and gene regulation of various
metabolites, and then to match the unique properties to
specialized roles. There is also a need for further study of
cell differentiation in spore-forming bacteria, with greater
knowledge enabling the identification and development of
the products of genus Bacillus towards wide-ranging
commercial applications.
Acknowledgments
The author would like to thank Khon Kaen University and
the Protein and Proteomics Research Group, Khon Kaen
University for their assistance.
REFERENCES
I. Abriouel, H., C. Franz, N. B. Omar, and A. Galvez. 2011.
Diversity and applications of Bacillus bacteriocins. FEMS
Microbiol. Rev. 35: 201-232.
2. Amara, N., B. P. Krom, G F. Kaufmann, and M. M. Meijler.
2011. Macromole.cular inhibition of quorum sensing: Enzymes,
antibodies and beyond. Chem. Rev. 111: 195-208.
3. Ansaldi, M., D. Marolt, T. Stebe. I. Mandic-Mulec, and D.
DubnatL 2002. Specific activation of the Bacillus quorwn-
sensing systems by isoprenylated pheromone variants. Mo/.
Microbiol. 44: 1561-1573.
4. Bais, H. P., R. Fall, and J. M. Vivanco. 2004. Bioconbul of
Bacillus subtilis against infection of Arabidopsis roots by
Pseudomonas syringae is facilitated by biofilm fonnation and
surfactin production. Plant P~iol. 134: 307-319.
5. Brotz., H., G Bierbawn, K. Leopold, P. E. Reynolds, and H. G
Sahl. I 998. The !antibiotic mersacidin inhibits peptidoglycan
synthesis by targeting lipid II. Antimicrob. Agents Chemother.
42: 154-160.
6. Chen, X. H ., A. Koumoutsi, R. Schol7., A. Eisenreich, K.
Schneider, I. Heinemeyer, et al. 2007. Comparative analysis of
the complete genome sequence of the plant growth-promoting
bacterium Bacillus amyloliquefaciens FZB42. Nat. BiotechnDI.
25: 1007-!014.
7. Chen, X. H., J. Vatei; J. Piel, P. Franke, R. Schol7., K.
Schneider, et al. 2006. Structural and functional characterization
of three polyketide synthase gene clusters in Bacillus
amyloliquefaciens FZB42. J &cteriol. 188: 4024-4036.
8. Christiaen, S., G Brackman, H. J. Nelis, and T. Coenye. 2011.
Isolation and identification of quorum quenching bacteria from
environmental samples. J Microbiol. Methods 87: 213-219.
9. Cooper, G R. and A Moir. 201 l. Amino acid residues in the
GerAB protein important in the function and assembly of the
alanine spore germination receptor of Bacillus subtilis 168. J
Bacteriol. 193: 2261-2267.
IO. Cutting, S. M. 2011. Bacillus probiotics. Food Microbiol. 28:
214-220.
II. Czajkowski, R. and S. Jafra. 2009. Quenching of acyl-
homoserine lactone-dependent quorum sensing by enzymatic
disruption of signal molecules. Acta Biochim. Pol. 56: 1-16.
12. Due, L. H., H. A. Hong, T. M. Barbosa, A. 0. Henriques, and
S. M. Cutting. 2004. Characterization of Bacillus probiotics
available for human use. Appl. Environ. Microbiol. 70: 2161-
2171.
13. Fuchs, S. W., T. W. Jaskolla, S. Bochmann, P. Kotter, T.
Wichelhaus, M. Karas, et al. 20 I I . Entianin, a novel subtilin-
like Ian ti biotic from Bacillus subtilis suhsp. spizizenii DSM 15029
with high antimicrobial activity. Appl. Errviron. Microbiol. 77:
1698-1707.
14. Fuqua, C. and E. P. Greenberg. 2002. Listening in on bacteria:
Acyl -homoserine lactone signalling. Nat. Rev. 3: 685-695.
15. Griffiths, K., J. Zhang, A. E. Cowan, J. Yu, and P. Setlow. 2011.
Gennination proteins in the inner membrane of dormant Bacillus
subtilis spores colocalize in a discrete cluster. Mot. Microbiol.
81: 1061-1077.
16. GouJd, G W. 1969. Germination, pp. 397-444. In G W. Gould
and A. Hurst (eds.). The bacterial spore, Academic Press,
London, England.
CLASSIFICATION OF BACIUUS BENERCIAL SUBSTANCES 1603
17. Guez., J. S., C. H. Millier, P. M. Danz.e, J. Buchs, and P.
Jacques. 2008. Respiration activity monitoring system (RAMOS),
an efficient tool to study the influence of the oxygen transfer
rate on the synthesis of lipopeptide by Bacillus subtilis
ATCC6633. J Biotechnol 134: 121-126.
18. He, H., L. A. Silo-Suh, J. Clardy, and J. Handelsman. 1994.
Zwittermicin A, an antifungal and plant protection agent from
Bacillus cereus. Tetrahedron Lett. 35: 2499-2502.
19. Herzner, A. M., J. Dischinger, C. Szekat., M. Josten, S. Schmitz,
A. Yakeleba, et al. 201 l. Expression of the !antibiotic meisacidin
in Bacillus amyloliquefaciens FZB42. PLoS ONE 6(7): e22389.
20. Higgins, D. and J . Dworkin. 2012. Recent progress in Bacillus
subtilis sporulation. FEMS Microbiol. Rev. 36: 131-148.
21. Hofemeister, J., B. Conrad, B. Adler, B. Hofemeister, J.
Feesche, N. Kucheryava, et al. 2004. Genetic analysis of the
biosynthesis of non-ribosomal peptide and polyketide-like
antibiotics, iron uptake and biofilm fonnation by Bacillus
subtilis AI/3. Mo/. Genet. Genomics 272: 363-378.
22. lgarashi, T. and P. Setlow. 2006. Transcription of the Bacillus
subtilis gerK operon, which encodes a spore germinant receptor,
and comparison with that of operons encoding other germinant
receptors. J Bacteriol. 188: 4131-4136.
23. lnglesby, T. V., T. O'Toole, D. A. Henderson, J. G Bartlett., M.
S. Ascher, E . Eitzen, et al. 2002. Anthrax as a Biological
Weapon. JAMA 287: 2236-2252.
24. Kanhere, A. and M. Vmgron. 2009. Horizontal gene transfers in
prokaiyotes show differential preferences for metabolic and
translational genes. BMC Evol. Biol. 9: 9
25. Keams, D. B., F. Chu, R. Rudner, and R. Losick. 2005. A
master regulator for biofilm formation by Bacillus subtilis. Mo/.
Microbiol. 55: 739-749.
26. Kinsinger, R. F., M. C. Shirk, and R. Fall. 2003. Rapid surface
motility in Bacillus subtilis is dependent on extracellular
surfactin and potassium ion. J &cteriol. 185: 5627-5631.
27. Kleerebez.em, M. and L. E. Quadri. 2001. Peptide pheromone-
dependent regulation of antimicrobial peptide production in
gram-positive bacteria: A case of multicellular behavior. Peptides
22: 1579-1596.
28. Kleerebez.em, M., L. E. Quadri, 0. P. Kuipers, and W. M. de
Vos. 1997. Quorum sensing by peptide pheromones and two
component signal-transduction systems in gram-positive bacteria.
Mo/. Microbiol. 24: 895-904.
29. Kloepper, J. W., C. M. Ryu, and S. Zhang. 2004. Induced
systemic resistance and promotion of plant growth by Bacillus
spp. Phytopathology. 94: 1259-1266.
30. Lee, H. and H. Y. Kim. 201 l. Lantibiotics, class I bacteriocins
from the genus Bacillus. J Microbiol. Biotechnol. 21: 229-235.
31. Li, H., X. Wang, M. Han, Z. Zhao, M. Wang, Q. Tang, et al.
2012. Endophytic Bacillus subtilis 22120 and its potential
application in control of replant diseases. A.fri. J Biotechnol. 11:
231-242.
32. Lopez., D., H. Vlamakis, R. Losick, and R. Koher. 2009.
Paracrine signaling in a bacteriwn. Genes Dev. 23: 1631-1638.
33. Maget-Dana, R., L. Thimon, F. Peypoux, and M. Ptack. 1992.
Surfactin/lturin A interactions may explain the synergistic effect
of surfactin on the biological properties of iturin A. Biochimie
74: 1047-1051.
34. Malone, C. L., B . R. Boles, and A. R. Horswill. 2007.
Biosynthesis of Staphylococcus aureus autoinducing peptides by
1604 Wiyada Mongkolthanaruk
using the Synechocystis DnaB Mini-Intein. Appl. Environ.
Microbial. 73: 6036-6044.
35. Mayville, P., Ci Ji, R. Beavis, H. Yang, M. Goger, R. P. Novick,
and T. W. Muir. 1999. Structure-activity analysis of synthetic
autoinducing thiolactone peptides from Staphylococcus aureus
responsible for virulence. Proc. Natl. Acad Sci. USA 96: 1218-
1223.
36. McPherson, D. C, H. Kim, M. Hahn, R. Wang, P. Grabowski,
P. Eichenberger, and A. Driks. 2005. Characterization of the
Bacillus subtilis spore morphogenetic coat protein CotO. J.
Bacterial. 187: 8278-8290.
37. Moir, A. 2006. How do spores genninate? J. Appl Microbial.
101: 526-530.
38. Moldenhauer, J., D. C. Ci Gotz, C. R. Albert, S. K. Bischof, K.
Schneider, R. D. Sussmuth, et al. 2010. The final steps of
bacillaene biosynthesis in Bacillus amyloliquefaciens FZB42:
Direct evidence for f!,y dehydration by a trans-acyltransferase
polyketide synthase. Angm11 Chern. Int. &I. 49: 1465-1467.
39. Mongkolthanaruk, W., Ci R. Cooper, J. S. P. Mawer, R. N.
Allan, and A. Moir. 2011. Effect of amino acid substitutions in
the GerAA protein on the function of the alanine responsive
germinant receptor of Bacillus subtilis spores. J. Bacterial. 193:
2268-2275.
40. Nicholson, W. L. 2002. Roles of Bacillus endospores in the
environment. Cell. Mol Life Sci. 59: 410-416.
41. Nicholson, W. L., N. Munakata, Ci Homeck, H.J. Melosh, and
P. Setlow. 2000. Resistance of Bacillus endospores to extreme
terrestrial and extraterrestrial environments. Microbial. Mol
Biol. Rev. 64: 548-572.
42 . Oman, T. J., J. M. Boettcher, H . Wang, X. N. Okalibe, and W.
A . Donk. 2011. Sublancin is not a !antibiotic but an S-linked
glycopeptides. Nat. Chern. Biol. 7 : 78-80.
43 . Ongena, M . and P. Jacques. 2007. Bacillus lipopeptides :
Versatile weapons for plant disease biocontroL Trends Microbial
16: 115-125.
44. Ongena, M~ J. Emmanuel, A. Adam, M . Paquot, A. Brans, B.
Joris, et al. 2007. Surfuctin and fengycin lipopeptides of Bacillus
subtilis as elicitors of induced systemic resistance in plants.
Environ Microbial. 9: 1084-1090.
45. Park, S. J., S. Y. Park, C . M. Ryu, S. H. Park, and J. K. Lee.
2008. The role of AiiA, a quorum-quenching enzyme from
Bacillus thuringiensis, on the rhizosphere competence. J.
M"1CTVbiol. Biotechnol. 18: 1518-1521.
46. Park, S . Y., S. J. Lee, T. K. Oh, J . W. Oh, B. T. Koo, D. Y.
Yum, and J. K. Lee. 2003. AhlD, an N-acylhomoserine
lactonase in Arthrobacter s p., and predicted homologues in
other bacteria Microbiology 149: 1541-1550.
4 7. Pelc:zar, P. L. and P. Setlow. 2008. Localiz.ation of the
germination protein GerD to the inner membrane in Bacillus
subtilis spores. J. Bacterial. 190: 5635-5641.
48. Pottathil, M., A. Jung, and B. A. Lazazzera. 2008. CSF, a
species-specific extracellular signaling peptide for communication
among strains of Bacillus subtilis and Bacillus mojavensis. J.
Bacterial. 190: 4095-4099.
49. Raaijmakers, J.M., I . Bruijn, 0 . Nybroe, and M. Ongena 2010.
Natural functions of lipopei:fides from Bacillus and Pseudomonas:
More than surfactants and antibiotics. FEMS Microbial. Rev.
34: 1037-1062.
50. Reiss, R., J. Thssen, and L. Thony-Meyer. 2011. Bacillus
pumilus laccase: A heat stable enzyme with a wide substrate
spectrum. BMC Biotechnol. 11: 9
51. Roche, D. M., J. T. Byers, D. S. Smith, F. Ci Glansdrop, D. R.
Spring, and M Welch. 2004. Communication blackout? Do
Nacylhomoserine-Iactone-degrading enzymes have any role in
quorum sensing? Microbiology 150: 2023-2028.
52. Romero, D., A. Vicente, R. Rakotoaly, S . Dufour, J . Veening, E.
Arrebola, et al. 2007. The iturin and fengycin families of
lipopeptides are key factors in antagonism of Bacillus subtilis
toward Podosphaera fasca. Mo/. Plant Microbe Interact. 20:
430-440.
53. Ryan, R. P. and J. M. Dow. 2008. Diffusible signals and
interspecies communication in bacteria Microbiology 154:
1845-1858.
54. Schneider, K., X . C. Chen, J. Yater, P. Franke, J. Nicholson, R.
Borriss, and R. D . Siissmuth. 2007. Macrolactin is the
polyketide biosynthesis product of the p/rs2 cluster of Bacillus
amyloliquefaciens FZB42. J. Nat. Prod. 70: 1417-1423.
55. Setlow, B~ A. E . Cowan, and P. Setlow. 2003. Germination of
spores of Bacillus subtilis with dodecylamine . J. Appl. M"u:robiol.
95: 637-648.
56. Set\ow, P. 2006. Spores of Bacillus subtilis: Their resistance to
and killing by radiation, heat and chemicals. J. Appl. Microbial.
101: 514-525.
57. Shank, E. A. and R. Kolter. 2011. Extracellular signaling and
multicellularity in Bacillus subtilis. Curr. Opin. Microbial. 14:
741-747.
58. Tam, N. K., N. Q. Uyen, H. A . Hong, L. H. Due, T. T. Hoa, C.
R. Serra, et al. 2006. The intestinal life cycle of Bacillus subtilis
and close relatives. J. Bacterial. 188: 2692-2700.
59. Uroz, S ., S. R. Chhabra, M . Camara, P. WIiiiams, P. Oger, and
Y. Dessaux. 2005. N-acylhomoserine lactone quorum-sensing
molecules are modified and degraded by Rhodococcus erythropo/is
W2 by both amidolytic and novel oxidoreductase activities.
Microbiology 151: 3313-3322.
60. Velho, R. V., D. G Caldas, L. F. Medina, S. M. Tsai, and A.
Brandelli. 201 l. Real-time PCR investigation on the expression
of sboA and ituD genes in Bacillus spp. Lett. Appl. Microbial.
52: 660-666.
61. Vollenbroich, D., Ci Pauli, M . Ozel, and J . Vater. 1997.
Antimycoplasma properties and application in cell culture of
surfactin, a lipopeptide antibiotic from Bacillus subtilis. Appl.
Errviron. Microbial. 63: 44-49.
62. Xu, D. and J. Cote. 2003. Phylogenetic relationships between
Bacillus species and related genera inferred from comparison of
3' end 16S rDNA and 5' end 16S-23S ITS nucleotide sequences.
Int. J. Syst. Evol. Microbial. 53: 695-704.
63. Zhou, Y., Y. L. Choi, M. Sun, and Z. Yu. 2008. Novel roles of
Bacillus thuringiensis to control of plant diseases. Appl.
Microbial. Biotechnol. 80: 563-572.
Bacillus subtilis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act [TSCA) I US EPA
TSCA Biotechnolocry
Horne
TSCA Biotechnology
Regulations
Filing a Biotechnology
Submission
TSCA Biotechnology
Notifications
Other EPA
Biotechnology
Activities
Other Government
Biotechnology Sites
Biotechnology
Publications
TSCA Biotechnology
Regional Contacts
U.S. ENVIRONMENTAL PROTECTION AGENCY
Biotechnology Program under the Toxic
Substances Control Act (TSCA)
Contact Us Search: Q All EPA ()This Area
You are here- EPA Hnmp w f hpmiral Cafetv X Pollirtinn Preventiort k Pollution Prevention & Toxics
+a Biote,chnofoav» Bacillus subtilis Final Risk Assessment
Bacillus subtilis Final Risk Assessment
ATTACHMENT I --FINAL RISK ASSESSMENT OF
BACILLUS SUBTILIS
(February 1997)
I. INTRODUCTION
Bacillus subtilis is a ubiquitous bacterium commonly recovered from water, soil, air, and
decomposing plant residue. The bacterium produces an endospore that allows it to endure extreme
conditions of heat and desiccation in the environment. B. subtilis produces a variety of proteases
and other enzymes that enable it to degrade a variety of natural substrates and contribute to
nutrient cycling. However, under most conditions the organism is not biologically active but exists
in the spore form (Alexander, 1977). B. subtilis is considered a benign organism as it does not
possess traits that cause disease. It is not considered pathogenic or toxigenic to humans, animals,
or plants. The potential risk associated with the use of this bacterium in fermentation facilities is
low.
History of Commercial Use and Products Subject to TSCA Jurisdiction
B. subtilis is one of the most widely used bacteria for the production of enzymes and specialty
chemicals. Industrial applications include production of amylase, protease, inosine, ribosides, and
amino acids. TSCA uses of proteases include leaning aids in detergents and dehairing and batting
in the leather industry. TSCA uses of amylases include desizing of textiles and starch modification
for sizing of paper (Erikson, 1976).
The Agency has reviewed, under TSCA, three PMNs of genetically modified B. subtilis for production
of a protease (P87-1030), alpha -amylase (P89-227), and lipase (P91-1154). EPA found that there
were no unreasonable risks associated with the use of these recombinant strains for enzyme
production in fermentation facilities.
II.IDENTIFICATION AND TAXONOMY
A. Overview
B. subtilis is a ubiquitous soil microorganism that contributes to nutrient cycling when biologically
active due to the various enzymes produced by members of the species. Although the actual
numbers in existence in the environment for thisspecies has not been determined, bacilli occur at
population levels of 106 to 107 per gram of soil (Alexander, 1977). However, unless a soil has
been recently amended with organic matter providing readily utilizable nutrients, the bacilli exist in
the endospore stage. It is thought that 60 to 100% of soil bacilli populations exist in the inactive
spore state (Alexander, 1977). Like most members of the genus, 8. subtilis is aerobic, except in
the presence of glucose and nitrate, some anaerobic growth can occur (Claus and Berkeley, 1986).
B. Taxonomy and Characterization
The genus Bacillus consists of a large number of diverse, rod -shaped Gram positive (or positive
only in early stages of growth) bacteria that are motile by peritrichous flagella and are aerobic.
Members of the genus are capable of producing endospores that are highly resistant to unfavorable
environment conditions (Claus and Berkeley, 1986). The genus consists of a diverse group of
organisms as evidenced by the wide range of DNA base ratios of approximately 32 to 69 mol% G
http://www.epa. gov/biotech_rule/puhs/fralfra009.htmi 1 /17/2014 9:32:97 AM]

Bacillus subtilis Final Risk Assessment I Biotechnology Program Linder Toxic Substances Control Act (TSCA) I US EPA
f C (Claus and Berkeley, 1986), which is far wider than that usually considered reasonable for a
genus (Norris et al., 1981).
8. subtilis is the type species of the genus. Historically, prior to the monographs of Smith in 1946
and 1952, 8. subtilis was a term given to all aerobic endospore-forming bacilli (Logan, 1988).
Numerous species that appeared in the early literature are no longer recognized as official species.
Former species designations that are now considered to be members of the species 8. subtilis
include B. aterrlmus, B. mesentericus, 8. niger, B. penis, B. vulgarus, 8. nigrificans, and B. natto
(Gibson, 1944 and Smlth et al., 1946 as cited by Gordon, 1973). Although in the past it has been
designated as a separate species, the latest edition of Bergey's Manual of Systematic Bacteriology
(Claus and Berkeley, 1986) listed 8. amyloliquefaciens as a member of the species B. subtilis.
However, recently it has again achieved the status of a separate species (Priest et al., 1987).
The Bacillus species subtilis, licheniformis, and pumilus are closely related and there has been
difficulty distinguishing among the three species that historically were grouped together as the
subtilis-group or subtilis-spectrum (Gordon, 1973). These three species clustered together (78%)
in the "su/tills" group in a numerical classification based on 118 unit characteristics of 368 strains
of Bacillus (Priest et al., 1988). However, this major cluster contained four subclusters that could
be identified as B. subtiiis, 8_ lichenffarmis, e_ pumulis, and B. amyloliquefaciens. Recent data in
the literature have suggested that it is possible to differentiate B. subtilis from B. llchenlformis and
B. pumulfs by the use of pyrolysis -gas chromatography (O'Donnell et al., 1980) or by the use of
API tests (Logan and Berkeley, 1981). In addition, B. subtilis and8. amyloliquefaciens show little
DNA sequence homology to each other (Seki et al., 1975; Priest, 1981) and can also be
distinguished from each other by pyrolysis -gas chromatography (O'Donnell et al., 1980) and by a
few phenotypic properties including the production of acid from lactose (Priest et al., 1987).
In conclusion, it appears that 8. subtilis can be distinguished from other closely related species.
However, because of changes in the classification of the genus, and the recent development of new
methods for taxonomic purposes, older strains may not actually be B. subtilis under present-day
definitions.
C. Related Species of Concern
There are several species of the genus that are known pathogens. These include B. anthracls which
is pathogenic to humans and other animals, and B. cereus which is a common cause of food
poisoning (Claus and Berkeley, 1986; Norris et al., 1981). B. thuringiensls, B. larvae, B.
lentlmorbus, B. popllliae, and some strains of 8. sphaericus are pathogenic to certain insects.
Other species in the genus are considered "opportunistic pathogens".
In a numerical classification using 118 characteristics of 368 species of Bacillus, the species 8.
thuringiensis, 8. cereus, and 8. mycoides clustered together at 89 - 92% similarity (Priest et al.,
1988). The 8. subtilis group joined the B. cereus group at 72% relatedness. There is no difficulty in
distinguishing between the toxin -producing strains of Bacillus and 8. subtilis.
III. HAZARD ASSESSMENT
A. Human Health Hazards
1. Colonization
B. subtilis is widely distributed throughout the environment, particularly in soil, air, and
decomposing plant residue. It has shown a capacity to grow over a wide range of temperatures
including that of the human body (Claus and Berkeley, 1986). However, B. subtflis does not appear
to have any specialized attachment mechanisms typically found in organisms capable of colonizing
humans (Edberg, 1991)_ Given its ubiquity in nature and the environmental conditions under which
it is capable of surviving, 8. subtilis could be expected to temporarily inhabit the skin and
gastrointestinal tract of humans, but it is doubtful that this organism would colonize other sites in
the human body (Edberg, 1991).
2. Gene Transfer
The transfer of gene sequences between strains of B. subtilis has been demonstrated when the
strains were grown together in soil (Graham and Istock, 1979). In addition, Klier et al. (1983)
demonstrated the ability of B. subtilis and B. thuringiensis to exchange high Frequency transfer
htm://www.epa_nov/biotech_nilelpubsgra/fra009.htrn[I/17/2014 9:32.07 AM]

Bacillus subtilis Final Risk Assessment Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
plasmids. Other studies have shown that B. subtilis has the ability to express and secrete toxins or
components of the toxins that were acquired from other microorganisms through such transfers of
genetic material. B. subtilis expressed subunits of toxins from Bordatella pertussis (Saris et at.,
1990a, 1990b), as well as subunits of diphtheria toxin (Hemila et al., 1989) and pneumolysin A
pneumococcal toxin (Taira et al., 1989). Although B. subtilis does not appear to possess indigenous
virulence factor genes, it is theoretically possible that it may acquire such genes from other
bacteria, particularly from closely related bacteria within the genus.
3. Toxin Production
A review of the literature by Edberg (1991) failed to reveal the production of toxins by B. subtilis.
Although it has been associated with outbreaks of food poisoning (Gilbert et al., 1981 and Kramer
et al., 1982 as cited by Logan, 1988), the exact nature of its involvement has not been
established. B. subtilis, like other closely related species in the genus, B. licheniformis, B. pumulis,
and 8. megaterium, have been shown to be capable of producing lecithinase, an enzyme which
disrupts membranes of mammalian cells. However, there has not been any correlation between
lecithinase production and human disease in B. subtilis.
B. subtilis does produce an extracellular toxin known as subtilisin. Although subtilisin has very low
toxigenic properties (Gill, 1982), this proteinaceous compound is capable of causing allergic
reactions in individuals who are repeatedly exposed to it (Edberg, 1991). Sensitization of workers
to subtilisin may be a problem in fermentation Facilities where exposure to high concentration of
this compound may occur. Exposure limits to subtilisin are regulated by Occupational Safety and
Health Administration (OSHA) (29 CFR 1900, et.)
4. Measure of the Degree of Virulence
B. subtilis appears to have a low degree of virulence to humans. It does not produce significant
quantities of extracellular enzymes or possess other virulence factors that would predispose it to
cause infection (Edberg, 1991). There are a number of reports where B. subtilis has been isolated
from human infections. Earlier literature contains references to infections caused by B. subtilis.
However, as previously stated,the term B. subtilis was synonymous for any aerobic sporeforming
bacilli, and quite possibly, many of these infections were associated with ,3. cereus. In a recent
British review article, Logan (1988) cites more recent cases of 8. subtilis infections in which
identification of the bacterium appeared reliable. Infections include a case of endocarditis in a drug
abuse patient; fatal pneumonia and bacteremia in three leukemic patients; septicemia in a patient
with breast cancer; and infection of a necrotic axillary tumor in another breast cancer patient.
Isolation of 8. subtilis was also made from surgical wound -drainage sites, frorn a subphrenic
abscess from a breast prosthesis, and from two ventriculo-atrial shunt infections (as cited by
Logan, 1988).
Reviews of Bacillus infections from several major hospitals suggest that 8. subtilis is an organism
with low virulence. Idhe and Armstrong (1973) reported that Bacillus infections were encountered
only twelve times aver a 6-1/2 year period. Species identification of these Bacillus infections was
not made. In another hospital study over a 6-yr. period, only two of the 24 cases of bacterernia
caused by Bacillus (of a total of 1,038 cases) were due to B. subtilis (as cited by Edberg, 1991).
Many of these patients were immunocompromised or had long term indwelling foreign bodies such
as a Hickman catheter.
B. subtilis has also been implicated in several cases of food poisoning (Gilbert et al., 1981 and
Kramer et al., 1982 as cited by Logan, 1988).
As previously mentioned, B. subtilis produces a number of enzymes, including subtilisin, for use in
laundry detergent products. There have been a number of cases of allergic or hypersensitivity
reactions, including dermatitis and respiratory distress after the use of these laundry products
(Norris et al., 1981).
5. Conclusions
B. subtilis is not a human pathogen, nor is it toxigenic like some other members of the genus. The
virulence characteristics of the microorganism are low. According to Edberg (1991) either the
number of microorganisms challenging the individual must be very high or the immune status of
the individual very low in order for infection with B. subtilis to occur.
http:llwww.epa.gov1biotech_rule/pubslfralfral709.htm[1/1712014 4:32:07 AM]

Bacillus subtilis Final Risk Assessment j Biotechnology Program Under Toxic Substances Control Act (TSCA) l US EPA
B. Environmental Hazards
1. Hazards t,QAnimals
B. subtilis has been isolated from a number of cases of bovine and ovine abortions, however, the
microorganism has neverbeen identified as the causal agent (Logan, 1988). Reports on association
of 8. subtilis with livestock abortions are fairly rare, and of much lower frequency than with other
Bacillus species, which are rare compared to al! other microorganisms, especially viruses and fungi.
B. subtilis has also been reported in 17 cases of bovine mastitis in which It was thought to he the
causal agent (Fossum et al., 1986). However, the limited number of cases of mastitis associated
with B. subtilis also is rare compared to mastitis caused by other microorganisms.
8. subtilis has also been shown to be capable of infecting and causing mortality of the 2nd instar
larvae of the mosquito, Anophelis culicifacies, which is the primary insect vector of malaria in
central India (Gupta and Vyas, 1989). B. subtilis was being investigated for use as a hiocantrol
agent in this study.
2. Hazards to Plants
B. subtilis is not considered to be a plant pathogen (7 CFR 330, at .; Claus and Berkeley,
1986). However, there are several reports in the literature that associate B. subtilis with certain
plant diseases. Kararah et al. (1985) produced soft rot of garlic cloves by injecting 8. subtilis into
them_ Bergey's Manual of Systematic Bacteriology notes that pectin and polysaccharides of plant
tissues can be decomposed by B. subtilis and that this microorganism can cause soft rut of potato
tubers (Claus and Berkeley, 1986). There are several abstracts obtained in a literature review that
suggests that B. subtilis may cause other plant diseases, however, no more information was
obtainable. One abstract reported that 8. subtilis was the cause of a broad open cancer ulcera on
Norway maples in forests in the Urals (Yakovleva et al., 1990). Another reported that an organism
tentatively identified as 8. subtilis was consistently isolated from glasswort (5alicorn/a) plants
suffering From a soft -rot disease (Stanghellini and Rasmussen, 1989).
3. Hazards to Other Microorganisms
8. subtilis has been shown to produce a wide variety of antibacterial and antifunga! compounds
(Katz and Demain, 1977; Korzybski et al,, 1978). It produces novel antibiotics such as difficidin
and oxydifficidin that have activity against a wide spectrum of aerobic and anaerobic bacteria
(Zimmerman et a1_, 1987) as well as more common antibiotics such as bacitracin, bacillin, and
bacillomycin B (Parry et al., 1983). The use of 8. subtilis as a biocontrol agent of fungal plant
pathogens is being investigated because of the effects of antiFungal compounds on Monilinia
fructicola (McKeen et al., 1986), Aspergilius flavus and A. parasiticus (Kimura and Hirano, 1988),
and Rhizoctonia (Loeffler et al., 1986).
Although B. subtilis produces a variety of antibiotic compounds in culture media, the importance of
antibiotic production in the environment is unknown (Alexander, 1977).
IV. EXPOSURE ASSESSMENT
A. Worker Exposure
8. subtilis is considered a Class 1 Containment Agent under the National Institute of Health (NIH)
Guidelines for Research Involving Recombinant DNA Molecules (U.S. Department of Health and
Human Services, 1986). This microorganism also falls under the Class 1 Containment under the
European Federation of Biotechnology guidelines (Frommer et al., 1989).
No data were available for assessing the release and survival specifically for fermentation facilities
using B. subtilis. Therefore, the potential worker exposures and routine releases to the
environment from large-scale, conventional fermentation processes were estimated on information
available from eight premanufacture notices submitted to EPA under TSCA Section 5 and from
published information collected from non -engineered microorganisms (Reilly, 1991). These values
are based on reasonable worst -case scenarios and typical ranges or values are given for
comparison.
During fermentation processes, worker exposure is possible during laboratory pipetting, inoculation,
sampling, harvesting, extraction, processing and decontamination procedures. A typical site
hrtp://www.epa.gov/biatech_rule/pubs/fraffra009.htrnj 1I17/2014 9:32:07 A.M]

Bacillus subtiiis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
employs less than 10 workers/shift and operates 24 hours/day throughout the year. NIOSH has
conducted walk-through surveys of several fermentation facilities in the enzyme industry and
monitored for microbial air contamination. These particular facilities were not using recombinant
microorganisms, but the processes were considered typical of fermentation process technology.
Area samples were taken in locations where the potential for worker exposure was considered to
be potentially greatest, i.e., near the fermentor, the seed fermentor, sampling ports, and
separation processes (either filter press or rotary drum filter). The workers with the highest
potential average exposures at the three facilities visited were those involved in air sampling. Area
samples near the sampling port revealed average airborne concentrations ranging from 350 to 648
cfu/m3. Typically, the Chemical Engineering Branch would not use area monitoring data to estimate
occupational exposure levels since the correlation between area concentrations and worker
exposure is highly uncertain. Personal sampling data are not available at the present time. Thus,
area sampling data have been the only means of assessing exposures for previous PMN
biotechnology submissions. Assuming that 20 samples per day are drawn and that each sample
takes up to 5 minutes to collect, the duration ofexposure for a single worker will be about 1.5
hours/day. Assuming that the concentration of microorganisms in the worker's breathing zone is
equivalent to the levels found in the area sampling, the worst -case daily inhalation exposure is
estimated to range up to 650 to 1200 cfu/day. The uncertainty associated with this estimated
exposure value is not known (Reilly, 1991).
B. Environmental and General Exposure
1. Fate of the Organism
8. subtiiis is a common saprophytic inhabitant of soils and is thought to contribute to nutrient
cycling due to the variety of proteases and other enzymes members of the species are capable of
producing. Growth normally occurs under aerobic conditions, but in complex media in the presence
of nitrate, anaerobic growth can occur (Claus and Berkeley, 1986). Under adverse environmental
conditions, B. subtiiis produces endospores that are resistant to heat and desiccation (Claus and
Berkeley, 1986). Specific data comparing the survivability of industrial and wildtype strains of B.
subtiles were not available in the existing literature. However, the ability of B. subtiiis to produce
highly resistant spores and grow under a wide range of conditions indicates that released strains
are likely to survive outside of containment.
2. Releases
Estimates of the number of 8. subtiiis organisms released during production are tabulated in Table
1 (Reilly, 1991). The uncontrolled/untreated scenario assumes no control features for the
fermentor offgases, and no inactivation of the fermentation broth for the liquid and solid waste
releases. The containment criteria required for the full exemption scenario assume the use of
features or equipment that minimize the number of viable cells in the fermentor off -gases. They
also assume inactivation procedures resulting in a validated 6log reduction of the number of viable
microorganisms in the liquid and solid wastes relative to the maximum cell density of the
fermentation broth.
TABLE 1. Estimated Number of Viable Bacillus subtilis
Organisms Released During Production
Uncontrolled/ Full
Release Media Untreated Exemption Release
(cfu/day) (cfu/day) (days/yr)
Air Vents 2x108 - lx1011 <2x108 - 1x1011 350
Rotary Drum Filter 250 250 350
Surface Water 7x1016 7x1010 90
http://www.epa.gnv/bintech_rule/pubs/fra/fia009.htm[ 1/17/2014 9:32:07 AM]

Bacillus subtilis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) J US EPA
Soil/Landfill 7x1018 7x1012 90
Source: Reilly, 1991
These are "worstcase" estimates which assume that the maximum cell density in the fermentation
broth for bacteria is 1011 cfu/ml, with a fermentor size of 70,000 liters, and the separation
efficiency for the rotary drum Filter is 99 percent.
3. Air
Specific data which indicate the survivability of 8. subtilis in the atmosphere after release are
currently unavailable. Survival of vegetative cells during aerosolization is typically limited due to
stresses such as shear forces, desiccation, temperature, and UV light exposure. However, its ability
to survive in a broad habitat range and produce endospores suggests that this organism may
survive after release. As with naturally -occurring strains, human exposure may occur via inhalation
as the organisms are dispersed in the atmosphere attached to dust particles, or lofted through
mechanical or air disturbance.
Air releases from fermentor offgas could potentially result in nonoccupational inhalation exposures
due to point source releases. To estimate exposures from this source, the sector averaging form of
the Gaussian algorithm described in Turner (1970) was used. For purposes of this assessment, a
release height of 3 meters and downward contact at a distance of 100 meters were assumed.
Assuming that there is no removal of organisms by controls/equipment for offgases, potential
human inhalation dose rates are estimated to range from 3.0 x 103 to 1.5 x 106 cfu/year for the
uncontrolled/untreated scenario and less than that for systems with full exemptions. It should be
noted that these estimates represent hypothetical exposures under reasonable worst case
conditions (Versar, 1992).
4. Water
The concentrations of 8. subtilis in surface water were estimated using stream flow values for
water bodies receiving process wastewater discharges from facilities within SIC Code 283 (drugs,
medicinal chemicals, and pharmaceuticals). The surface water release data (cfu/day) tabulated in
Table 1 were divided by the stream flow values to yield a surface water concentration of the
organism (cfu/I). The stream flow values for SIC Code 283 were based on discharger location data
retrieved from the Industrial Facilities Dischargers (IFD) database on December 5, 1991, and
surface water flow data retrieved from the RXGAGE database. Flow values were obtained for water
bodies receiving wastewater discharges from 154 indirect (facilities that send their waste to a
PQTW) and direct dischargers facilities that have a NPDES permit to discharge to surface water).
Tenth percentile values indicate flows for smaller rivers within this distribution of 154 receiving
water flows and 50th percentile values indicate flows for more average rivers. The flow value
expressed as 7Q10 is the lowest flow observed over seven consecutive days during a l0year
period. The use of this methodology to estimate concentrations of B. subtilis in surface water
assumes that all of the discharged organisms survive wastewater treatment and that growth is not
enhanced by any component of the treatment process. Estimated concentrations of 8. subtilis in
surface water for the uncontrolled/untreated and the full exemption scenarios are tabulated in
Table 2 (Versar, 1992).
TABLE 2. Bacillus subtilis Concentrations in Surface Water
Receiving
Flow Stream Flow Organisms
(MLD*) (cfu/l)
Mean 7Q10 Mean 7Q10
littp://www.epa.eov/biotech_Me/pubs/frafra009.htm[l /17/2014 9:32:07 AM]

Bacillus subtilis Final Risk Assessment Biotechnology Program Under Toxic Substances Control Act (TSCA) US EPA
Uncontrolled/Untreated
loth Percentile 156 5.60 4.5x108 1.25x1010
50th Percentile 768 68.13 9.11x107 1.03x109
Full Exemption
loth Percentile 156 5.60 4.5x102 1.25x104
50th Percentile 768 68.13 9.11x101 1.03x103
*MLD = million liters per day
Source: Versar, 1992
5, Soil
The natural habitat for 8. subtilis is soil. Therefore, longterm survival in soil may be expected to
occur. Human exposures via dermal and ingestion mutes, and environmental exposures [i.e., to
terrestrial, avian, and aquatic organisms (via runoff)] may occur at the discharge site because of
the establishment of B. subtilis within the soil.
6. Summary
Although direct monitoring data are unavailable, worst case estimates do not suggest high levels of
exposure of 8. subtilis to either workers or the public resulting from normal fermentation
operations.
V. INTEGRATION OF RISK
A. Discussion
Bacillus subtilis is a ubiquitous, saprophytic, soil bacterium which is thought to contribute to
nutrient cycling due to its ability to produce a wide variety of enzymes. This latter feature of the
microorganism has been commercially exploited for over a decade. B. subtilis has been used for
industrial production of proteases, amylases, antibiotics, and specialty chemicals. The Agency has
reviewed three submissions for production of enzymes using genetically modified B. subtilis and
found no unreasonable risks to human health or the environment from the use of this
microorganism in fermentation facilities.
Historically, B. subtilis was a term given to all aerobic endospore-forming bacilli_ Later, B. subtflis
and two closely related species, B. licheniformis, and 8. pumilus, were grouped taxonomically into
what was known as the subtilis-group. However, recently methods have been developed that allow
B. subtilis to be distinguished from these other species.
B. subtilis is not a frank human pathogen, but has on several occasions been isolated from human
infections. Infections attributed to B. subtilis Include bacteremia, endocarditis, pneumonia, and
septicemia. However, these infections were found in patients in compromised immune states. There
must be immunosuppression of the host followed by inoculation in high numbers before infection
with B. subtilis canoccur. There also have been several reported cases of food poisoning attributed
to large numbers of 8. subtilis contaminated food. B. subtilis does not produce significant
quantities of extracellular enzymes or other factors that would predispose it to cause infection.
Unlike several other species in the genus, B. subtilis is not consider toxigenic. 8_ subtilis does
produce the extracellular enzyme subtilisin that has been reported to cause allergic or
hypersensitivity reactions in individuals repeatedly exposed to it.
Overall, 8. subtilis has a low degree of virulence. Although the possibility of human infection is not
non-existent, it is low in the industrial setting where exposure to the bacterium is expected to be
low and where highly immunocompromised individuals would not be present. In an industrial
setting with the use of proper safety precautions, good laboratory practices, and proper protective
hrtp:llwww_epa.govlbiatech_ rule/pubslfra/fra009.htm[1/ 17/2014 9:32:07 AM]

Bacillus subtilis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
clothing and eyewear, the potential for infection of workers should be quite low. The only human
health concern for workers in the fennentation facility is the potential for allergic reactions with
chronic exposure to subtilisin. As previously stated, OSHA has established an exposure limit to
subtilisin which must be met in the industrial setting.
Likewise, the ecological hazards associated with the use of B. subtilis are low. There are several
reports in the literature on the association of B. subtilis with abortions in livestock. However, these
few reports indicate that this association must be fairly rare, and typically, the animals were
immunocompromised. In addition, B. subtilis has not been shown to be a causal agent and is not
considered an animal pathogen. Likewise, B. subtilis is not considered a plant pathogen. Although
it produces enzymes such as polygalacturonase and cellulase that are sometimes associated with
the ability to produce soft rot in plant tissue, there are many organisms that are capable of
producing a soft rot when injected beneath the outer protective epidermal layers.
The use of B. subtilis in an industrial setting should not pose an unreasonable risk to human health
or the environment. First, human health and environmental hazards of B. subtilis are low. Second,
the number of microorganisms released from the fennentation facility is low. In addition, B. subtllis
is ubiquitous in the environment, and the releases expected from the fermentation facilities will not
significantly increase populations of this bacterium in the environment.
In conclusion, the use of B. subtilis in fermentation facilities for the production of enzymes or
specialty chemicals has low risk. Although not completely innocuous, the industrial use of B. subti/is
presents low risk of adverse effects to human health or the environment.
B. Recommendations
Bacillus subti/is is recommended for the tiered exemption.
VI. REFERENCES
7 CFR 330, .et~., as amended.
29 CFR 1900, .et~-, as amended.
Alexander, M. 1977. Introduction to Soil Microbiology. John Wiley and Sons, Inc., New York.
Claus, D. and R.C.W. Berkeley. 1986. Genus Bacillus Cohn 1872, pp. 1105-1139. ln: P.H.A.
Sneath, et al. (eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins Co.,
Baltimore, MD.
Edberg, S.C. 1991. US EPA human health assessment: Bacillus subtilis. Unpublished, U.S.
Environmental Protection Agency, Washington, D.C.
Erikson, R.J. 1976. Industrial applications of the bacilli: A review and prospectus, pp. 406-419. ln:
D. Schlesinger (ed.), Microbiology. American Society for Microbiology, Washington, DC.
Fossum, K., H. Kerikstad, M. Binde, and K.-E. Pettersen. 1986. Isolations of Bacillus subti/is in
connection with bovine mastitis. Nordisk Veterinaermedicin 38:233-236.
Frommer, W., B. Ager, L. Archer, B. Brunius, C.H. Collins, R. Donikian, C.F. Frontali, S. Hamp, E.H.
Houwink, M.T. Kuenzi, P. Kramer, H. Lagast, S. Lund, J.L. Mahler, F. Normand-Plessier, K.
Sargeant, G. Tuijnenburg Muijs, S.P. Vranch, R.G. Werner. 1989. Safe biotechnology III. Safety
precautions for handling microorganisms of different classes. Appl. Microbiol. Biotechnol. 30:541-
552.
Gill, D.M. 1982. Bacterial toxins: A table of lethal amounts. Microbiol. Rev. 46:86-94.
Gordon, R.E. 1973. The genus Bacillus. Agricultural Handbook No. 427 . Agricultural Research
Service, U.S. Department of Agriculture, Washington, DC.
Graham, J.B., and C.A. !stock. 1979. Gene exchange and natural selection cause Bacillus subtllls to
evolve in soil culture. Sci. 204:637639.
Gupta, D.K. and M. Vyas. 1989. Efficacy of Bacillus subtilis against mosquito larvae (Anophelis
http://www.epagov/biotech _ rule/pubs/fra/fra009.htm[l/17/2014 9:32:07 AM]
Bacillus subtilis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
culicfacies). Zeitschrift fuer Angewandte Zoologie 76(1):85-91.
Hemila, H., L.M. Glode, and I. Paiva. 1989. Production of diphtheria toxin CRM228 in Bacillus
subtilis. Fed. Eur. Micro biol. Soc. Lett. 65: 193-198
Ihde, D.C. and D. Armstrong. 1973. Clinical spectrum of infection due to Bacillus species. Amer. J.
Med. 55:839-845.
Kararah, M.A., F.M. Barakat, M.S. Mikhail, and H.M. Fouly. 1985. Pathophysiology in garlic cloves
inoculated with Bacillus subtilis, Bacillus pumilus, and Erwinia carotovora. Egyptian J. Phytopathol.
17(2): 131-140.
Katz, E. and A.C. Demain. 1977. The peptide antibiotics of Bacillus: Chemistry, biogenesis, and
possible functions. Bacteriol. Rev. 41:449-474.
Kimura, N. and S. Hirano. 1988. Inhibitory strains of Bacillus subtills for growth and aflatoxin
production of aflatoxigenic fungi. Agric. Biol. Chem. 52(5):1173-1179.
Klier, A., C. Bourgouin, and G. Rapoport. 1983. Mating between Bacillus subtilis and Bacillus
thuringiensis and transfer of cloned crystal genes. Mol. Gen. Genet. 191:257-262.
Korzybski, T., Z. Kowszyk-Gindifer, and W. Kurylowicz. 1978. Antibiotics isolated from the genus
Bacillus (Bacillaceae), pp. 1529-1661. ln: Antibiotics -Origin, Nature and Properties, Vol. III.
American Society of Microbiology, Washington, DC.
Loeffler, W., J.S.-M. Tschen, N. Vanittanakom, M. Kugler, E. Knorpp, T.-F. Hsieh, and T.-G. Wu.
1986. Antifungal effects of bacilysin and fengymycin from Bacillus subtilis F-29-3: A comparison
with activities of other Bacillus antibiotics. J. Phytopathol. 115(3):204-213.
Logan, N.A. 1988. Bacillus species of medical and veterinary importance. J. Med. Microbial. 25:157-
165.
Logan, N.A ., and R.C.W. Berkeley. 1981. Classification and identification of the genus Bacillus using
API tests, pp. 106-140. In: R.C.W. Berkeley and M. Goodfellow (eds.). The Aerobic Endospore-
Forming Bacteria: Oassification and Identification. Academic Press, Inc., London.
McKeen, C.D., C.C. Reilly, and P.L. Pusey. 1986. Production and partial characterization of
antifungal substances antagonistic to Monilinia fructicola. Phytopathol. 76(2): 136-138.
Norris, J.R., R.C.W. Berkeley, N.A. Logan, and A.G. O'Donnell. 1981. The genera Bacillus and
Sporolactobaclllus, pp. 1711-1742. In: M.P. Starr et al. (eds.), The Prokaryotes: A Handbook on
Habitats, Isolation, and Identification of Bacteria, Vol. 2. Springer-Verlag, Berlin.
Obi, S.K.C. 1980. Lecithinase and toxin production in Bacillus spp. Zentralbl. Bakteriol. 1 ABT. Orig.
A Med. Mikrobiol. Infektionskr. Parasitol. 246(3) 415-422.
O'Donnell, A.G., J.R. Norris, R.C.W. Berkeley, D. Claus, T. Kanero, N.A. Logan, and R. Nozaki.
1980. Characterization of Bacillus subtilis, Badl/us pumilus, Bacillus licheniformis, and Bacillus
amyloliquefadens by pyrolysis gas-liquid chromatography, deoxyribonucleic acid-deoxyribonucleic
acid hybridization, biochemical tests, and API systems. Internat. J. Syst. Bacterial. 30:448-459.
Parry, J.M., P.C.B. Turnbull, and J.R. Gibson. 1983. A Colour Atlas of Bacillus Species. Wolfe
Medical Publications, Ltd., London.
Priest, F.G. 1981. DNA homology in the genus Bacillus, pp. 33-57. In: R.C.W. Berkeley and M.
Goodfellow (eds.), The Aerobic Endospore-Forming Bacteria: Classification and Identification.
Academic Press, Inc., London.
Priest, F.G., M. Goodfellow, L.A. Shute, and R.C.W. Berkeley. 1987. Bacillus amyloliquefaciens sp.
nov. nom. rev. Intemat. J. Systematic Bacterial. 37:66-71.
Priest, F.G., M. Goodfellow, and C. Todd. 1981. The genus Bacillus: A numerical analysis, pp-91-
103. In: R.C.W. Berkeley and M. Goodfellow (eds.), The Aerobic Endospore-Forming Bacteria:
Classification and Identification. Academic Press, Inc., London.
http://www.epa.gov/biotech _ rule/pubs/fra/fra009 .htrn[ 1/17/2014 9:32 :07 AM]
Bacillus subtilis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) { US EPA
{
Priest, F.G., M. Goodfellow, and C. Todd. 1988. A numerical classification of the genus Bacillus. J.
Gen. Microbiol. 134: 1847-1882.
Reilly, B. 1991. Analysis of Environmental Releases and Occupational Exposure in Support of
Proposed TSCA 5(h)(4) Exemption. Unpublished, U.S. Environmental Protection Agency,
Washington, D.C.
Saris, EJ,, U. Airaksinen, S. Nurmiharju, K. Runeberg-Nyman, and I. Palva. 1990. Expression of
Bordatella-pertussis toxin subunits in Bacillus subtilis. Biotechnol. Lett. 12:873-878.
Saris,P., S. Taira, U. Airaksinen, A. PaIva, M. Sarvas, I. Palva, and K. Runeberg-Nyman. 1990.
Production and secretion of pertussis toxin subunits in Bacillus subtilis. Fed. Eur. Microbioi, Soc.
Microbiol. Lett. 56:143-148.
Seki, T., T. Oshirna, and Y. Oshima. 1975. Taxonomic study of Bacillus by deoxyribonucleic acid -
deoxyribonucleic acidhybridization and interspecific transformation. Internat. J. Syst. Bacterial.
25:258-270.
Stanghellini, M.E. and S.L. Rasmussen. 1989. Two new diseases of Salicornia sp. caused by Bacillus
subtilis and Macrophomina phaseolina. Annual Meeting of the American Phytopathological Society,
Pacific Division, June 20-21, 1989. Phytopathology 79(8):912.
Taira, S., E. Jalonen, J.C. Paton, M. Sarvas, and K. Runeberg-Nyman. 1989. Production of
pneumonlysin, a pneumococcai toxin, in Bacillus subtilis. Gene 77:211-218.
Turner, B. 1970. Workbook of Atmospheric Dispersion Estimates. U.S. Environmental Protection
Agency, Research Triangle Park, NC.
U.S. Department of Health and Human Services. 1986. Guidelines for research involving
recombinant DNA molecules; Notice. 51 FR 16958, May 7, 1986.
Versar. 1992. Screening level exposure assessment of Bacillus species for 5(h)(4) exemption under
the proposed biotech rule. Unpublished, U.S. Environmental Protection Agenry, Washington, D.C.
Yakovleva, L.M., P.V. Derevyankin, and R.I. Gvozdyak. 1990. Bacteriosis in the Norway maple.
Mikrobioloicheskii Zhumal (Kiev) 52(4):60-64.
Zimmerman, S.B., C.D. Schwartz, R.L. Monaghan, B.A. Pleak, B. Weissberger, E.C. Gilfillan, 5.
Mochales, S. Hernandez, S.A. Currie, E. Tejera, and E.O. Stapley. 1987. Difficidin and oxydiffrcidin:
Novel broad spectrum antibacterial antibiotics produced by Bacillus subtilis. J. Antibiotics
40(12):1677-1681.
EPA Home I Privacy and S-surity Notim { Contact Us
http: //www. epa. gov)biotech_rule f pubs/fra1fraD09.htm
Print }]s-Is
Last updated on ?9'/?27?/?2012
http://www,epa.govlbiotech_rule/pubs./&a/fra009.htm{ 1 /17/20t4 9:32:07 AM]

Bacillus Licheniformis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) US EPA
TSCA Biotechnology
Home
TSCA Biotechnology
Regulations
Filing a Biotechnology
Submission
TSCA Biotechnology
Notifications
Other EPA
Biotechnology
Activities
Other Government
Biotechnology Sites
Biotechnology
Publications
TSCA Biotechnology
Regioriat Contacts
U.S. ENVIRONMENTAL PROTECTION AGENCY
Biotechnology Program under the Toxic
Substances Control Act (TSCA)
Contact Us Search: ©All EPA ()This Area
You are here: LPA Home Chemical Safety & pollution Prevention w Pollution PreventjpQ$e_Toxics x einreehnolOpy
W► Bacillus Lirhenifomnts Final Risk Assessment
Bacillus Licheniformis Final Risk Assessment
ATTACHMENT I --FINAL RISK ASSESSMENT OF
BACILLUS LICHENIFORMI$
(February 1997)
I. INTRODUCTION
Bacillus licheniformis is a saprophytic bacterium that is widespread in nature and thought to
contribute substantially to nutrient cycling due to the diversity of enzymes produced by members
of the species. It has been used in the fermentation industry for production of proteases, amylases,
antibiotics, and specialty chemicals for over a decade with no known reports of adverse effects to
human health or the environment. This species is easily differentiated from other members of the
genus that are pathogenic to humans and animals. There are several reports in the literature of
human infections with B. Iicheniformis, however, these occurred in immunosuppressed individuals
or following trauma, There are no indications that 8_ Iicheniformis is pathogenic to plants.
However, there are numerous reports in the literature of an association between 8. Iicheniformis
and abortions in livestock. In most reports, there were predisposing factors which may have
resulted in immunosuppression of the affected animals. Since B. licheniformis is ubiquitous in the
environment and appears to be an opportunistic pathogen in compromised hosts, the potential risk
associated with the use of this bacterium in fermentation facilities is low.
History of Commercial Use and Products Subject to TSCA Jurisdiction
8. licheniformis has been used in the fermentation industry for over a decade for production of
proteases, amylases, antibiotics, or specialty chemicals. The ATCC Catalogue of Bacteria and
Phages lists strains which are capable of producing alkaline proteases, alpha -amylases,
penicillinases, pentosanases, bacitracin, proticin, 5'-inosinic acid and inosine, citric add, and
substituted L-tryptophan (Gherna et al., 1989). statistics from ten years ago (Eveleigh, 1981),
indicated that industrial microbial fermentation was responsible for production of 530 tons of
protease and 320 tons of alpha -amylase on an annual basis. According to Eveleigh (1981), the
main industrial protease was one produced by B. Iicheniformis for use as a cleaning aid in
detergents. Other TSCA uses for proteases include dehairing and batting in the leather industry
and TSCA uses of alpha -amylase include desizing of textiles and starch modification for sizing of
paper (Erikson, 1976).
EPA has reviewed, under TSCA, a genetically modified strain of B. Iicheniformis used for the
production of a hydrolase enzyme (P87-1511), and two recombinant strains for production of
alpha -amylase (P89-1071, and P92-SO).
II. IDENTIFICATION AND TAXONOMY
A. Overview
Bacillus Iicheniformis is a ubiquitous bacterium thought to be of importance in the environment as
a contributor to nutrient cycling due to the production of protease and amylase enzymes (Claus
and Berkeley, 1986). Although the actual numbers in existence in the environment for this species
have not been determined, in general, bacilli occur at population levels of 106 to 10' per gram of
soil (Alexander, 1977). B. Tereus is isolated most frequently from soils; however, this is thought to
be due to its ability to crowd -out other species in enrichment culture rather than reflecting an
actual predominance in soils (Norris et al., 1981). Unless a soil has been recently amended with
http://www.epa_gov/biotech_rule/pubs/ti-dfra005.htrni htm[ 1/17/20i 4 9:42:02 AM]

Bacillus Lichenifonnis Final Risk Assessment ! Biotechnology Program Under Toxic Substances Control Act (TSCA) ! US EPA
organic matter which provides for readily utilizable nutrients For vegetative cells, the bacilli exist
predominately as endospores. It is thought that between 60 to 100 % of soil populations of Bacillus
exist in the inactive spore state and that these endospores are capable of surviving for many years
(Alexander, 1977).
B. Taxonomy and Characterization
The genus Bacillus consists of a large number of diverse, rod -shaped Gram positive (or positive
only in early stages of growth) bacteria which are capable of producing endospores that are
resistant to adverse environmental conditions such as heat and desiccation (Claus and Berkeley,
1986). Typically, the cells are motile by peritrichous flagella and are aerobic_ The genus consists of
a diverse group of organisms as evidenced by the wide range of DNA base ratios of approximately
32 to 69 mol% G + C (Claus and Berkeley, 1986) which is far wider than usually considered
reasonable for a genus (Norris et al., 1981).
B. licheniforrnis is ubiquitous in nature, existing predominately in soil as spores. Unlike other bacilli
that are typically aerobic, B. licheniformis is facultatively anaerobic, allowing for growth in
additional ecological niches. The microorganism is usually saprophytic. Its production of proteases
and ability to break down complex polysaccharides enables it to contribute substantially to nutrient
cycling (Claus and Berkeley, 1986). Certain members of the species are capable of denitrification;
however, their importance in bacterial denitrification in the environment is considered to be small
asthe bacilli typically persist in soil as endospores (Alexander, 1977)_
The Bacillus species B. subtilis, 8, licheniformis, and B. pumulis are closely related, and
historically, there has been difficulty distinguishing among the three species. Gordon (1973), who
conducted much of the pioneering work on the taxonomy of the genus, referred to these three
species as the subtilis-group or subtilis-spectrum.
More recent work has suggested that 8. ficheniformis is one of the better defined Bacillus species.
The species is genetically homogeneous based on DNA -DNA hybridization studies (Claus and
Berkeley, 1986). In addition, Seki et al. (1975) demonstrated that DNA -DNA hybridization studies
correlated well with species identification using conventional taxonomic characteristics such as
those in Bergey's Manual of Systematic Bacteriology (Claus and Berkeley, 1986). Based on numeric
taxonomic analyses, Priest et al. (1988) placed B. licheniformis in a unique phenotypic cluster
positioned close to, and between, B. subtilis and B. purnilus. Independently, similar but
unpublished work done for EPA by the Microbial Systematics Section at the National Institute of
Dental Research provided a tight cluster of 8. licheniformis strains. As in the Priest et al. (1988)
study, most strains clustered at the 92% level, but strains at the edges overlapped into the
adjacent duster, a small group of B. pumilus. Two B. pumilus strains also were embedded in the B.
licheniformis portion of the identification matrix. However, other studies have shown that 8.
licheniformis could be fairly readily differentiated from other species in the genus by the use of API
diagnostic test kits (Logan and Berkeley, 1981). In addition, B. licheniformis was also easily
distinguishable from other closely related members of the genus using pyrolysis gas -liquid
chromatography (O'Donnell et al., 1980.)
C. Related Species of Concern
There are several species of the genus which are known pathogens. These include B. anthracis
which is pathogenic to humans and other animals, and B. cereus which is a common cause of food
poisoning (Claus and Berkeley, 1986; Norris et al., 1981). B. thuringiensis, S. larvae, B.
fentimorbus, B. popiliae, and some strains of B. sphaericus are pathogenic to certain insects.
Other species in the genus can be opportunistic pathogens of humans or animals.
In a numerical classification using 118 characteristics of 368 species of Bacillus, the species B.
thuringiensis, 8. cereus, and B. mycoides clustered together at 89 - 92% similarity (Priest et al.,
1988). The B. subtilis group, to which 8. licheniformis belongs, joined the B. cereus group at 72%
relatedness. Therefore, there is no difficulty in distinguishing between the toxin -producing strains
of Bacillus and B. licheniformis.
III. HAZARD ASSESSMENT
A. Human Health Hazards
1. Colonization
htip://www.epa.vav/biotech_nsle./pubsifra/fra005 . htm [11l 712014 9:42:02 AM]

Bacillus Lichenifortnis Final Risk Assessment l Biotechnology Program Under Toxic Substances Control Act (TSCA) l US EPA
Bacillus licheniformis is a ubiquitous organism and likely enters the human digestive system many
times a day. While data regarding its ability to survive in the human gastrointestinal tract are
sparse, it is likely that the spores will pass through without causing harm. Outside the
gastrointestinal tract, the
organism would likely be a temporary Inhabitant of skin. Although it can grow over a wide range of
temperatures including that of the human body (Claus and Berkeley, 1986), it is unlikely that this
microorganism will colonize humans to any large degree. Contact with the microorganism,
therefore, would generally be relegated to soil and other environmental sources.
2. Gene Transfer
While the species itself does not appear to have virulence factor genes, the genus Bacillus is known
to be able to acquire plasmids from other bacteria in the environment. There is evidence to
suggest that other species of Bacillus, such as B. subtilis, actively exchange genetic information in
the soil (Graham and Istock, 1979). It is, therefore, theoretically possible for 8. licheniformis to
acquire the ability to produce toxins or other virulence factors; however, this has not been
demonstrated.
3. Toxin Production
A review of the literature by Edberg (1992) failed to reveal toxigenic substances produced by 8.
licheniformis. While there have been cases of acute, selfiimited gastroenteritis associated with the
isolation of large numbers of this species, a toxic or direct effect on intestinal epithelia has not
been demonstrated. It is difficult to ascertain whether the species in these reported cases, which
are quite limited in number, actively participated in the infection or were isolated in conjunction
with an unidentified pathogen. Obi (1980) reported that a number of species of the genus Bacillus,
including B. licheniformis, 8_ subtilis, 8. megaterium, and B. pumilus, were able to produce a
lecithinase. Lecithinase is an enzyme that can disrupt the cell membrane of mammalian cells.
However, there has not been acorrelation with production of this lecithinase and human disease.
4. Measure. of the Degree of Virulence
While not innocuous, B. licheniformis appears to have a very low degree of virulence. It does not
produce significant quantities of extracellular enzymes and other factors likely to predispose it to
cause infection. The species has been isolated a number of times from human infections. The
literature (cited below) suggests that there must be immunosuppression or trauma in order for
infection with this species to occur. Farrar (1963) divided human infections by species of Bacillus
into the following groups: (1) local infections of a closed space, such as the eye, in which the
organism is inoculated in high numbers secondary to trauma, (2) mixed infections in which the
species of Bacillus is found in the company of other organisms with higher virulence properties, and
(3) disseminated infections, usually in profoundly immunosuppressed individuals, in which the
species is recovered from multiple sites, usually including the blood stream.
Reviews of Bacillus infections from several major hospitals have indicated the relative lack of
virulence of B. licheniformis. For example, Ihde and Armstrong (1973) reviewed cases at Memorial
Sloan Kettering Cancer Hospital over a 6-1/2 year period. Unidentified species of Bacillus were
isolated in twelve cases of infection, two of which were felt to be serious. Banerjee et al_ (1988),
reviewing all Bacillus bacteremia cases during a sixyear period from 1978 to 1986, found 18 febrile
patients experiencing 24 episodes of bacteremia. B. licheniformis was isolated from one case. Of
these 18 patients, 15 had lymphoma or leukemia and three had breast cancer. Nine of the patients
had neutrophil counts of less than 1000. Seven of these patients had an indwelling Hickman
catheter in place. Scanning and transmission electron microscopy from one of the Hickman
catheters showed Bacillus organisms growing in a biofilm inside the Hickman catheter. By
comparison, during the same period, there were 1,038 cases of bacteremia.
In a review article, Logan (1988) reported several infections produced by B. licheniformis. One
case was an ophthalmitis, a corneal ulcer, Following trauma (Tabbara and Tarabay, 1979). Other
cases included septicemia and bacteremia, and peritonitis with bacteremia in a patient with an
upper small bowel perforation (Sugar and McCloskey, 1977). In the literature, there is also
circumstantial evidence implicating B. licheniformis as a cause of food poisoning (Gilbert et al.,
1981; Kramer et al., 1982). Fuchs et al. (1984) and Pessa et al. (1985) described Bacillus
infections associated with intravenous catheters.
http:Uwww.epa.govfbiotech_rnlelpubslfra/fra005.htm[ 1/17/2014 9:42:02 AM]

Bacillus Licbenitormis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
In a 10 year review of records at the YaleNew Haven Hospital, B. licheniformis was isolated four
times as a cause of infection (Edberg, 1992), In two patients the species was associated with eye
trauma; in one patient it was associated with a silicone -based implant; and in the fourth patient it
was associated with metastatic lung cancer.
5. Overall Assessment of Viiu ilenr.e
Edberg (1992) concluded that the virulence characteristics of B. licheniformis are very low. He
stated that in order to achieve an infection, either the number of microorganisms must be very
high or the immune status of the host low. While the possibility of infection with B. licheniformis is
Tow, it is not nonexistent.
6. Other J-lazards
Due to its ubiquitous presence as spores in soil and dust, B. licheniformis is widely known as a
contaminant of food (Norris et al., 1981). It is a common spoilage organism of milk (Mostert et al.,
1979; Foschino et al., 1990), packaged meats (Bell and DeLacy, 1984), and some canned goods
(Norris et al., 1981). However, it is typically not thought to be a causal agent of food poisoning. B.
licheniformis has also been shown to be a contaminant of pharmaceutical tablets (Nandapurkar et
al., 1985.)
7. Conclusions
B. licheniformis is not a human pathogen nor is it toxigenic. It is unlikely to be confused with
related species that are However, if challenged by large numbers of this microorganism,
compromised individuals or those suffering from trauma may be infected.
B. Environmental Hazards
1. Hazards to Animals
There are numerous reports in the literature on the association of B. licheniformis with livestock
abortions (for a more detailed account, see McClung, 1992). In a recent review article, Logan
(1988) stated that isolations of 8. licheniformis from bovine and ovine abortions appear quite
regularly in the Veterinary Record by the Veterinary Investigation Service and the Scottish
Investigation Service, especially after wet summers when the silage is of low quality. Ryan (1970)
reported the isolation of B. licheniformis in two cases of cattle abortion. Although it was not
possible to attribute this microorganism as the causal agent, attempts to demonstrate other
infectious agents yielded negative results. likewise, Mitchell and Barton (1986) also reported
isolation of only B. licheniformis in three cases ofbovine abortion. The presence of the B.
licheniformis in fetal stomach contents suggests that the bacterium is capable of entering the
bloodstream of the adult animals and crossing the placenta to the fetus.
Johnson et al. (1983) reported the death of 15 calves due to B. licheniformis infection in a herd in
Scotland. In all cases, no viruses or bacteria other than B. licheniformis were isolated from the
stomach contents and internal organs. However, this herd apparently was debilitated by (1) an
earlier infection with BVD (bovine viral diarrhea) virus which is known to cause immunosuppression
in cattle, and (2) a severe vitamin A deficiency from poor quality, moldy hay. The authors
speculated that the feeding for three months on poor quality hay had exposed the calves to a
heavy challenge of B. licheniformis both through ingestion and inhalation.
According to a veterinary diagnostician in this country, the incidence of bovine abortion caused by
members of the Bacillus genus (both 6. licheniformis and B. cereus grouped together) was 3.5% of
the total abortions and stillbirths examined (8,962) over a 10-year period in South Dakota
(Kirkbride, 1993). The total number of abortions and stillbirths caused by all bacteria was 14.49%.
Bacillus ranked second in frequency of occurrence, after Actinornyces pyrogenes. The fact that
abortions associated with Bacillus species are less common compared to other microorganisms,
particularly viruses and fungi, has resulted in very little research being conducted to investigate
i•■- =mm 1 whether B. licheniformis is the actual causal agent in these cases. The veterinary diagnostics
�•■ laboratories in this country make attempts to isolate any and ail microorganisms present in the
aborted fetuses which are sent to them for inspection_ However, there is no determination of
whether the organism(s) isolated are the etiological agents and often there is little background
information supplied as to whether there were predisposing factors which may have led to
compromised immune systems in the animals.
http://www.epa.gov/biotech_mle/pubWfralfra005.htrn[l/I 7/2014 9:42:02 AM1

13aciiius Licheniformis Final Risk Assessment ! Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
B. licheniformis has also been reported to be associated with abortions in swine (Kirkbride et al.,
1986). Members of the genus Bacillus have also been associated with abortions in sheep (Mason
and Munday, 1968; Smith and Frost, 1968), however, in both these latter reports, species
identification was not made.
There are also reports in the literature of associations of B. licheniformis with bovine mastitis
(Logan, 1988) and goat mastitis (Kalogridou-Vassiliadou, 1991).
In addition, Wright et al. (1978) reported a water -borne B. licheniformis infection in laboratory
mice which resulted in depressed hemoglobin content, white cells and platelet counts.
Many of the reports on livestock abortion have suggested that 8. licheniformis is a causal agent.
This has been shown to be the case for B. cereus where inoculation of the microorganism resulted
in cattle abortion (Wohlgemuth et al., 1972). As yet, no one has confirmed B. licheniformis as the
actual etiological agent in animal abortions. This literature also suggests that in these cases of B.
licheniformis infection, the livestock was in a compromised immune state. According to Kirkbride
(1993), the immune reaction at the junction of the maternal and fetal placentas is suppressed,
most likely to prevent rejection of the fetus. Consequently, opportunistic microorganisms, even
with low virulence, have the ability to multiply and cause lesions, and result in abortion.
2., Haz_ards to Plants
No reports in the literature were encountered that suggested that 8. licheniformis is a plant
pathogen. There was no mention of any plant pathogenic activity in Bergey's Manual of Systematic
Bacteriology (Claus and Berkeley, 1986) nor in the U.S. Department of Agriculture list of pathogens
under the Federal Plant Pest Act (7 CFR 330, 2.t
3. Hazards Posed to Other Microorganisms
B. licheniformis is capable of producing several antimicrobial compounds. It produces the
antibiotics licheniformin (Callow and Hart, 1946), bacitracin (Johnson et al., 1945), and at least
one other antibiotic from a certain strain, 2725 (Woolford, 1972). Sacitracin is active mainly
against Gram positive bacteria, whereas the antibiotic from strain 2725 is active against various
Gram positive and Gram negative species (Woolford, 1972). These antibiotics have been shown to
be produced in culture, however, the importance of antibiotic production in regulating the soil
community and the significance in the environment is unknown (Alexander, 1977).
B. licheniformis has been shown to be inhibitory to the growth of various fungi and has recently
been investigated for its use as a biocontrol agent of several fungal pathogens. Shigemitsu et al.
(1983) noted malformation of Fusarium oxysporum f. sp. cucumerinum caused by metabolite(s)
produced by B. licheniformis when the organisms were cultured together. Scharen and Bryan
(1981) also showed that metabolites of B. licheniformis produced in culture were antagonistic to
Pyrenophora Ceres, the cause of net blotch of barley. When applied to the leaves of barley
seedlings, 8. licheniformis established itself and prevented infection by the fungus. Likewise, 8.
licheniformis was shown to be antagonistic to Pyrenophora tritici-repentis which causes wheat tan
spot (Mehdizadegan, 1987). Singh and ❑wivedi (1987) reported that B. licheniformis reduced the
growth of Sclerotium rolfsil sacc. (the causal agent of foot rot ofbarley) by 310I0 in mixed culture.
The metabolites alone produced by the bacilli in culture were also inhibitory to the pathogen. In
addition, B. licheniformis was shown to be antagonistic to Phymatotrichum omnivorum, the cause
of cotton root rot (Cook et al., 1987). Although B. licheniformis and/or products produced by the
microorganism are inhibitory to the growth of numerous other microorganisms in the environment,
due to the widespread nature of this bacterium, it is unlikely that any perturbations in microbial
community structure would occur by the potential release of additional numbers of these
microorganisms to the environment from fermentation facilities operating under the conditions of
the exemption.
4. Conclusions.
The issue of livestock abortions is the most serious environmental hazard identified for 8.
licheniformis. However, it has not been scientifically established that 8. licheniformis is the
causative agent. B. licheniformis appears to be an opportunistic pathogen that may create
problems in immunocompromised livestock. However, livestock abortions associated with Bacillus
species are infrequent compared to other microorganisms.
http:liwww.epa.eov/biotech_ruleipubs/fra/fra005.htm[ I/17/2014 9:42:02 AM]

Bacillus Licheniformis Final Risk Assessment I Biotechnology Program Under "Foxic Substances Control Art (TSCA) l 1JS EPA
IV. EXPOSURE ASSESSMENT
A. Worker Exposure
B, licheniformis is considered a Class 1 Containment Agent under the National Institute of Health
(NIH) Guidelines for Research Involving Recombinant DNA Molecules (U.S. Department of Health
and Human Services, 1985). This microorganism also falls under the Class 1 Containment under
the European Federation of Biotechnology guidelines (Frommer et al., 1989).
No data were available for assessing the release and survival specifically for fermentation Facilities
using B. licheniformis. Therefore, the potential worker exposures and routine releases to the
environment from large-scale, conventional fermentation processes were estimated on information
available from eight premanufacture notices submitted to EPA under TSCA Section 5 and from
published information collected from non -engineered microorganisms (Reilly, 1991). These values
are based on reasonable worst -case scenarios and typical ranges or values are given for
comparison.
During fermentation processes, worker exposure is possible during laboratory pipetting, inoculation,
sampling, harvesting, extraction, processing and decontamination procedures. A typical site
employs less than 10 workers/shift and operates 24 hours/day throughout the year. NIOSH has
conducted walk-through surveys ofseveral fermentation facilities in the enzyme industry and
monitored for microbial air contamination. These particular facilities were not using recombinant
microorganisms, but the processes were considered typical of fermentation process technology.
Area samples were taken in locations where the potential for worker exposure was considered to
be potentially greatest, i.e., near the fermentor, the seed fermentor, sampling ports, and
separation processes (either filter press or rotary drum filter). The workers with the highest
potential average exposures at the three facilities visited were those involved in air sampling. Area
samples near the sampling port revealed average airborne concentrations ranging from 350 to 648
cfu/m3. Typically, the Chemical Engineering Branch would not use area monitoring data to estimate
occupational exposure levels since the correlation between area concentrations and worker
exposure is highly uncertain. Personal sampling data are not available at the present time. Thus,
area sampling data have been the only means of assessing exposures for previous PMN
biotechnology submissions. Assuming that 20 samples per day are drawn and that each sample
takes up to 5 minutes to collect, the duration of exposure for a single worker will be about 1.5
hours/day. Assuming that the concentration of microorganisms in the worker's breathing zone is
equivalent to the levels found in the area sampling, the worst -case daily inhalation exposure is
estimated to range up to 650 to 1200 cfu/day. The uncertainty associated with this estimated
exposure value is not known (Reilly, 1991).
B. Environmental and General Exposure
1. Fate of the Organism
B. licheniformis is a common saprophytic inhabitant of soils and is capable of producing endospores
when vegetative growth conditions are unfavorable. Unlike most bacilli, growth occurs under
anaerobic conditions as well as aerobic, and occurs at temperatures as high as 55C (Claus and
Berkeley, 1986). The endospores produced by 6. licheniformis resist severe heat treatment (Claus
and Berkeley, 1986). Specific data comparing the survivability of industrial and wildtype strains of
B. licheniformis were not available in the existing literature. However, the ability of E. licheniformis
to produce highly resistant spores and grow under a wide range of conditions indicates that
released strains are likely to survive outside of containment.
2. Releases
Estimates of the number of B. licheniformis organisms released during production are tabulated in
Table 1 (Reilly, 1991). The uncontrolled/untreated scenario assumes no control features for the
fermentor offgases, and no Inactivation of the fermentation broth for the liquid and solid waste
releases. Thecontainment criteria required for the full exemption scenario assume the use of
features or equipment that minimize the number of viable cells in the fermentor off -gases. They
also assume inactivation procedures resulting in a validated 6log reduction of the number of viable
microorganisms in the liquid and solid wastes relative to the maximum cell density of the
fermentation broth.
http://www.epa.gov/biotech_rule/pubs/fralfra005.hnnf 1 /17/2014 9:42:02 AM]

Bacillus t.icheniformis Final Risk Assessment l Biotechnology Program Under Toxic Substances Control Act (TSCA) l US EPA
TABLE 1. Estimated Number of Viable Bacillus licheniformis
Organisms Released During Production
Uncontrolled/ Full
Release Media Untreated Exemption Release
(cfu/day) (cfu/day) (days/yr)
Air Vents 2x108 - lx1011 z2x108 - 1x1011 350
Rotary Drum Fitter 250 250 350
Surface Water 7x1016 7x1010 90
Soil/Landfill 7x1018 7x1012 90
Source: Reilly, 1991
These are "worstcase" estimates which assume that the maximum cell density in the fermentation
broth for bacteria is 1011 cfu/ml, with a fermentor size of 70,000 liters, and the separation
efficiency for the rotary drum filter is 99 percent.
3. Air
Specific data which indicate the survivability of B. licheniformis in the atmosphere after release are
currently unavailable. Survival of vegetative cells during aerosolization is typically limited due to
stresses such as shear forces, desiccation, temperature, and UV light exposure. However, its ability
to survive in a broad habitat range and produce endospores suggests that this organism may
survive after release. As with naturally -occurring strains, human exposure may occur via inhalation
as the organisms are dispersed in the atmosphere attached to dust particles, or lofted through
mechanical or air disturbance.
Air releases from fermentor offgas could potentially result in nonoccupational inhalation exposures
due to point source releases. To estimate exposures from this source, the sector averaging form of
the Gaussian algorithm described in Turner (1970) was used. For purposes of this assessment, a
release height of 3 meters and downward contact at a distance of 100 meters were assumed.
Assuming that there is no removal of organisms by controls/equipment for offgases, potential
human inhalation dose rates are estimated to range from 3.0 x 103 to 1.5 x 106 du/year for the
uncontrolled/untreated scenario andless than that for systems with full exemptions. It should be
noted that these estimates represent hypothetical exposures under reasonable worst case
conditions (Versar, 1992).
4. Water
The concentrations of B. licheniformis in surface water were estimated using stream flow values for
water bodies receiving process wastewater discharges from facilities within SIC Code 283 (drugs,
medicinal chemicals, and pharmaceuticals). The surface water release data (cfu/day) tabulated in
Table 1 were divided by the stream flow values to yield a surface water concentration of the
organism (cfu/I). The stream flow values for SIC Code 283 were based on discharger location data
retrieved from the Industrial Facilities Dischargers (IFD) database on December 5, 1991, and
surface water Flow data retrieved from the RXGAGE database. Flow values were obtained for water
bodies receiving wastewater discharges from 154 indirect (facilities that send their waste to a
POTW) and direct dischargers facilities that have a NPDES permit to discharge to surface water).
Tenth percentile values indicate flows for smaller rivers within this distribution of 154 receiving
water flows and 50th percentile values indicate flows for more average rivers. The flow value
expressed as 7Q10 is the lowest flow observed over seven consecutive days during a l0year
period. The use of this methodology to estimate concentrations of 8. licheniformis in surface water
http:/fwww.epa.gov/biotech_rula/pubs/fi-alfra005.htrn[ 1 / 17/2014 9:42:02 AM}

Bacillus Licheniformis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) l US EPA
assumes that all of the discharged organisms survive wastewater treatment and that growth is not
enhanced by any component of the treatment process. Estimated concentrations of B. licheniformis
in surface water for the uncontrolled/untreated and the full exemption scenarios are tabulated in
Table 2 (Versar, 1992).
TABLE 2. Bacillus licheniformis Concentrations in Surface Water
Receiving
Flow Stream Flow Organisms
(MLD*) (cfu/I)
Mean 7Q10 Mean 7Q10
Uncontrolled/Untreated
loth Percentile 156 5.60 4.5x108 1.25x101°
50th Percentile 768 68.13 9.11x107 1.03x109
Full Exemption
loth Percentile 156 5.60 4.5x102 1.25x104
50th Percentile 768 68.13 9.11x101 1.03x103
*MLD = million liters per day
Source: Versar, 1992
5. Soil
The natural habitat for B. licheniformis is soil. Therefore, longterm survival in soil may be expected
to occur. Human exposures via dermal and ingestion routes, and environmental exposures [i.e., to
terrestrial, avian, and aquatic organisms (via runoff)] may occur at the discharge site because of
the establishment of B. licheniformis within the soil.
6. Summary
Although direct monitoring data are unavailable, worst case estimates do not suggest high levels of
exposure of 8. licheniformis to either workers or the public resulting from normal fermentation
operations.
V. INTEGRATION OF RISK
A. Discussion
Bacillus licheniformis is a ubiquitous, saprophytic, soil bacterium which is thought to contribute to
nutrient cycling due to its ability to produce a wide variety of enzymes. This latter feature of the
microorganism has been commercially exploited for over a decade. B. licheniformis has been used
for industrial production of proteases, amylases, antibiotics, and specialty chemicals with no known
reports of adverse effects to human health or the environment. The Agency has reviewed three
submissions for production of enzymes using genetically modified B. licheniformis.
Although the genus Bacillus is rather heterogenous based on a wide range of DNA base ratios (32
to 69 mol% G + C), the species B. licheniformis is rather homogeneous based on DNA -DNA
http://w►wv.epagov/biotech_ rule/pubs/fra/fra0O5.han(1/17/2014 9:42:02 AM]

Bacillus Licheniformis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
hybridization studies. Historically, B. licheniformis and two closely related species, B. subtilis, and
B. pumilus, were grouped taxonomically into what was known as the subtilis-group. However,
recently methods have been developed that allow B. /icheniformis to be differentiated from these
other species.
B. licheniformis is not a frank human pathogen, but has on several occasions been isolated from
human infections. Diseases attributed to B. licheniformis include bacteremia, opthalmitis following
trauma, and there are reports of food poisoning based on circumstantial evidence. However, the
literature suggests that there must be immunosuppression of the host, or there must be trauma
(especially to the eye) followed by inoculation in high numbers, before infection can occur. B.
licheniformis does not produce significant quantities of extracellular enzymes or other factors that
would predispose it to cause infection. Unlikeseveral other species in the genus, B. licheniformis
does not produce toxins. Overall, B. licheniformis has a low degree of virulence. Although the
possibility of human infection is not non-existent, it is low in the industrial setting where highly
immunocompromised individuals would not be present. Infection might be a possibility following
trauma, but in the industrial setting with the use of proper safety precautions, good laboratory
practices, and proper protective clothing and eyewear, the potential for infection of workers should
be quite low.
Likewise, the ecological hazards associated with the use of B. licheniformis are low. There are
various reports in the literature suggesting that B. licheniformis is a cause of abortion in livestock.
However, Koch's postulates have not been satisfied demonstrating that this microorganism was the
causal agent. In most these cases, infections with B. licheniformis occurred in animals already in an
immunocompromised state resulting from either (1) infection with other organisms or (2) poor
nutrition. Apparently, there is immunosuppression associated with maternal and fetal placentas in
pregnant livestock, whereby opportunistic microorganisms are capable of causing infection and
lesions in fetuses. Although B. licheniformis has not been shown to be an etiological agent of
livestock abortion, it has been associated with a number of cases. Even so, the association of B.
licheniformls with livestock abortion is quite small compared to the total number of abortions in
livestock caused by all other microorganisms, particularly viruses and fungi.
The use of B. licheniformis for industrial production of enzymes should not pose environmental
hazards. First, the number of microorganisms released from the fermentation facility is low. In
addition, B. licheniformis is ubiquitous in the environment, and the releases expected from
fermentation facilities operating under the conditions of this exemption will not significantly
increase populations of this microorganism in the environment. Therefore, although B. licheniformis
may be associated with livestock abortions, the use of this microorganism in fermentation facilities
will not substantially increase the frequency of this occurrence, even if a scenario for high exposure
to B. licheniformis released from the fermentation facility to livestock could be envisioned.
In conclusion, the use of B. licheniformis in fermentation facilities for production of enzymes or
specialty chemicals presents low risk. Although not completely innocuous, B. licheniformis presents
low risk of adverse effects to human health or the environment.
B. Recommendations
B. licheniformis is recommended for the tiered exemption.VI. REFERENCES
7 CFR 330, ~ ~-, as amended.
Alexander, M. 1977. Introduction to Soil Microbiology. John Wiley and Sons, Inc., New York.
Banerjee, C., C.I. Bustamante, R. Wharton, E. Tally, and J.C. Wade. 1988. Bacillus infections in
patients with cancer. Arch. Intern. Med. 148:17691774.
Bell, R.G. and K.M. Delacy. 1984. Influence of NaCl, NaN02, and oxygen on the germination and
growth of Bacillus licheniformis, a spoilage organism of chub-packed luncheon meat. J. Appl.
Bacterial. 57:523-530.
Callow, R.K. and P.D. Hart. 1946. Antibiotic material from Bacillus licheniformis (Weigmann,
emend. Gibson) active against species of mycobacteria. Nature 157:334.
Claus, D. and R.C.W. Berkeley. 1986. Genus Bacillus Cohn 1872, pp. 1105-1139. In: P.H.A. Sneath
et al. (eds.), Bergey's Manual of Systematic Bacteriology, Vol. 2. Williams and Wilkins Co.,
http://www.epa.gov/biotech_rule/pubs/fra/fra005 .htm[1 /17/2014 9:42:02 AM]
Bacillus Licheniformis Final Risk Assessment Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
Baltimore, MD.
Cook, C.G., K.M. El-zik, LS. Bird, and M.L. Howell. 1987. Effect of treatment with Bacillus species
on cotton root traits, yield, and Phymatotrichum root rot. Proceedings of the Beltwide Cotton
Production Research Conferences, Jan. 4-8, Dallas, TX.
Edberg, S.C. 1992. US EPA human health assessment: Bacillus licheniformis. Unpublished, U.S.
Environmental Protection Agency, Washington, D.C.
Erickson, R.J. 1976. Industrial applications of the bacilli: a review and prospectus, pp. 406419. In:
D. Schlesinger (ed.), Microbiology. American Society for Microbiology, Washington, DC.
Eveleigh, D.E. 1981. The microbial production of industrial chemicals. Scientific American 245:155-
178.
Farrar, W.E. 1963. Serious infections due to nnonpathogenic" organisms of the genus Bacillus. Am.
J. Med. 34: 134.
Foschino, R., A. Galli, and G. Ottogalli. 1990. Research on the microflora of UHT milk. Ann.
Microbial. Enzymol. 40:47-60.
Frommer, W., B. Ager, L. Archer, B. Brunius, C.H. Collins, R. Donikian, C.F. Frontali, S. Hamp, E.H.
Houwink, M.T. Kuenzi, P. Kramer, H. Lagast, S. Lund, J.L. Mahler, F. Normand-Plessier, K.
Sargeant, G. Tuijnenburg Muijs, S.P. Vranch, R.G. Werner. 1989. Safe biotechnology III. Safety
precautions for handlingmicroorganisms of different classes. Appl. Microbial. Biotechnol. 30:541-
552.
Fuchs, P.C., M.E. Gustafson, J.T. King, et al. 1984. Assessment of catheter associated infection risk
with the Hickman right atrial catheter. Infect. Control 5:226230.
Gherna, R., P. Pienta, and R. Cote. 1989. American Type Culture Collection, Catalogue of bacteria
and phages. American Type Culture Collection, Rockville, MD.
Gilbert, R.J., P.C.B. Turnbull, J.M. Parry, and J.M. Kramer. 1981. Bacillus cereus and other Bacillus
species: Their part in food poisoning and other clinical infections, pp. 297314. In: R.C.W. Berkeley
and M. Goodfellow (ed.), The Aerobic Endosporeforming Bacteria. Academic Press Inc., London.
Gordon, R.E. 1973. The genus Bacillus. Agricultural Handbook No. 427. Agricultural Research
Service, U.S. Department of Agriculture, Washington, DC.
Graham, J.B., and C.A. !stock. 1979. Gene exchange and natural selection cause Bacillus subtilis to
evolve in soil culture. Sci. 204:637639.
Ihde, D.C., and D. Armstrong. 1973. Clinical spectrum of infection due to Bacillus species. Am. J.
Med. 55:839845.
Johnson, W.S., G.K. Maclachlan, and G.F. Hopkins. 1983. An outbreak of sudden death and
respiratory disease in weaned calves due to Bacillus licheniformis infection and subsequent
abortions and stillbirths. Proceedings of the Third International Symposium of the World Association
of Veterinary Laboratory Diagnosticians, Volume 1, June 13-15, Ames, IA.
Johnson, B.A., H. Anker, and F.L. Meleney. 1945. Bacitracin: A new antibiotic produced by a
member of the B. subtilis group. Sci. 102:376-377.
Kalogridou-Vassiliadou, D. 1991. Mastitis-related pathogens in goat milk. Small Ruminant Res.
4:203-212.
Kirkbride, C.A. 1993. Bacterial agents detected in a 10-year study of bovine abortions and
stillbirths. J. Vet. Diagn. Invest. 5:64-68.
Kirkbride, C.A., J.E. Collins, and C.E. Gates. 1986. Porcine abortion caused by Bacillus sp. J. Amer.
Vet. Med. Assoc. 188:1060-1061.
Kramer, J.M., P.C.B. Turnbull, G. Munshi, and R.J. Gilbert. 1982. Identification and characterization
http://www.epa.gov/biotech _ rule/pubs/fra/fraOOS.htm[l/17/2014 9:42:02 AM]
Bacillus Licheniformis Final Risk Assessment I Biotechnology Program Under Toxic Substances Control Act (TSCA) I US EPA
of Bacillus cereus andother Bacillus species associated with food poisoning, pp. 261286. In: J.E.L.
Corry, D. Roberts, and F.A. Skinner (ed.), Isolation and identification methods for food poisoning
organisms. Society for Applied Bacteriology technical series no. 17. Academic Press, Inc., London.
Logan, N.A. 1988. Bacillus species of medical and veterinary importance. J. Med. Microbial. 25:157-
165.
Logan, N.A. and R.C.W. Berkeley. 1981. Classification and identification of the genus Bacillus using
API tests, pp. 106-140. In: R.C.W. Berkeley and M. Goodfellow (eds.), The Aerobic Endospore-
Forming Bacteria: Classification and Identification. Academic Press, Inc., London.
Mason, R.W. and B.L. Munday. 1968. Abortion in sheep and cattle associated with Bacillus spp.
Australian Veterinary J. 44:297-298.
McClung, G. 1992. Ecological hazard assessment of Bacillus licheniformis for the 5(h)(4)
exemptions in the proposed biotechnology rule. Unpublished, U.S. Environmental Protection
Agency, Washington, D.C.
Mehdizadegan, F. and F.J. Gough. 1987. Partial characterization of compounds produced by
Pseudomonas fluorescens and Bacillus licheniformis antagonistic to Pyrenophora tritici-repentis, the
cause of wheat tan spot. Phytopathol. 77:1720.
Mitchell, G. and M.G. Barton. 1986. Bovine abortion associated with Bacillus /icheniformis.
Australian Veterinary J. 63:160-161.
Mostert, J.F., H. Luck, and R.A. Husmann. 1979. Isolation, identification, and practical properties of
Bacillus species from UHT and sterilized milk. S. Afr. J. Dairy Sci. 11: 125-132.
Nandapurkar, S.N. 1985. Bacteria isolated from the pharmaceutical preparation: I. Tablets. Indian
J. Hosp. Pharm. 22: 131-139.
Norris, J.R., R.C.W. Berkeley, N.A. Logan, and A.G. O'Donnell. 1981. The genera Bacillus and
Sporolactobacillus, pp. 1711-1742. In: M. P. Starr et al. (eds.), The Prokaryotes: A Handbook on
Habitats, Isolation, and Identification of Bacteria, Vol. 2. Springer-Verlag, Berlin.
O'Donnell, A.G., J.R. Norris, R.C.W. Berkeley, D. Claus, T. Kanero, N.A. Logan, and R. Nozaki.
1980. Characterization of Bacillus subtilis, Bacillus pumilus, Baclf/us licheniformis, and Bacillus
amyloliquefadens by pyrolysis gas-liquid chromatography, deoxyribonucleic acid-deoxyribonucleic
acidhybridization, biochemical tests, and API systems. Intemat. J. Systematic Bacteriol. 30:448-
459.
Obi, S.K.C. 1980. Lecithinase and toxin production in Bacillus spp. Zentralbl. Bakteriol. 1 ABT. Orig.
A Med. Mikrobiol. Infektionskr. Parasitol. 246(3):415422.
Pessa, M.E., and R.J. Howard. 1985. Complications of HickmanBroviac catheters. Surg. Gynecol.
Obstet. 161:257260.
Priest, F.G., M. Goodfellow and C. Todd. 1988. A numerical classification of the genus Bacillus. J.
Gen. Microbiol. 134:1847-1882.
Reilly, B. 1991. Analysis of Environmental Releases and Occupational Exposure in Support of
Proposed TSCA 5(h)(4) Exemption. Unpublished, U.S. Environmental Protection Agency,
Washington, D.C.
Ryan, A.J. 1970. Abortion in cattle associated with Bacillus licheniformis. The Veterinary Record
86:650-651.
Scharen, A.L. and M.D. Bryan. 1981. A possible biological control agent for net blotch of barley.
Phytopathol. 71:902-903.
Seki, T., T. Oshima, and Y. Oshima. 1975. Taxonomic study of Bacillus by deoxyribonucleic acid-
deoxyribonucleic acid hybridization and interspecific transformation. Intemat. J. Systematic
Bacterial. 25:258-270.
http:/ /www.epa.gov/biotech _ rule/pubs/fra/fra005 .htm[ I /I 7/2 014 9 :42:02 AM]
Bacillus Licheniformis Final Risk Assessment 1 Biotechnology Program Under Toxic Substances Control Act (TSCA) i US EPA
Shigemitsu, H., K. Hirano, M. Kohno, H. Ishizaki, and H. Kunoh. 1983. Effect of Bacillus
licheniformis on Fusarium oxysporum f. sp. cucumerinum. Trans. Mycological Society of Japan
24:477-486.
Singh, R.K. and R.S. ❑wivedi. 1987. Studies on biological control of Sclerotium rolfsii sacc. causing
foot rot of barley. Acta Botanica Indica. 15:160-164.
Smith, I.Q. and A.3. Frost. 1968. The pathogenicity to pregnant ewes of an organism of the genus
Bacillus. Australian Veterinary J. 44:17-19.
Sugar, A.M. and R.V. McCloskey. 1977. Bacillus licheniformis sepsis. J. Am. Med. Assoc. 238:1180.
Tabbara, K.F., and N. Tarabay_ 1979. Bacillus licheniformis comeal ulcer. Am. J. Ophthalmol.
87(5):717719.
Turner, 8. 1970. Workbook of Atmospheric Dispersion Estimates. U.S. Environmental Protection
Agency, Research Triangle Park, NC.
U.S. Department of Health and Human Services. 1986. Guidelines for research involving
recombinant DNA molecules; Notice. 51 FR 16958, May 7, 1986.
Versar. 1992. Screening level exposure assessment of Bacillus species for 5(h)(4) exemption under
the proposed biotech rule. Unpublished, U.S. Environmental Protection Agency, Washington, D.C.
Wohlgemuth, K., E.J. Bicknell, and C.A. Kirkbride. 1972. Abortion in cattle associated with B.
cereus. J. Amer. Vet. Med. Assoc. 161:1688-1690.
Woolford, M.K. 1972. The semi large-scale production, extraction, purification, and properties of an
antibiotic produced by Bacillus licheniformis strain 2725.3. Appl. Bacterial. 35:227-231.
Wright, D.J.M., D.J. Frost, and P. Eaton. 1978. Water -borne Bacillus licheniformis infection in mice.
Laboratory Animals 12:149-150.
EPA Horne I Privacy and Security Noticq I Contact Us
http://www.epa .gov/ b i otech_rule/ pubs/fra/fra 005. htm
Print As -Fs
Last updated on ?9?/2272/?2012
http://www.epa.gov/hiotech_rule/pubs/fru/fra005.htm[l/I 7/2014 9:42:02 AMj

48 FAMILY I. BACILLACEAE
reflects former or current activity of vegetative Bacillus cells
there. Indeed, the ease with which strains of a close relative of
Bacillus pu·milus and strains of Bacillus thuringiensi,s have been
isolated from pristine environments along the Victoria Land
coast of Antarctica (Forsyth and Logan, 2000; and unpublished
observations) cannot easily be explained as widespread and
chance contamination ·with endospores from another source;
the organisms almost certainly undergo some multiplication in
those em-ironments. Bacillus Jv:marwli was found as both spores
and vegetative cells at geothermal sites in Antarctica where soil
temperatures ranged from 3.4 °C to 62.5 °C; the proportions
of sporulated cells tended to be higher at the temperature
extremes and lower at temperatures approaching the optimum
growth temperature (50 °C) of the organism (Logan et al.,
2000). Isolation of organisms showing special adaptions to
the environments in which they are found, such as acidophily,
alkaliphily, halophily, psychrophily, and thermophily, suggests
that these organisms must be metabolically active in these
niches, but it tells us little about the importances of their roles in
the ecosystems, and nothing about their interactions v.-ith other
members of the flora. Bacillus tlumnantarnicus, which warrants
transfer to Geobacillw; (see Species Incertae &dis, below), was
also found in the geothermal soil of Cryptogam Ridge, Mount
Melbourne, Antarctica (Nicolaus et al., 1996), a site from which
Bacillus Jumarioli was isolated (Logan et al., 2000).
Many &cillus species will degrade biopolymers, with versa-
tilities ~'31'}-ing according to species, and it is therefore assumed
that they have important roles in the biological cycling of carbon
and nitrogen; it is further assumed that their activities in food
spoilage and biodegradation reflect the contamination of these
materials by endospores derived from dusts and other vehicles.
Valid though these assumptions may be, the ever-increasing
diversity of known Bacillus species and their apparent primary
habitats implies that such generalizations may deserve reconsid-
eration in some cases, and that certain species may have quite
specialized activities.
Habitats. Although isolates of many of the established
species have been derived from soil, or from environments
that may have been contaminated directly or indirectly by
soil, the range of isolation sources is very l\-ide, and includes,
in addition to temperate, acidic, neutral and alkaline soils,
fresh and marine waters, foods and clinical specimens: air
(Bacillus carboniphilus), arsenic-rich sediments (Bacillus arsen-
iciselenatis, Bacillus selenitireducens) and arsenic-contaminated
mud and water (Bacillus indicus, Bacillus macyae), bauxite-pro-
cessing waste (Bacillm vedden), brine (Bacillus haloalkaliphi-
lus), compost (Bacillus circulans, Bacillus coagulans, Bacillus
lichenifonnis, Bacillus sphaericus, Bacillus subtilis), emperor
moth caterpillars ("phane~; Bacillus cereus, Bacillus circulans,
Bacillus liclumiformis, Bacillus megaterium, Bacillus mycoides,
Bacillus pumilus, Bacillus subtilis}, feathers (Bacillus cereus,
Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis), feces
(Bacillus alkalophilus, Bacillus badius, Bacillus cohnii, Bacillus
jle,:·us, Bacillus halodurans, Bacillus megaterium, Bacillus pseudo-
finnus), geothermally heated soils (Bacillus fumarioli, Bacil-
lus luciferensis, Bacillm schlegelii), honey bee and greater wax
moth frass (Bacillus cereus, Bacillus megaterium, Bacillus sphaeri-
cus), inner tissues of plants (Bacillus amyloliquefaciens, Bacillus
cereus, Bacillus erulophJticus, Bacillus insolitus, Bacillus licheni-
f0771lis, Bacillus megaterium, Bacillus pumilus, Bacillus subtilis),
invertebrates (Bacillm oleronius, Bacillus sphaerirn,s, Bacillus
thurin.giensis), leather (Bacillus cereus, Bacillus firm-us, Bacil-
lus lichenifm-mis, Bacillus tTUgaterium, Bacillus pumilus, Bacil-
lus sphacricus, Bacillus subtilis), milk ( Bacillus cereus, Bacillus
coagulans, Bacillus lichenif ormis, Bacillus smithii, Bacillus sporo-
thennodurans, Bacillus weihenstephanensis), naturally heated
'Waters (Bacillus methanolicus, Bacillus okuhidensis), poultry
litter and manure (Bacillus cereus, Bacillus Jastidiosus, Bacil-
lus halodurans, Bacillus pumilus, Bacillus sttbtilis), paper and
paperboard (Bacillus amyloliquefaciens, Bacillus cereus, Bacil-
lus circulans, Bacillus coagulans, Bacillus firmus, Bacillus Jlexus,
Bacillus halodurans, Bacillus liclzeniformis, Bacillus megaterium,
Bacillus mycoides, Bacillus pumilus, Bacillus sphaericus, Bacillus
subtilis, Bacillus thuringiensis), recycled paper pulp (Bacillus
pumilus), seaweed (Bacillus algicola), saline and hypersaline
emironments (Bacillus akawphilus, Bacillus firmzts, Bacillus
halodenitrificans, Bacillus halophilus, Bacillus megaterium), sew-
age and wastewater treatment processes (Bacillus funiculus,
Bacillus thermocloacae), sheep fleece (Bacillus cereus, Bacillus
thuringiensis), silage (Bacillus coagulans, Bacillus siralis), soda
lakes (Bacillus agaradhaerens, Bacillus cohnii, Bacillus psezukfir-
mus, Bacillus vedden), solfatara (Bacillus tusciae), gemstones
(Bacillus badius, Bacillus cereus, Bacillus circulans, Bacillus
coagulans, Bacillus firmus, Bacillus lentus, Bacillus licheniformis,
Bacillus mycoides, Bacillus subtilis; Khan et al., 2001), stone
surfaces of ancient monuments (Bacillus licl1eniformis, Bacil-
lus megaterium, Bacillus mycoides, Bacillus subtilis; Turtura et aL,
2000), subterranean soil and water (Bacillus infernus, Bacillus
subterraneus), and wall paintings (Bacillus decolorationis, Heyr-
man et al., 2003a; Bacillus barbaric-us, Taubel et al., 2003).
Most species of Bacillus are heterotrophic organisms that
have been isolated on complex organic media. Relatively few
attempts have been made to isolate aerobic endospore-formers
which can utilize inorganic sources of carbon and energy, or to
demonstrate that established heterotrophic species are capable
offacultative autotrophy. The two thermophiles Bacillus schlegelii
and Bacillus tmciae remain the only species in the genus shown
to be facultatively chemolithoautotrophic. Nitrogen fixation is
well established for certain species in Paenibacillus (Paenibacil-
lus az.otofixans, Paenihacill·us macerans, Paenibacillus poZvmyxa), but
less is knm11n about Bacillus species utilizing atmospheric nitrogen;
although Bacillus e.daphicus and Bacillus mucilagi1WSUS were iso-
lated on nitrogen-free media, these species are actually mem-
bers of Paenibacillus (see Species Incertae Sedis, below). However,
several studies have demonstrated nitrogen fixation by strains
of Bacillus cereus, Bacillus lichenij(m11is, &cillus megaterimn, Bacil-
lus sphaericus, and unidentified strains (some of which may have
been Paenibacillusspecies) isolated from rhizospheres and phyl-
loplanes, and from endophytic sites and mycorrhizae (Rozycki
et aL, 1999). A comparative phylogenetic study, however, con-
cluded that nitrogen fixation among aerobic endospore-form-
ers is restricted to certain species of Paenibacillus (Achouak
et al., 1999). Nitrogen-fixing Bacillus and Paeni.bacillw; growing
in the rhizosphere may help to promote plant growth; other
ways in which aerobic endospore-formers may promote the
growth of plants include (Chanway, 2002): the production of
phytohormones, increasing nutrient availability (to the plant or
to other, nitrogen-fixing, bacteria; Zlotnikov et al., 2001), the
suppression of ethylene production by the plant in its rhizo-
sphere, interactions l\-ith symbiotic bacteria and fungi (1\·ledina
et al., 2003), enhancement of root nodulation, and biological
control of plant pathogens by various mechanisms including
GENUS I. BACILLUS 49
the production of antibiotics. In one study, Bacillus subtilis
and Bacillus mycuides were found to dominate the rhizosphere
of tea bushes (Pandey and Palni, 1997), a strain of the latter
species having antifungal activity. Epiphytic Bacillus strains can
have protective roles in the phyllosphere (Collins and Jacob-
sen, 2003; Jock et al., 2002). Representatives of several species,
including Bacillus amyloliquefaciens, Bacillus cereus, Bacillus endo-
phyticus, Bacillus insolitus, Bacillus licheniformis, Bacillus megate--
rium, Bacillus pumilus and Bacillus subtilis, have been isolated
from the inner tissues of healthy plants, including cotton,
grape, pea, spruce and sweet corn, and some strains appear to
have important roles in growth promotion and plant protection
(Rel,a et al., 2002). These endophytes and epiphytes can have
potential as agents for the biocontrol of plant diseases, and
their spores offer advantages in the formulation of such prepa-
rations (Emmert and Handelsman, 1999). Hosford (1982) and
Leary et al. (1986), on the other hand, reported Bacillus species
showing pathogenicity for plants.
Bacillus species are known to have roles in the postharvest
processing and flavor development of cocoa (Schwan et al.,
1995), coffee (Silva et al., 2000), tobacco (English et al., 1967)
and vanilla (Roling et al., 2001), in the production of natural
fibers and other vegetable products, in several traditional fer-
mented foods based on leaves and seeds (and poultry eggs)
(often dominated by Bacillus subtilis, Wang and Fung, 1996;
Beaumont, 2002; Sarkar et al., 2002), and in composting (Blanc
etal., 1999; Strom, 1985).
Bacillus species may cause deterioration of hides intended
for leather production (Birbir and Ilgaz, 1996). Keratinolytic
strains of Bacillus cemus, Bacillus licheniformis, Bacillus pumilus,
Bacillus subtilis and of unidentified Bacillus species have roles in
the degradation of feathers in poultry waste and may be found
in the plumage of many bird species (Burtt and Ichida, 1999;
Kim et al., 2001). Bacillus species also play a part in the degrada-
tion of chitin. This acti,.,ity has been demonstrated for strains
of Bacillus circulans, Bacillus coa~ns, Bacillus lentus, Bacillus
lichenifurmis, Bacillus megaterium, Bacillus pumilus and Bacillus thu-
ringiensis (Clements et al., 2002), and strains of Bacillus amyloliq--
uefaciens, Bacillus cemus, Bacillus megaterium, Bacillus sphaericus
and Bacillus subtiliswhich utilize the chitin in crustacean wastes
have been isolated (Sabry, 1992; Wang and H",ang, 2001). The
,ruue of chitinolysis to insect-pathogenic strains of Bacillus thur-
ingiensis is evident, and an exochitinase from a strain of" B thu-
ringiensis subsp. pakistam~ ,1,as found to be toxic to 11edes aeg;ypti
lanae (Thanthiankul et al., 2002). Chitinases from a soil isolate
of Bacillus amyloliquejaciens have been found to have antifungal
properties (Wang et al., 2002). (The two species Bacillus chitin-
olyticus and Bacillus ehimensis, which were isolated using a chitin
medium, are members of Paenibacillus).
Strains of several Bacillus species have been found to accu-
mulate metal ions non-enzymically by adsorption to their cell
surfaces and this can be of importance in waste treatment and
natural environments: Bacillus licheniformis cells can accumulate
cerium, cobalt and copper ions from aqueous and simulated
waste solutions (Hafez et al., 2002), Bacillus subtilis may accumu-
late aluminum, cadmium, iron and zinc, and aluminosilicates
(Urrutia and Beveridge, 1995), and an unidentified Bacillus
strain bound chromium, copper and lead ions (Nourbakhsh
et al., 2002). Bacillus megaterium biomass \\'<lS found to bioreduce
ions of the precious metals gold, palladium, platinum, rhodium
and silver (Lin et al., 2001). Bacillus arseniciselenatis and Bacil-
lus selenitreducens can use oxyanions of the two highly toxic ele-
ments arsenic and selenium as terminal electron acceptors in
anaerobic respiration, and the environmental impact of such
activity is becoming appreciated (Stolz and Oremland, 1999).
The trichome-forming bacteria "Anisomitus", "Arthromitus",
"Entomitus", "Coleomitus", "Af.etahacterium, and "SprTTospirillum",
which occur in the alimentary tracts of animals, and which have
been reported to form endospores, were listed as Genera Incer-
tae Sedis in the First Edition of this i\1anual (Claus and Berke-
ley, 1986). Since that time, molecular methods have allowed
considerable progress to be made in the taxonomy of some of
these organisms. A cultivable • Arthromitus" strain from sow bug
or wood louse (Porcellio scaber) has been identified as Bacillus
cereus, and similar organisms have been isolated from moths,
roaches and termites 0orgensen et al., 1997; Margulis et al.,
1998). Bacillus oleronius \\'<lS first isolated from the hindgut of
the termite Reticulitermes santonensis, and cellulolytic strains
of the Bacillus cereus group and Bacillus -megaterium have been
found in the gut of another termite, motermopsis angusticollis
(Wenzel et al., 2002). An "Arthromitus~ -like endospore has been
reported from a Miocene termite preserved in amber (Wier
et al., 2002). On the other hand, several nonculturable, seg-
mented, filamentous bacteria from chickens, mice, rats and
trout have been shown to represent a distinct subline of the
Clostridium subphylum, which has been proposed as "Candida-
tus Arthromitus" (Snel et al., 1995; Urdaci et al., 2001). Strains
of "Metahacterium polyspora" from guinea pig cecum are closely
related to the extremely large, viviparous, intestinal symbionts
of the surgeonfish, Epulopiscium species, and also belong to the
Cwstridiumsubphylum (Angert etal., 1996).
Cellular fatty acids. This approach \\'<lS recently reviewed by
Kampfer (2002). On the basis of the fatty acid compositions
of 19 Bacillus sensu lato species Kaneda (1977) recognized six
groups (A-F). All except group D were found to contain major
amounts of branched chain acids, while within groups A--C only
insignificant amounts (<3%) of unsaturated fatty acids were
found; these latter three groups could be separated on the basis
of their predominant fatty acids. Group A contained species
now allocated to Bacillus (Bacillus circulans, Bacillus lichen.iformis,
Bacillus megaterium, Bacillus pumilus and Bacillus subtilis ), Brroiha-
cillus and Paenihacillus and contained C 14'° an,ci<o (26--60%) and
C 15'° 00 (13-30%) acids, with chain lengths of 14-17. Among
group B strains, now all allocated to Paen-ihacillus, C 1M ='"'"' acid
predominated (39--62%), with chain lengths between 14 and
17. In group C species, now accommodated in Geobacillus, C 15'°
00 acid ,1,as predominant. In group D, now A.licyclohacillus, a
unique fatty acid pattern with up to 70% cyclohexane fatty acids
of chain length 17-19 \\,as found. Group E comprised members
of the Bacillus cereus group of species which, unlike the other
groups, always had small proportions (7-12%) of unsaturated
fatty acids present; the predominant fatty acids (19--21 % ) were
of the C 15'°'-"' type. The psychrophiles Bacillus (now Sporosarrina)
glohispm11s and Bacillus insolit11s formed group F and contained
large proportions (17-28%) of unsaturated fatty acids, and the
predominant branched-chain fatty acids in these two species
were C,s,oamei.so acids. The work ofKaneda was largely confirmed,
and \\'<lS supplemented, by the comprehensh·e study of Kampfer
(1994). For many species of Bacillus sensu strido (but except-
ing Bacillus badim, the Bacillus cere-us group, Bacillus circuhms,
O'Donnell Formulas, Flora eaLsnce, Bacillus Laicroayurus Bod Strain, Sp Capsules - iHcrb.com
Trusted Brands. Heaitny Rewards.
- US (English) -
Vest Sellers ELM!! Speciaj6 Trials Frbles
Free shioring for orders over S7Q
My Account j View Cart & Check Out
Items In Can Total $0.00
LReorder
# A BCDE F G 11 I J K L MN OP ORS T U V W X Y Z
Suoptcrnents
Bath & Beauty
Grocery
O'Donnell Formulas, Flora Balance
Categories s Supplements s Probiotics, Acidophilus Stabilized Probiotics
Related Products View All
4 11.5 Q1)
$17.57 trA-1 if:i 8
O'Donnell Formulas, Flora
W. Balance. Powder. leo g (3.5
Product Overview
Description
- Vegetable Capsules
• Non -Dairy
• Dietary Supplement
Kids & Babies
Other
Links
Visit Manutadure tS we1Sile
O'Donnell Formulas, Flora Balance, Bacillus Laterosporus Bod
Strain, 60 Capsules
SRP: S24.95
Our Price: $17.57
Savings of. $7.38 (30% Off)
`! +5 19 Renews
In Stock: Yes
1 W'Rdd to can ' 0 Wish List
Free 1-5 Dav Domestic Ship;,iry for orders °vet5a9 C�
Jarrow Formulas,
Lactoferrin, 250 mg, 60
Capsules
10 Like this . itirTweet;
Specifications
¢_n
Shipping Weight 0.18 lbs (0.08 kg)
• Product Code: FOB-90002
• UPC Code: 027189900020
• Package Quantity: 60 Capsules
Dimensions: 2.1 x 2.1 x 3.6 in, 0.15
lbs (0.07 kg)
u p 21st Century Health Care,
Lycopene, Maximum
Strength, 25 mg, 60 Tablets
$23.37 '�,:; .r 82 - y7.09 ***Of* 63
Expiration Date: Jui 2018
Reasons to Shop at iHerb1
Page 1 oul of 3
O'Donnell Formulas, Flora
Balance, The Ultimate
Acidophiius Plus Bacillus
$13.44 :Tis`fi3 13
Flara-Balance will help maintain a healthy colon and will repopulate the intestine with beneficial bacteria, thus enhancing the immune system.
Flora-Batanc a is a revolutionary dietary supplement containing Bacillus Laterosporus (ROD strain), a viable microorganism, formulated under a
registered secret process with patents issued. Patent # 5455028
Suggested Use
Adults: Every morning, 20 minutes before eating, take two capsules with water. After one or two months, reduce usage by half.
Children: One capsule as described above. If you wish. capsules may be opened and contents dissolved in water.
Other Ingredients
Rice hour, vegetable capsule.
Trusted
Store
hnpJ/µviw.iherb.com/O-Drnutell-Formulas-Flent-Balance-ltaeillus-Lateraspnrus-Bod-Strain-60-CapsuleVe078[ 11t 713014 1.2:58fl8 pm]

O'Donnell Formulas, Flora Balance, Bacillus Lau: ospon=s Bod Strain. 60 Capsules - iHerb.com
Warnings
Store at room temperature, or refrigerate-
Do not use if seal is broken.
Supplement Facts
Serving Size: 2 Capsules
Servings Per Container: 30
Amount Per Serving % Daily Value
Total Carbohydrate <1 g 1 I'
Bacillus Laterosporus (BOO strain) 70 meg t
(Minimum of 1 million microorganisms)
'Percent Daily Values are based on a 2,000 calorie diet.
t Daily Value not established.
Customer Reviews
**5s *5 (19 Reviews)
5 Stars r - (15)
4 Stars { (0)
3 Stars R (2)
2 Stars (0)
1 Stars 5 (2)
,`Fci4la�so�aa� `
Ii+Nrtp Yt y.
71=STU : 17 JAN
About Us
Contact Us
Shipping
Help Topics
international OFaceboak ®iHerb Library iHerb Rewards
Customer Privacy ©Twitter iHerb Blog Affiliates
Mobile Apps ECGoogle+ IDPinterest Careers
Terms of Use D YouTube TInstagram Suppliers
iHerb.com COCopyright 1997-2014 iHerb inc. All rights reserved.
iriertKE is a regIstered trademark of iHertt, Inc Trusted Brands. Healthy Rewards and Me itierb.cam Trustee Brands, Healthy Rewards. Logo are trademarks of iHerb_ Inc
Fnday, Jan 17. 2014, 9 57 PST
Disclaimer Statement& made. cr product& 5:•L3 through tits websile. have rid teen evaluated by the United Slates Fool and Drug Admvsisbatiars.
They are ncl intended to diagnose, irsat ours or preA7tnr any disc Read more .
•
http:fikow •.iherb.eomfp-Donnctl-Formulas-Flora-Balance-Baallus-LatrnBporus-Bad-Strain-60-Capsuics'60741/17/20t4 12.58:08 PM)

308 FAMILY IV. PAENIBACILLACEAE
FIGURE 34. Type strain of Breuibacillus laterosporus grown on trypticase
soy agar for 24-36h, showing creamr-white and smooth colonies with
irregular margins. Bar= 2mm. Photograph prepared by NA Logan.
acids may be accompanied by the production of small amounts of
alkali , but, these generally are not easily detected by the routine
test methods, and some characters may prove to be inconsistent
when retested. Growth temperatures "ary considerably, and the
descriptions of the individual species should be consulted.
Habitats. Most Brevibacillus strains have been isolated from
the natural emironment, particularly soils, where they appear
to be saprophytes, but there have been some isolations from
human clinical specimens and from human illness, and Breviha-
cillus laterospurus has long been associated \\ith insect pathoge-
nicity. As with Bacillus, the spores of these organisms may readily
sunive distribution from these natural emironments to a wide
variety of other habitats, and some strains ha,·e been found as
contaminants in foods and pharmaceutical products. For more
information on endospores in the environment, see the chap-
ter on Bacillus. Breuibacillus species may be isolated following
heat treatment of specimens in order to select for endospores.
Although the presence of their spores in a given environment
does not necessarily indicate that the organisms are metaboli-
cally active there, repeated and independent isolations from
such a habitat make it reasonable to assume that vegetative
Brroibacillus cells are, or have been, active there. Isolates iden-
tified as Bacillus brevis prior to the allocation of many strains
bearing this name into the new and re'l-ived species Breuibacillus
agri, Breuihacillus l,o,-st,eumsis, Brevibacillus centrosporus, Brei.ribacil-
lus choshinensis, Brevihacillus JQT7llosus, Brcuihacillus (now Aneurini-
bacillus) migulanu,s, Bre11ibacillus paralmwis, Brevihacillus reus,-..eri,
Aneurinibacillus danicus, and strains assigned to Bacillus brevis by
authors unaware that these nomenclatural changes had been
proposed (between 1995 and 2004), may or may not be authen-
tic strains of this species or even members of the genus. This
should be borne in mind when reading the accounts of habitats
in which strains have been found. Isolations of strains identified
as Bacillus brevis ha,·e been reported from tannery processing
(Birbir and Ilgaz, 1996), black crusts on open-air stone mon-
uments in Italy (Turtura et al ., 2000), and soil contaminated
\\ith hexachlorocyclohexane. A Bacillus brevis strain secreting
an extracellular•cellulase was found in another soil (Singh and
Kumar, 1998), and Wenzel et al. (2002) found a cellulolytic
Brevibacillus breois in the gut of the termite 7-ootennopsis angusti-
collis. Isolates identified as Brevihacillus brevis have been reported
from the airborne dust of schools and children's daycare cen-
ters (Andersson et al., 1999), food packaging products of paper
and board (Pirttijarvi et al., 2000), the submerged rhizosphere
of the seagrass Vallisneria americana ( wild celery) in an estuarine
environment (Kurtz et al., 2003), and in the humus of Norway
spmce (Picea ahies) (Elo et al., 2000).
The sources of the type strains of the species Bre!!ihacillus
agri, Breuihacillus borst,ebmsis, Breuibacillus choshinensis, Brei.•ihacil-
lus f=us, Brroibacillus ginsengisoli, and Brevihacillus reus-..eri
were soils. Foodstuffi; are readily contaminated by soil organ-
isms; Breuihacillus cent:rospQTUS has been isolated from spinach
and Brevibacillus parabrevisfrom cheese. Breuibacillus centrospQTUS
has also been found in estuarine seagrass rhizosphere (Kurtz
et al., 2003). Strains of Brei.tibacillus invocatus and Brevibacillus
agri were repeatedly isolated from a pharmaceutical fermenter
plant and its antibiotic raw product over a period of several
months (Logan et al., 2002). Breuibacillus agri has also been iso-
lated from sterilized milk, a gelatin processing plant, clinical
specimens, and a public water supply where it was implicated in
an outbreak of\\-aterborne illness (Logan et al., 2002). Breviha-
cillus centrosporus was isolated from a bronchio-alveolar lavage,
Breuibacillus parabrevis was found in a breast abscess, and both
species have been isolated from human blood (Logan et al.,
2002). The original strain of the thermophilic species Breuibacil-
lus themwrube,-v.'as isolated from mushroom compost (Craveri
et al., 1966; Guicciardi et al., 1968), Brevibacillus borstelensis or a
close relative of this species was found to be a prominent mem-
ber of the flora of hot synthetic compost (Dees and Ghiorse,
2001), and a hydrogen sulfide decomposing strain of Brevihacil-
lu.s Jormosus has been isolated from pig feces compost (Nakada
and Ohta, 2001). The geothermal soil of the northwest slope of
Mount Melbourne, a volcano in Antarctica, yielded strains of a
moderately thermophilic and moderately acidophilic species,
Brli!IWacillus leuicki~ that were isolated in small numbers along
with Bacillus Jumarwli (Allan et al., 2005; Logan et al., 2000).
The specific epithet of Brevihacillus laterosporu.s is derived
from the organism's unique sporangial morphology. It pro-
duces parasporal bodies (PBs) that displace the spore later-
ally in the sporangium (Figure 32); these bodies have been
described as resembling canoes or the keels of ships. Montaldi
and Roth (1990) examined sporangia by thin-section trans-
mission electron microscopy and found three kinds of PB: i)
a large one, associated with the spore, and of similar volume
to it, with a lamellar structure of sequentially smaller layers, ii)
a smaller globular or angular one of 100-200nm in diameter
that appeared at the same time as the lamellar PB but which
was not attached to the spore in anyway, and iii) a striated, rod-
shaped PB with diameter of at least 200 nm. Brevihacillu.s latero-
sporus was originally isolated from water (Laubach, 1916), but
McCray (1917) isolated other strains with sporangial morpholo-
gies similar to the Laubach et al. strain from the diseased larvae
of bees. White (1920), who had named his bee larvae isolates
as Bacillus mpheus in 1912 but had not described them, recog-
nized the similarity between the two species. Bacillus orpheus is
thus a S}'l10n}m of Brli!IWacillus laterosporus. Endosporeformers
named Bacillus pulvifaciens by Katznelson (1950) were isolated
from diseased honeybee larvae (including cases of powdery
scale). Gordon et al. (1973) thought that they might form a
connection between Bacillus /.an•ae and Bacillus laterosporus and
Enhanced Hydrocarbon Biodegradation by a Newly Isolated
Bacillus subtilis Strain
Nelly Cbristovaa.*, Borjana Tuleva3 , and Boryana Nikolova-Damyanovab
a Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str, bl 26,
1113 Sofia, Bulgaria. E-mail: [e-mail]
b Institute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str,
bl 9, 1113 Sofia, Bulgaria
* Author for correspondence and reprint requests
Z. Naturforsch. 59c, 205-208 (2004); received August 8, 2003
Toe relation between hydrocarbon degradation and biosurfactant (rhamnolipid) pro-
duction by a new Bacillus subtilis 22BN strain was investigated. The strain was isolated for
its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-
active compound at high concentrations (1.5 -2.0 g 1-1). Biosurfactant production was de-
tected by surface tension lowering and emulsifying activity. The strain is a good degrader of
both hydrocarbons used v..ith degradability of 98.3 ± 1 % and 75 ± 2% for n-hexadecane and
naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination
of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated
with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first
report of Bacillus subtilis strain that degrades hydrophobic compounds and at the same time
produces rhamnolipid biosurfactant.
Key words: Hydrocarbon Degradation, Biosurfactants, Bacillus subtilis
Introduction
Organic compounds with limited water solubil-
ity are biodegraded very slowly because of their
low availability to microbial cells. The availability
of slightly soluble organic compounds can be en-
hanced by microbially produced surfactants which
increase aqueous dispersion by many orders of
magnitude (Zhang and Miller, 1992). In many
cases, biosurfactants also stimulate the biodegra-
dation of organic compounds. For example, alkane
degradation is stimulated by rhamnolipids (Zhang
and Miller, 1992), sophorose lipids (Oberbremer
and Muller-Hurtig, 1990) and phospholipids (Kap-
peli and Fmnerty, 1979).
The objective of this research was to investigate
whether the enhanced degradation of hydrocar-
bons by the newly isolated strain Bacillus subtilis
22BN could be related to the registered during
growth on n-hexadecane and naphthalene secre-
tion of rhamnolipid surfactant.
Material and Methods
Microorganism and medium
The strain Bacillus subtilis 22BN used in this
study was isolated from hydrocarbon contami-
nated industrial waste water samples. The organ-
ism was selected by means of enrichment culture
techniques for its ability to grow on n-hexadecane
and/or on naphthalene as single sources of carbon
and energy. The selected strain was identified on
the basis of Gram reaction, cell morphology and
several physiological and biochemical tests follow-
ing directions of the latest edition of Bergey's
Manual (Holt et al., 1994). The composition of the
mineral salt medium used in this study was de-
scribed by Tuleva et al. (2002).
Growth conditions
Batch growth experiments were performed in
500-ml Erlenmeyer flasks containing 100 ml min-
eral salt media, pH 7.2. The carbon source, n-hexa-
decane and crystalline naphthalene (Aldrich
Chemical Co., Steinheim, Germany), was added at
the concentration of 20 g 1-1 . The experiments
were started by inoculation with 5% log phase cul-
ture pregrown on meat peptone broth. All cultures
were performed in the dark at 28 ± 1 °C in an or-
bital incubator at 130 rpm. Control flasks without
bacteria were incubated in the same conditions to
quantify losses due to abiotic processes. As no sig-
nificant losses (less than 1 % ) were found in the
abiotic flasks, it is therefore assumed that losses
0939-5075/2004/0300-0205 $ 06.00 © 2004 Verlag der Zeitschrift ftir Naturforschung. Tubingen · http://www.znaturforsch.com · D
206 N. Christova et al. · Hydrocarbon Degradation by a New Bacillus subtilis Strain
are entirely due to biodegradation. Growth was
monitored by measuring the A 610•
Detection of biosurfactant activity
Samples of the culture media of the selected
strain were centrifuged at 8000 x g for 20 min. Sur-
face tension (ST) of the supernatant fluid of the
culture was measured by the ring method using
automatic Wilhelmy tensiometer (Biegler Elec-·
tronic, Mauerbach, Austria). The emulsifying ac-
tivity of the supernatant culture was estimated by
adding 0.5 ml of sample fluid and 0.5 ml of kero-
sene to 4.0 ml of distilled water. The tube was vor-
texed for 10 s, held stationary for 1 min, and then
visually examined for turbidity of a stable emul-
sion.
Blue agar plates containing cetyltrimethylam-
monium bromide (CTAB) (0.2 mg m1-1 ; Sigma
Chemical Co., Poole, UK) and methylene blue
(5 µg m1-1) were used to detect extracellular an-
ionic glycolipid production (Siegmund and Wag-
ner, 1991). Biosurfactants were observed by the
formation of dark blue halos around the colonies.
Detection and quantification of rhamnolipids
The surface active compound was extracted by
liquid-liquid extraction with 3 volumes diethyl
ether from the supernatant fluid which was previ-
ous acidified with HO to pH 2. The organic ex-
tracts were analyzed by thin layer chromatography
(TLC) on silica gel 60 plates (5553, Merck). Chro-
matograms were developed with chloroform/
methanol/acetic acid (15:5:1 v/v/v) and visualized
by orcinol/sulfuric acid staining as described by
Itoch et al. (1971) using rhamnolipids RLL
(½6H4809) and RRLL (C32Hss0n) from Pseu-
domonas aeruginosa as reference substances
(Jeneil Biosurfactant Company, Saukville, USA).
Further identification of the sugar moiety after
acidic hydrolysis confirmed it as rhamnose.
The orcinol assay (Chandrasekaran and Be-
miller, 1980) was used for direct assessment of the
amount of glycolipids in the sample. The rhamno-
lipid concentrations were calculated from standard
curves prepared with L-rhamnose and expressed
as rhamnose equivalents (RE) (mg m1-1).
Determination of residual n-hexadecane and
naphthalene concentrations
Biodegradation was measured as substrate dis-
appearance. Residual n-bexadecane and naphtha-
lene were extracted from whole cultures \vith two
volumes of n-hexane and analyzed by gas chroma-
tography using Hewlett-Packard gas chromato-
graph model 5890 equipped with a 30 m HP-5 cap-
illary column and a flame ionization detector.
Cell surface hydrophobicity test
The bacterial adhesion to hydrocarbons
(BATH) assay was used to determine changes in
cell surface hydrophobicity during growth on mini-
mal salt medium with 2% n-hexadecane or 2%
naphthalene (Rosenberg et al., 1980).
Analysis of naphthalene intermediates
The concentration of hydroxylated aromatic
metabolites from naphthalene degradation was
determined by the method of Box (1983) which
uses the Folin-Ciocalteu reagent. As it was as-
sumed that the major metabolite is salicylic acid,
a standard curve was prepared with sodium salicy-
late and the concentration of hydroxylated meta-
bolic intermediates was estimated as salicylate
equivalents in mg 1-1 .
Results and Discussion
Detection of the surface active compound
The newly isolated strain B. subtilis 22BN
formed halos on blue agar plates which detect the
production of extracellular anionic glycolipids
(Siegmund and Wagner, 1991). In the thin-layer
chromatogram one glycolipid spot was revealed
after the orcinol/sulfuric acid staining at Rr 0.86
corresponding to the reference monorhamnolipid
RLL from Pseudomonas aemginosa. Further iden-
tification of the sugar moiety after acidic hydroly-
sis confirmed it as rhamnose. It is interesting to
point out that the rhamnolipid is produced during
growth on hydrophobic carbon sources (hexade-
cane and naphthalene), while synthesis of the well
characterized lipopeptide biosurfactant surfactin
from B. subtilis or B. licheniformis is inhibited by
hydrophobic substrates (Cooper et al, 1981).
Surfactant production
B. subtilis 22BN produced rhamnolipid biosur-
factant on both hydrocarbons we used (n-hexade-
cane and naphthalene). In both cases, the surface
tension (ST) of the medium decreased at the be-
ginning of exponential growth. During growth on
n-hexadecane (Fig. I), there was a drop from 71 to
N. Christova et al. - Hydrocarbon Degradation by a New Bacillus subtilis Strain 207
1.6 - 16
L4
02
0
E 1 0
c
9a
06
a
122
i0
a
30ZG
10 A-
u
10
✓ tl
30
s0
70
60
90
100
tl 1 2 3 4 5 B 7
Time (days)
Fig 1. Binsurfactant production and n-hexadecane deg-
radation by Bacillus subtilis 22BN grown on mineral salt
medium with 2% n-hexadecane as substrate. Incubation
was done at 28 °C with shaking at 130 rpm. OD, optical
density. Biosurfactant levels are expressed as rhamnosc
equivalents (RE). Biodegradation is expressed as % re-
sidual n-hcxadccanc. Values arc averages from tripli-
cate flasks.
38.3 niN m--1 within 24 h of cultivation, then for
only 4 h ST declined to 21-1 mN m-' finally reach-
ing a minimum of 19 mN m-' within 48 h of incu-
bation. This finding was consistent with the results
obtained by Zhang and Miller (1992). They indi-
cated that low rhamnolipid concentrations cause
sharp lowering in the surface tension and may be
this is the dominant mechanism to enhance octa-
decane dispersion. Growth on naphthalene was
not accompanied by such dramatic changes in ST
(Fig. 2). It decreased to 355 mN m`' within 4 d of
cultivation reaching a minimum of 26.8 mN m-1
and did not decline further on. In addition, the
drop in the surface tension in both cases was ac-
companied by the formation of stable emulsions
of the cell -free culture broth with kerosene. which
indirectly implies the production of a biosurfactant
or a mixture of biosurfactants. High levels of
rhanntolipid at a concentration of 1.5 and 2.0 g 1 -1
were estimated in the stationary phase during
growth on n-hexadecane and naphthalene. respec-
tively.
so
_110
ra
70 —
30�
10 - to
o 0 Ili —.10a
a 4 a it is 20 20
Erma 1aay5l
Ftg.2. Biosurfactant production and naphthalene degra-
dation by Bacillus subtilis 22BN grown on mineral salt
medium with 2% naphthalene as substrate. Incubation
was done at 28 °C with shaking at 130 rpm- OD, optical
density. Biosurfactant levels are expressed as rhamnose
equivalents (RE). Biodegradation is expressed as % re-
sidual naphthalene. Values are averages from triplicate
flasks.
Hydrocarbon degradation
The impact of biosurfactant production was as-
sessed when the kinetics of degradation of the two
hydrocarbon substrates were analyzed. As seen in
Fig. 1, the biodegradation of n-hexadecane rapidly
increased after 3 d of cultivation and the biodegra-
dation percentage values (mean ± SD%, n = 3)
after 4 d of cultivation were already 79 t 2% and
at the end of the incubation (7 d) only 1.7 ± 1%
of the initial n-hexadecane was present.
Polycyclic aromatic hydrocarbons are utilized
only in the dissolved state. So, naphthalene degra-
dation by B. subtilis 22BN (Fig. 2) was slower com-
pared to that of n-hexadecane and accumulation
of naphthalene degradation metabolites was ob-
served when the concentration of rhamnoipids in
the medium increased sharply. The major metab-
olite, salicylic acid, reached maximal value of
450 mg 1-1 in the beginning of stationary growth
and did not change till the end of incubation, The
biodegradation percentage values (mean ± SI]%u,
n = 3) after 5 d of cultivation were 32 ± 4% and
at the end of the incubation (24 d) 75 ± 2% of the
initial naphthalene was biodegraded_

208 N. Christova et al. · Hydrocarbon Degradation by a New Bacillus subtilis Strain
These results suggest that in both cases hy-
drocarbon degradation was related with the accu-
mulation of the rhamnolipid biosurfactant in the
medium. By increasing the solubility of the hy-
drophobic substrates it facilitated their transport
to the microbial cells and enhanced their metabo-
lism. When n-hexadecane was used as the sub-
strate, concentration of the rhamnolipid of 0.4 g 1-1
was enough to allow access of the cells to the sub-
strate and to achieve 80% biodegradation within
only 4 d. If the strain utilized naphthalene higher
concentration of the biosurfactant (1.4 g 1-1) was
necessary to achieve 65% biodegradation for a
longer period (12 d) of cultivation.
Cell hydrophobicity during growth on
hydrocarbons
It has been suggested previously that cell sur-
face hydrophobicity is an important factor in pre-
dicting adhesion to surfaces (van Loosdrecht et al.,
1987). Thus, cell hydrophobicity was used as a
measure of potential cell affinity for hydrophobic
substrates and was determined by bacterial adher-
ence to hydrocarbon (BATH) assay.
Box J. D. (1983), Investigation of the Folin-Ciocalteu
phenol reagent for the determination of polyphenolic
substances in natural waters. Water Res. 17, 511-525.
Chandrasekaran E. V. and Bemiller J. N. (1980), Constit-
uent analyses of glycosaminoglycans. In: Methods in
Carbohydrate Chemistry (Whistler R. L., ed.). Aca-
demic Press, New York, pp 89-96.
Cooper D. G., Donald C. R., Duff S. J. B., and Kosaric N.
(1981), Enhanced production of surfactin from Bacil-
lus subtilis by continuous product removal and metal
cation addition. Appl. Environ. Microbial. 42, 408-
421.
Holt J. G., Krieg N. R., Sneath P. H. A., Staley J. T., and
Williams S. T. (1994), Bergey's Manual of Determina-
tive Bacteriology. Williams & Wilkins, Baltimore.
Itoch S., Honda H., Tomita F., and Suzuki T. (1971},
Rhanrnolipid produced by Pseudomonas aeruginosa
grown on 11-paraffin. J. Antibiot. 24, 855-859.
Kappeli 0. and Fmnerty W.R. (1979), Partition of al-
kane by an extracellular vesicle derived from hexade-
cane-grown Aci11erobacter. J. Bacterial. 140, 707-712.
van Loosdrecht M. C., Lyklema J., Norde W., Schraa G.,
and Zehnder A. J.B. (1987), The role of the bacterial
cell wall hydrophobicity in adhesion. Appl. Environ.
Microbial. 53, 1893-1897.
During growth on n-hexadecane, the transition
from exponential to stationary growth was accom-
panied by an important increase in cell surface
hydrophobicity (from 41 ± 3% to 75 ± 1 %,
respectively). When grown on naphthalene, the
hydrophobicity of B. subtilis 22BN cells changed
from 27 ± 5% in the early exponential phase to
66 ± 3% in the late exponential phase and then
to 79 ± 2% in the beginning of stationary growth.
These results are consistent with those of Zhang
and Miller (1994) who described that cell hydro-
phobicity can be induced to change in the presence
of a combination of both rhamnolipid and a
slightly soluble substrate. The importance of cell
surface properties for the biodegradation of hy-
drophobic organic substrates was indicated pre-
viously by Rosenberg and Rosenberg (1981). They
showed that the rate of hydrocarbon degradation
by the bacterial cells was dependent on cell affin-
ity. Cells with high affinity for hydrocarbons uti-
lized hexadecane more effectively than those with
low affinity. Similarly in this study, development
of higher hydrophobicity correlated with increased
utilization of both hydrocarbon substrates.
Oberbremer A. and Muller-Hurtig R. (1990), Effect of
the addition of microbial surfactants on hydrocarbon
degradation in a soil population in a stirred reactor.
Appl. Microbial. Biotechnol. 32, 485-489.
Rosenberg M., Gutnick D., and Rosenberg E. (1980),
Adherence of bacteria to hydrocarbons: a simple
method for measuring cell surface hydrophobicity.
FEMS Microbial. Lett. 9, 29-33.
Rosenberg M. and Rosenberg E. (1981), Role of adher-
ence in growtll of Acinetobacter calcoaceticus RAG-1
on hexadecane. J. Bacteriol.148. 51-57.
Siegmund I. and Wagner E (1991), New method for de-
tecting rhamnolipids excreted by Pseudomonas spe-
cies during growth on mineral agar. Biotechnol. Tech.
s, 265-268.
Tuleva B. K., Ivanov G. R., and Christova N. E. (2002),
Biosurfactant production by a new Pseudomonas pu-
tida strain. Z. Naturforsch. 57c, 356-360.
ZhangY. and Miller R. M. (1992), Enhanced octadecane
dispersion and biodegradation by a Pseudomonas
rhamnolipid surfactant (biosurfactant). Appl. En\'i-
ron. Microbial. 58. 3276-3282.
Zhang Y. and Miller R. M. (1994), Effect of Pseudomo-
nas rhamnolipid biosurfactant on cell hydrophobicity
and biodegradation of octadecane. Appl. Environ.
Microbial. 60, 2101-2106.
©Invent Enterprises
Vikas Nagar, Lucknow, INDIA
editor1jeb.co.in
Full paper available on: wvrw.seb.co.in
J. Environ. Biol.
33, 985-989 (2012)
ISSN:0254-8704
CODEN: JEBIDP
Biodegradation of Benzo[a]pyrene by the mixed culture of Bacillus
cereus and Bacillus vireti isolated from the petrochemical industry
Author Details
Ramya Mohandass
(Corresponding author)
Department of Genetic Engineering, SRM University Kattankulathur, Chennai -603 023, l nd is
e-mail: ramyamohandass@gmail_com
Paltabi Rout
Department of Biotechnology, SRM University, Chennai -603023, India
Sonia Jiwal
Department of Biotechnology, SRM University, Chennai - 603 023. India
Chltrambafam Sasiltala
Department of Genetic Engineering, SRM University, Chennai - 603 023, India
Publication Data
Paper received:
08 February 2011
Rsvrsedrerd:
08 Judy 2011
Accepted:
O6August2011
In troduction
Abstract
Polycyclic aromatic hydrocarbons are a.group of compounds that pose threat to humans and animal life.
Methods to reduce the amount of PAHs in the environment are continuously being sought. The bacterial
consortium capable of utilizing benzo(a)pyrene as the sole source of carbon and energy was isolated from
petrochemical soil. The tsolateswere ideated as 6aallus °emus and Bacr7lus virmti based on morphological
characterization, and 16S rtJNAgene sequence anatysis.About 58.98 %of benzo(a)pyrene at concentration
of 500 mg I•' in mineral salts medium were removed by bacterial consortium. GC mass spectral analysis
showed the presence of metabolite cis-�4-(14 ydroxypyren$yt)-2-oxobul-3ersoic acid. The results indicate
that the bactefiatoonsorfiuun is a new bacterial resource for biodegrading benzo(a)pyrene and might be used
for bioremedlationof sites heavily contaminated by benzo[a]pyrene and its derivatives.
Key words
8enzo(a) pyrene, Bacillus cereus, Bacillus viref , Biodegration, Mixed culture
Polycyclic aromatic hydrocarbons are a class of toxic
environmental pollutants consisting of two or more fused benzene
rings. They contain two or more fused aromatic rings in linear,
angular or duster arrangements.(Cemiglia, 1992). They are
generated continuously by the incomplete combustion of organic
matter, volcanic eruptions anti forest fires. They are also distributed
into the environment due to hurnan activities such as cigarette
smoking, automobile exhaust, and the processing, production and
spillage of petroleum (t hauhan et aL, 2008). PAHs are highly
toxic, mutagenic and carcinogenic. They do not degrade easily
under natural conditions, PAHs are hydrophobic compounds and
their persistence in the environment is due to theirlow water solubility
(Cerniglia, 1992). The solubility of PAH decreases and
hydrophobicity increases with an increase in number of fused
benzene rings. The volatility decreases with an increasing number
of fused rings (Wilson and Jones,1993). High molecular weight
PAI-Is such as benzola]pyrene, benzo[a]fluoranthene,
benzo(j)fkxoranthene, benzo(k)ffuoranthene and indeno(1,2,3-
cd)pyrene are potential carcinogens.
Benzo[a]pyrene (BaP), a potycydic aromatic hydrocarbon
(PAH) containing five fused benzene rings, is considered to be one
of the 16 PAt1s defined as prioritypoiutants by the US Environmental
Protection Agency (EPA) due to its carcinogenicity, and acute toxicity
(Boonchan etal., 2000). Benzo[a]pyrene may be removed from
the environment through the biodegradattve actions of bacteria and
fungi. BaP has been detected in a variety of environmental samples
(Juhasz and Naidu, 2000; Kanaly and Harayama, 2000). From
Journal of Environmental Biology •November 2012 •

986
contaminated soil and water, BaP enters into the food chain and
metabolized to its genotoxicform which then interacts with nucleic
a:id and proteins fomiing reactive mocromolecular adducls resulting
in BaP ilduced toxicity, mutagenesis c11d catinogenesis in mammals.
Studies have shown that there is a great diversity of microorganisms
which are capable of degrading low molecular weight PAHs such
as naphthalene, pyrene and phenanthrene. Relatively, few
organisms have been observed to degrade high molecular weight
PAHs. It has been reported that Mycobacterium vanbaa/enii PYR-
1 (Moody et al., 2004), Bacillus subtilis (Hunter et al., 2005) and
Sphingomonas yanoikuyae JAR02 (Rentz et al., 2008) are involved
in the degradation of BaP. There is limited information regarding the
bacterial degradation of high molecular weight PAHs in both
environmental samples and pure or mixed cultures (Kanaly and
Harayama, 2000). It has been reported that benzo[a]pyrene
biotransfonnation by bacteria occurs under cometabolic conditions
(Juhasz and Naidu, 2000). BaP cannot be utifized both as a carbon
and an energy source for single microorganisms (Cerniglia, 1992),
it is necessary that a growth substrate be supplied to initiate growth
of the organism and to induce the production of catabo6c enzymes.
Studies have shown that low molecular weight hydrocarbons are
metabolized by pure strains and biodegradation of high molecuar
weight hydrocarbons requires the combined efforts of different
populations (Kan~ et al., 2000). PAH compounds are transformed
throughout a series of different metabolk: reactions by microorganisms
in the real environment Therefore, the study of PAH metabolism by
bacterial consortia is important It will provide new insights for
improving future studies on bioremediation of environmental
pollutants.Aim of this study was isolation of the bacteria whiptoould
utilize the benzo(a)pyrene as its carbon source from petrochemical
contaminated soil and identification of the degraded-products of
benzo(a)pyrene by the bacterial consortium canied out in gas
chromatography-mass spectrometry.
Materials and Jlethods
Soil samp6ng : Soil sanplesweracollectedfromsoa con~ated
with oil refinery wastes of petroleum industry (Ctlennai Petroleum
Corporation Limited) situated in Che~. Tamil Nada, India It was
stored at -20"C.
Isolation and identifj.catlon otbenzo(a)pyrene degrading
bacteria: The soil samples were colle$dfrom three sites in a
petroleum industry situated in Chennai. 0.25 g of PAH
contarrinated soft-was suspended, in 50 ml of mineral salt medium.
Into the suspension 0.$ pgml-1 of benzo(a)pyrene were added
as enrichment substrate and. the suspension were incubated
with shaking·at 120 rpm at 30oC in the dark for 14 days. After 14
days, 100 µI of inoculum was spread on MSM-agar plate
supplementedwilfl benzo{a]pyrene (0.5 mg ml-1} as sole source
of carbon and energy and incubated for 48 h at 37oC.The
morphologically distinct bacterial colonies which appeared on
MSM-agar plates were aseptically picked and streaked on minimal
salt medium agar plates to obtain pure culture. All isolates were
Journal of Environmental Biology .November 2012•
Mohandass et al.
stored at -20°C as the liquid cultures containing 70% glycerol
(v/v).
Total genomic DNA was extracted according to the method
introduced by Kanaly and Harayama, (2000). The 16S rDNA
gene fragment was amplified by PCR using the set of primers: 16S-
8F {5'-GAGAGTTTGATCCTGGCTCAG-3') and 16S-1495R (5'-
C GGCTACCTTGTTACGA CTTC-3'). The PCR conditions (35
cycles of 3 min at 94 "C, 1 min at 94°C, 1 min50"C, 2 min at 72°C
and 2 min at 72°C} were performed in a thermal~ (Mastercycler
Gradient, Eppendorf, USA).The amplified products were eluted
from the gel and sequenced usingABI ~ Xi..fGenticAnalyser,
Applied Biosystems ,USA). Thff6SfONA sequence of the isolates
were compared using the BLAST program at the National Ce!Jter
for Biotechnology lnform.-i (NCBI, httpr//www.ncbi.QlRLmll.gov/)
and the sequences analysis deposited at Genbank with the
accession numbers.HM435450 ·~MM135451.
Blodegradatiortotbenzo{alJ>ytene : lheoonsortium was added
into MSM-BaP {0,5mg ml-1). Each culture flask containing 20 ml of
1nediwlplus bacHlriarmocula '8 x 10-1 CFU mt1) was placed on a
rotary shaker ( 120 ·rpm) at 30 oC in the dark. MSM containing
tk.Smg mt1 BaP,!>ut~outthe bacterial consortium, was used as a
control. Samples were extracted from the experimental system at 0,
2S and-35 days.
Extraction and identification of benzo[a)pyrene
metabolites: To obtain enough quantities of the possible
metabolites for separation and identification with GCMS, the
extraction of BaP was performed according to Li et al. (2008) and
l{analy and Harayama {2000).Afterdesired period of incubation,
samples from biodegradation experiments were used for the
extraction of benzo[a]pyrene. The contents from each culture
vials were centrifuged at 4000 rpm for 10 min and the particles
were allowed to deposit for 5 min, and 20 ml of supernatant was
transferred to separating funnels and extracted twice with an equal
volume ofdichloromethane. The organic extracts were pooled
and dried over anhydrous Na2S0 4 • The extracts were condensed
by evaporation of the dichloromethane under a stream of nitrogen,
and remains were dissolved in 1 ml hexane. Analyses of cultures
by GC-mass spectroscopy (MS) were performed by using a
Shimadzu GC 2010 instrument fitted with a fused silica capillary
column {DB-5; 30m by 0.25mm; J & W Scientific). The temperature
program was as follows: 50"C for 2min, followed by an increase
at a rate of 6°C min-1 to 300"C. The injection volume was 2 µI, and
the carrier gas was helium (flow rate: 1.7ml min-1). The mass
selective detector was operated in the scan mode to obtain spectral
data for compound identification (benzo(a)pyrene molecular ion
at rn/z 252).
Results and Discussion
Isolation of benzo(a)pyrene degrading bacteria : The
enriched soil samples obtained from the contaminated sites of
petrochemical industry contained two morphologically distinct
Biodegradation of benzo pyrene using bacterial strain
colonies. The isolate 1 was Gram positive, facultative anaerobic
and motile bacterium. It appeared as large rods and was creamy in
color. Endo spores fed in central position. Itshowed negative results
for oxidase and urease tests. However, the cells of isolate 2 were a
Gram negative, facultative anaerobic, motile and round -ended rod.
It appeared as circular, raised and dark cream color. Endo spores
be in central position.
Identification of bacterial strains: The bacteria; sbrainscapable
of degrading benzo(alpyrene were identified using 16S rDNA gene
sequencing from which the nucleotide sequences of the strains
were derived 16S rDNA gene fragments (1.5 kb) were amplified
from the total DNA of the isolates (Fig.1). Blast results of the nucleotde
sequence showed that isotatei had 99% identity with Bacillus cereus
and isolate 2 had 99% identity with Bacillus vireti. The nucleotide
sequences of these two bacterial strains were submitted to the Gen
Bank with the accession numbers HM135450 and HM135451.
Mineralization of benzo[a]pyrene and identification of its
metabolites: By the end of the experimental process, 58.98% of
BaP had been degraded by bacterial isolates_ During the
benzo[alpyrene (0.5mg mr) mineralization process, metabolites
formed by 25 d and 35 d were analyzed using GC -mass spectral
analysis (Table 1). The chromatogram of BaP showed a peak at
retention time of 44.36 with ni/z (mass/charge) ratio of 252 (Fig.
2a), which corresponds tattle molecular weight of benzo[a]pyrene.
In the chromatogram taken after 25th d, the original peak was not
seen and new peak with retention lime 22.9 was formed. Mass
spectra showed mass ion at rr lz value of 206 with characterized
fragment ions as 191 and 57 (Fig.2b). After incubation for 35 d, one
1
2
3
ammilMo
trr
4-
1,5kb
Lane 1 1kb DNA Marker. Lane 2: The amplified product of isolate 1; Lane
3: The emptied product of isolate 2
Fig. 1 PCR ampfifeation of genorrac DNA of bacterial isolates by using
16S r DNA primers
987
metabolite of benzo(a)pyrene (0.5mg mr) was identified with mass
ion at mlz of 314 by MS (Fig.2c). With the reference information of
standard compounds and reported benzo[aipyrene degradation
pathway, this compound was proposed to be cis-4-(7-
hydroxypyren-8-yl)-2-oxobut- 3enoic acid (Fig. 2c) and
benzo[a]pyrene itself (Fig. 2a), respectively, The compounds
corresponding to the other peaks were unidentified. Formation of
cis-4-( iydroxypyren-&yl)-2-oxobut-3enoic acid vies consistent with
meta bssianofthe hydroxylated Balo molecule (Scthriekler eta1.,1996).
Up to now, only a few strains capable of degrading
benzo[a]pyrene have been reported, such as i&germicidestrain
B836 (Gibson et al., 1975), Mycobacterium vanbaalehii PYR-1
(Moody et al., 2004), Bacillus subtilis (hunter et aL, 2005),
Sphingomonas paucimabr7is strain EPA 505 (Ye et al., 1996),
Sphingomonas yaooikuyae 3AR02 (Rentz et al., 2008),
Stenotrophomonas INN 10,010 (Boonchan et al., 2002)
and Rhodanobader BPG 1 (kanaly et at, 2002). E arty observation
of benzo[a]pyrenedegradation was -made with mutant Beeemickia
strain 138136 and Psegdorrict,as sp. strain NCIB 9816 grown on
succinate plus biphenyl and succinate plus salicylate (Barnsley,
1975). It had been observed that 25% of benzo[a]pyrene was
mineralized by bacterial consortium (Stenotrophomonasmaltophika
VUN 10,010 and'Peak/Ilium janthineflum) to COz over 49 days,
accompantead by transient accumulation and disappearance of
intermediates as detected by high pressure liquid chromatography.
Luo et al. (2009) reported that 44.07% of the 10 ppm
benzo[a]pyrene was degraded after 14 d incubation by bacterial
consortium which consists of Ochrobarrrr sp., Stenodophomonas
maftophiiia and Pseudomonas fluorescens. in previous studies, it
had been reported that Mycobacterium strain RJGII-135, isolated
from a coal gasification site, and was able to degrade (14C)
benzo[a}pyrene, producing metabolites cis-4-(7-hydraxypyren-8-
y1)-2-oxobut-3enoic acid, methylated 4,5-chrysenedicarboxytic
acid, cis-7,8-dihydrodiol-BaP and 7,8-dihydro-pyrene-8-carboxylic
add. In this study, bacterial consortium (Bacillus cen3us and Bacillus
Wet) isolated from pet roieum contaminated inated soii was able tio transform
58.98% of benzofalpyrene. The formations of cis-4-(7-
hydroxypyren-8-yl)-2-oxobut-3enoic acid intermediates were
found. This may be due to meta fission of the hydroxylated
benzo[a]pyrene. The defined bacterial consortium provides a more
versatile and effective way to clean up environmental pollution
(Mrozik and Piotrowska-Seget, 2010). Nficrobial cooperation may
promote broader and more efficient in -situ degradation of complex
pollutant mixtures. Additional studies have to be made to reveal the
optimum conditions (nutrients, temperature etc.) required to maximum
benzo(a)pyrene degradation by bacterial consortium. With a better
understanding of the degradation process of PAH by bacteria,
strategies will be developed for the removal and containment of
carcinogenic PAH from contaminated ecosystems and the reduction
of health risks associated with exposure to PAH.
Jounraf of Environmental Biology *November 2012*

988
10—
% Relative Abundance
1^_5
100 110 120 130
Mohandass et at
53
140 150 160 170 18(1 190 200 210 220 230 240 250
hat 1LX. `
11 s.# t91 )
% Relative Abundance
100
% Relative Abundance
mix
1 lai
I: F- f + . •--r r .w-r-r•r "I
' S PO 00 I 11► 171i l 0 Igo lie sco
mli
(C)
197
314
GO 73 125 258
109 3 29 '13 2 6
• 30 1 153 1= 18.5 244 278 I 306 I
" 14 30 50 70 90 110 130 150 170 190 210 230 250 270 290 310 330
mlz
Fig. 2- Mass spectra of the meta3 olIt s of benzorajpyrene transformed by the bacterial consortium at (a) 0 days, (h) 25 days and {c) 35 days
Table-1: GC mass spectral analysis of benzo[a]pyreae metabolites during its degradation by bacterial consortium
Metabolite
Molecular ion Retention time(min) Fritz of fragrnents ions
cis-447-hydroxypyren$y1}2-oxabut-3enpic acid
314 31.8 306, 286, 278, 258, 244, 213
Journal of Environmental Biology *November 2012s

Biodegradation of benzo pyrene using bacterial strain
Acknowledgment
Authors like to thank SRM University for providing all infrastrucrure
facilities to perform this research v.urk.
References
Barnsley, E.A.: The bacterial degradation offluoranthene and benzo(a)pyrene.
Can. J. Microbial., 21, 1004-1008 (1975).
Boonchan, S., M.L. Britz and G.S. Stanley: Degradation and mineralization
of high-molecular-weight polycyclic aromatic hydrocarbons by defined
fungal-bacterial cocultures. Appl. Environ. Microbial., 66, 1007-1019
(2000).
Chauhan, A., Fazlurrahman, J.G. Oakeshott and R.K. Jain: Bacterial
metabolism of polycyclic aromatic hydrocarbons: Strategies for
bioremediation: Indian J. Microbial., 48, 95-113(2008).
Cerniglia, C.E.: Biodegradation of polycyclic aromatic hydrocarbons.
Biodegradation, 3, 351-368(1992).
Gibson, D.T., V. Mahadevan, RM. Jerina, H. Yagi and H.J.C. Yeh: Oxidation
of the carcinogens benzo(a)pyrene and benzo(a)anthracene to
dihydrodiols by a bacterium. Science, 189, 295-297(1975).
Hunter, RD., S.I.N. Ekunwe, D. Dodor, H. Hwang and L. Ekunwe: Bacillus
subtilis is a potential degrader of pyrene and benzo(a)pyrene. Inf. J.
Environ. Res. Public Hlth., 2, 267-271(2005).
Juhasz, A.L. and R. Naidu: Bioremediation of high molecular weight
polycyclic aromatic hydrocarbons: A review of the microbial
degradation of benzo(a)pyrene. Int Biodeter. Biodegrad., 45, 57--88
(2000).
Kanaly, R.A. and S. Harayama: Biodegradation of high-molecular-weight
989
hydrocarbons by bacteria. J. Bacterial., 182, 2059-2067 (2000).
Kanaly, R.A., S.Harayama and K.Watanabe: Rhodanobacter sp.strain
BPC1 in a benzo(a)pyrene-mineralizing bacterial consortium. Applied
Environ. Microbial., 68, 5826-5833 (2002)
Li, X., X. Lin, P. Li, W. Liu, L. Wang, F. Ma and K.S. Chukwuku:
Biodegradation of the low concentration of polycyclic aromatic
hydrocarbons in soil by microbial consortium during incubation. J.
HaL Mater., 172, 601-5 (2009).
Luo, Y.R, Y. Tian, X. Huang, C. Yan, H.S. Hong, G.H. Lin and T.L. Zheng:
Analysis of community structure of a microbial consortium capable
of degrading benzo[a]pyrene by DGGE. Marine Pollu. Bull., 58,
1159-1163 (2009).
Moody,J.D., J.P. Freeman, P.P. Fu and C.E. Cerniglia; Degradation of
benzo(a)pyrene by Mycobacterium vanbaalenii PYR~1. Appl. Environ.
Microbial., 70, 340-345 (2004),
Mrozik, A. and z. Piotrowska-Seget Bioauginentation as a $trafegy for
cleaning up of soils contaminated with ~ compounds: llicrobiol.
Res., 165, 363-375(20je}.
Rentz, J.A., P .J.J. Alvarez and J.j... Schnoor: Benzo{a]pyrene degradation
by Sphingomonas yanoikuyae JA-R02. Environ. Pollut, 151, 669-
6TT (2008).
Schneider, J., R. GrosseF, K . ..{ayasimhulu, W. Xue and D. Warshawsky:
Degradation pf Pyrene, Ben$}anthracene, and Benzo(a)pyrene by
Mycobacterium sp.. Strain RJGll-135, isolated from a former coal
gasilication site, Appl EnvirlJIJ. Microbial., 62, 13-19 (1996).
.Wilson, S,C. and K.C. Jones: ilioremediation of soil contaminated with
PAHs: A review . .Environ. Pollu., 81, 229-249 (1993).
, Ye, B., M.A. Siddiqi,J(E. Maccubbin, S. Kumar and H.C. Sikka: Degradation
of polynuclear aromatic hydrocarbons by Sphingomonas paucimobilis.
Environ. Sci. Tedmal., 30, 136-142 (1996).
Journal of Environmental Biology .November 2012•
Protecting Groundwater for Health
Protecting Groundwater for Health
Managing the Quality of Drinking-water Sources
Edited by Oliver Schmoll, Guy Howard,
John Chilton and Ingrid Chorus
;t t · -I World Health
~-.,§ Organization
~
mm
Publishing
LONDON• SEATTLE
Published on behalf of the World Health Organization by
1W A Publishing, Alliance House, 12 Caxton Street, London SWIH OQS, UK
Telephone: [phone]; Fax: [phone]; Email: [e-mail]
www.iwapuhlishiog.com
First published 2006
0 World Health Organi7.ation (WHO) 2006
Printed by TJ International (Ltd), Padstow, Cmnwall, UK
Apart from any fair dealing for the puq,oses of research or private study, or criticism or review, as permitted under
the UK Copyright, Designs and Patents Aet (1998), no part of this publication may be reproduced, stored or
transmitted in any form or by any means, without the prior permission in writing of the publisher, or, in the case of
photog,-aphic reproduction, in accordance with the terms of licences issued by the Copyright Licensing Agency in the
UK. or in accordance with the terms oflicenses issued by the appropriate reproduction rights organization outside the
UK Enquiries concerning reproduction outside the terms stated here should be sent to lW A Publishing at the address
printed above.
The publisher makes no representation, express or implied, with regard to the accuracy of the information comained
in this book and cannot accept any legal responsibility or liability for errors or omissions that may be made.
Disclaimer
The opinions expressed in this publication are those of the authors and do not necessarily reflect the views or policies
of the International Water Association or the World Health Organization. 1W A, WHO and the editors will not accept
responsibility for any loss or damage suffered by any person acting or refiainiog from acting upon any material
contained in this publication
In addition, the mention of specific manufacturers' products does not imply that they are endorsed or
recommended in preference to others of a similar nature that are not mentioned Errors and omissions excepted, the
names of proprietary products are distinguished by initial capital letters.
The designations employed and the presentation of the material in this publication do not imply the expression of
any opinion whatsoever on the part of the World Health Organi7.ation concerning the legal status of any country,
territory, city or area or of its authorities, or concerning the delimitation of its frontiers or bmmdaries. Dotted lines on
maps represent approximate border lines for which there may not yet be full agreement.
The views expressed in Chapter 1 are those of the authors and do not necessarily reflect the views or policies of
the US EPA
British Library Cata/og11ing-in-P11h/icotion Data
A CIP catalogue record for this book is available from the British Library
WHO Library Catalog11ing-in-Publication Data
Protecting ground ·water for health.
I.Potable water 2 Water pollution -prevention and control 3.Water supply 4.ltisk management -methods I.World
Health Organi7.ation.
ISBN 92 4 154668 9 (NLM classification: WA 675)
ISBN13: 9781843390794 (IW A Publishing)
Contents
Structure of this book ........................................................................................................... xi
Acknowledgements .............................................................................................................. xii
Acronyms and abbreviations ............................................................................................. :rvii
SECTION I: SCIENTIFIC BACKGROUND
1 Groundwater and public health --·······--------------·l
I. I Groundwater as a source of drinking water ............................................................ 4
1.2 The public health and soci~conomic context of groundwater protection ............. 7
1.3 Groundwater quantity ............................................................................................. 8
1.4 Disease derived from groundwater use ................................................................... 9
1.5 Groundwater in the context of international activities to reduce water-related
disease .................................................................................................................. 13
1.6 Groundwater in the WHO Guidelines for Drinking-water Quality ....................... 15
L 7 References ............................................................................................................ 16
2 Groundwater occurrence and bydrogeological environments ..... ____ 21
2.1 Groundwater in the hydrological system .............................................................. 22
2.2 Groundwater occurrence and movement .............................................................. 26
2.3 Groundwater discharge and recharge ................................................................... 34
2.4 Groundwater flow systems ................................................................................... 36
2.5 Geological environments and aquifer types .......................................................... 38
2.6 References ............................................................................................................ 46
vi Protecting Groundwater for Health
3 Pathogens: Health relevance, transport and attenuation _______ 49
3.1 Microbial pathogens and microbial indicator organisms ...................................... 50
3.2 Distribution of pathogens and faecal indicators in groundwater ........................... 55
3.3 Transport and attenuation of microorganisms in the underground ....................... 60
3.4 References ............................................................................................................ 76
4 Chemicals: Health relevance, transport and attenuation ____ ................ 81
4.1 Subsurface transport and attenuation of chemicals ............................................... 82
4.2 Natural inorganic constituents .............................................................................. 90
4.3 Nitrogen species ................................................................................................... 99
4.4 Metals ................................................................................................................. 102
4.5 Organic compounds ............................................................................................ 105
4.6 Pesticides ............................................................................................................ 125
4.7 Emerging issues .................................................................................................. 129
4.8 References .......................................................................................................... 131
S Socio-economic, institutional and legal aspects in groundwater assessment
and protection .......... ---.. ····-····---------------139
5. I Socio-economic status: issues of poverty and wealth ......................................... 140
5.2 Population and population density ...................................................................... 144
5.3 Community participation and consultation ......................................................... 144
5.4 Land tenure and property rights .......................................................................... 147
5.5 Valuing and costing groundwater protection ...................................................... 149
5.6 Setting goals and objectives -how much will be protected? .............................. 151
5. 7 Institutional issues .............................................................................................. 152
5.8 Legal framework ................................................................................................ 153
5.9 References .......................................................................................................... 154
SECTION II: UNDERSTANDING 1HE DRINKING-WATER CATCHMENT
6 Collecting information for characterising the catchment and assessing
pollution potential---------------·--····· .............. 159
6.1 Types of information and access to it ................................................................. 160
6.2 The need for collaboration .................................................................................. 166
6.3 Sufficiency and quality of information-dealing with uncertainty ..................... 169
6.4 Summary-how to proceed ................................................................................ 171
6.5 References .......................................................................................................... 174
7 Characterisation of the socio-economic, institutional and legal setting •...•.. 175
7.1 Defining socio-economic status ....................................................•..................... 175
7.2 Institutional and stakeholder analysis ................................................................. 180
7.3 Managing stakeholder discussions -levelling the playing field ......................... 185
7.4 Developing public participation ......................................................................... 185
7.5 Analysis ofland-use and groundwater use for policy development .................... 188
7.6 Valuing groundwater protection ......................................................................... 191
7. 7 Checklist ............................................................................................................. 193
7.8 References .......................................................................................................... 195
8 Assessment of aquifer pollution vulnerability and susceptibility to the
impacts of abstraction __________ ....................................... 199
8.1 Defining, characterising and mapping groundwater vulnerability ...................... 200
8.2 Information needs and data sources for vulnerability assessment ...................... 213
8.3 Estimating groundwater recharge ....................................................................... 219
Contents vii
8.4 Natural hydrochemical and geochemical environments ..................................... 223
8.5 Characterising groundwater abstraction ............................................................. 225
8.6 Susceptibility of groundwater resources to degradation ..................................... 228
8. 7 Checklist ............................................................................................................. 236
8.8 References .......................................................................................................... 239
9 Agriculture: Potential hazards and information needs _______ 243
9. I Use of manure and fertilisers .............................................................................. 244
9.2 Disposal of animal carcasses .............................................................................. 252
9.3 Animal feedlots .................................................................................................. 253
9.4 Use of wastewater and sewage sludge on land and in aquaculture ..................... 255
9.5 Use ofpesticides ................................................................................................. 257
9.6 Irrigation and drainage ........................................................................................ 263
9. 7 Checklist ............................................................................................................. 265
9.8 References .......................................................................................................... 270
10 Human excreta and sanitation: Potential hazards and information needs-275
I 0.1 Contaminants of concern from sanitation systems .............................................. 2 77
10.2 Types of sanitation and their potential to contaminate groundwater .................. 280
10.3 Assessing the risks to groundwater ..................................................................... 298
10.4 Analytical indication ofhuman excreta and sewage in groundwater .................. 300
I 0.5 Checklist ............................................................................................................. 301
10.6 References .......................................................................................................... 304
11 Industry, mining and military sites: Potential hazards and information
needs-------------·-----------309
11.1 Industrial activities ............................................................................................. 310
11.2 Mining activities ................................................................................................. 318
11.3 Militaiy facilities and activities .......................................................................... 328
11.4 Checklist ............................................................................................................. 333
11.5 References .......................................................................................................... 336
12 Waste disposal and landfill: Potential hazards and information needs -·····339
12.1 Types of solid waste ........................................................................................... 340
12.2 Waste storage, treatment and disposal sites ........................................................ 344
12.3 Factors governing contamination of groundwater by disposal of waste ............. 345
12.4 Assessing groundwater contamination associated with waste sites .................... 354
12.5 Checklist ............................................................................................................. 356
12.6 References .......................................................................................................... 360
13 Traffic and transport: Potential hazards and information needs ____ J63
13.1 Groundwater pollutants from traffic ................................................................... 364
13.2 Traffic-and transport-related activities polluting groundwater .......................... 367
13.3 Pathways of pollutants into groundwater.. .......................................................... 368
13.4 Checklist ............................................................................................................. 369
13.5 References .......................................................................................................... 372
viii Protecting Groundwater for Health
SECTION III: SITUATION ANALYSIS
14 Assessment of groundwater pollution potential ---·-·--·-·----···---·-··375
14.1 The overall assessment process .......................................................................... 376
14.2 Components of assessment of pollutant loading ................................................. 3 78
14.3 Outcome of assessing pollution potential ........................................................... 389
14.4 Using groundwater quality monitoring to support the assessment ...................... 391
14.5 The Barbados case study .................................................................................... 391
14.6 The Perth case study ........................................................................................... 401
14.7 References .......................................................................................................... 408
15 Establishing groundwater management prioriti~--------··-411
15.1 Ensuring the suitability ofinformation .... : ......................................................... .412
152 Prioritising pollutants in groundwater with respect to urgency of management
responses ............................................................................................................ 414
15.3 Selection of management options ....................................................................... 419
15.4 Documentation and reporting ............................................................................. 425
15.5 References .......................................................................................................... 426
SECTION IV: APPROACHES TO DRINKING-WATER SOURCE PROTECTION
MANAGEMENT
16 Water Safety Plans: Risk management approaches for the delivery of safe
drinking water from groundwater soorces-------------431
16.1 End-product testing and the need for a risk management approach .................... 433
16.2 Scope of Water Safety Plans ............................................................................... 433
16.3 Preliminary steps for developing Water Safety Plans ........................................ .436
16.4 Hazard analysis ................................................................................................... 438
16.5 System assessment .............................................................................................. 440
16.6 Control measures ................................................................................................ 443
16.7 Operational monitoring ....................................................................................... 449
16.8 Corrective actions ............................................................................................... 450
16.9 Verification ......................................................................................................... 451
16.10 Supporting programmes ..................................................................................... 452
16.11 Documentation ................................................................................................... 453
16.12 References .......................................................................................................... 462
17 Groundwater protection zones---·-----····-······-····-·· ....... ______________________ 465
17 .1 The concept of a zone of protection ................................................................... .466
17.2 Delineating protection zones .............................................................................. 467
17.3 Fixed radius and travel time approaches ............................................................ .471
17.4 Approaches using vulnerability assessments ..................................................... .4 76
17 .5 A risk assessment approach for delineating protection zones ............................ .4 77
17.6 Prioritising schemes for groundwater protection ................................................ 480
17. 7 Managing land use and human activities in protection zones ............................ .483
17.8 Monitoring and verification of protection zones ................................................ .490
17.9 References .......................................................................................................... 491
18 Sanitary completion of protection works around groundwater sources ...... 493
18.1 Sanitary completion and health ........................................................................ ..494
18.2 The needs for effective control measures in sanitary completion ...................... .495
18.3 Control measures in sanitary completion: Planning and design ......................... .496
18.4 Control measures in sanitary completion: Construction and materials ............... 502
Contents ix
18.5 Control measures in sanitary completion: Operation and maintenance .............. 503
18 .6 Assessment of sanitary completion and establishing priority risk factors ........... 505
18 . 7 Control measures for sanitary completion of groundwater sources .................... 511
18.8 References .......................................................................................................... 513
19 Hydrological management .. ·--·--·--·--·----------517
19.1 Managing abstraction to prevent saline intrusion ............................................... 518
19.2 Managing abstraction to control induced pollution ............................................ 521
19 .3 Management of artificial recharge and wastewater use ...................................... 522
19.4 Bank infiltration ................................................................................................. 528
19.5 Validation of artificial recharge and bank infiltration schemes .......................... 529
19.6 Monitoring and verification ofcontrol measures for hydrological management 530
19.7 References .......................................................................................................... 533
SECTION V: APPROACHES TO POLLUTION SOURCE MANAGEMENT
20 Policy and legal systems to protect groundwater-------·-·--·---537
20.1 Groundwater protection policies ......................................................................... 538
20.2 Legislative framework for groundwater protection ............................................ 546
20.3 Consultation and participation ............................................................................ 548
20.4 Land use planning and management ................................................................... 551
20.5 Tools for pollution control.. ................................................................................ 553
20.6 Enforcement ....................................................................................................... 558
20. 7 Management plans for disasters and incidents .................................................... 5 59
20.8 References .......................................................................................................... 561
21 Agriculture: Control and protection. ______________ ,563
21.1 Pathogen management on agricultural laod ........................................................ 565
21.2 Nutrient management on agricultural land ......................................................... 569
21.3 Management of wastewater and human excreta used on land and in
aquaculture ......................................................................................................... 573
21.4 Nutrient and pathogen management on grazing land .......................................... 5 75
21.5 Management of animal feeding operations and dairies ....................................... 577
21.6 Pesticide management ........................................................................................ 579
21.7 Irrigation water management and drainage ......................................................... 581
21.8 Monitoring and verification of measures controlling agricultural activities ....... 582
21.9 References .......................................................................................................... 5 85
22 Bumau excreta and sanitation: Control and protection .... _________ .... _ .. , ••. 587
22.1 Balancing investment decisions .......................................................................... 588
222 Selecting the right sanitation technology ............................................................ 590
22.3 Measures for controlling risks from on-site sanitation ....................................... 591
22.4 Controlling risks from septic tanks and aquaprivies ........................................... 600
22.5 Measures for prevention and control of sewer leakage ....................................... 60 I
22 .6 Control measures for se\\'age treatment .............................................................. 605
22.7 Monitoring and verification of measures controlling sanitation systems ............ 607
22.8 References .......................................................................................................... 609
x Protecting Groundwater for Health
23 Industry, mining and military sites: Control and protection _____ 6J3
23.1 Industrial and military sites ................................................................................ 615
23.2 Mining ................................................................................................................ 623
23.3 Monitoring and verification of measures controlling industry, mining and
military sites ....................................................................................................... 627
23.4 References .......................................................................................................... 630
24 Waste disposal and landfill: Control and protection -------····-631
24.1 Waste control ...................................................................................................... 63 I
24.2 Siting and planning oflandfills ........................................................................... 636
24 .3 Design strategies for landfills ............................................................................. 63 7
24.4 Operation and maintenance oflandfills .............................................................. 645
24.5 Public participation and education ...................................................................... 646
24.6 Monitoring and verification of measures controlling waste disposal and
landfill ................................................................................................................ 64 7
24.7 Refereoces .......................................................................................................... 650
25 Traffic and transport: Control and protection ----------653
25. l Planning and regulations .................................................................................... 655
25.2 Runoff control .................................................................................................... 656
25.3 Design and maintenance of protective structures ................................................ 657
25.4 Minimising usage ofhannful chemicals ............................................................. 658
25.5 Accidental spillage and disposal ......................................................................... 659
25.6 Monitoring and verification of measures controlling traffic and transport ......... 659
25. 7 References .......................................................................................................... 662
lnde:i .................................................................................................................. 663
Structure of this book
This book is a tool for developing strategies to protect groundwater for health by
managing the ()way of drinking -water sources. For this purpose it provides different
points of entry. As illustrated m the Figure below. the book consists of five sections.
SECTION I
SCIENTIFIC BACKGROUND
SECTION IIl
SITUATION ANALYSIS
SECTION IV �\
APPROACHES TO
DRINKING -WATER SOURCE
PROTECTION MANAi,
SECTION iI
IJNDERSTAN'DING T}1E
DRINKING -WATER
CATCHMENT
SECTION V
APPROACHES TO
POLLUTION SOURCE
MANAGEMENT
Structure of Protecting Groundwater for Health

xii Protecting Groundwater for Health
Section I covers the scientific background needed to wderstand which pathogens and
chemicals are relevant to human health, how they are transported in the underground and
how they may be reduced, removed or retarded (Chapters 3 and 4). The criteria for
inclusion of agents in this overview are their relevance to human health and their
relevance in growdwater. Fmther the concept of groundwater recharge areas is
introduced in Chapter 2, and basic hydrological and hydrogeological background
information is provided. The section is concluded by Chapter 5 which introduces socio-
economic and institutional considerations relevant to developing the protection of
groundwater resources.
Section U provides background information for characterizing and understanding the
drinking-water catchment The chapters in this section explain how conditions and
human activities in the catchment may lead to the occurrence of pathogens or haz.ardous
substances in groundwater. The section begins with general guidance on collecting
information (Chapter 6). Chapter 7 discusses assessing the socio-economic and
institutional setting as a necessmy basis for choosing and implementing feasible
management actions. Chapter 8 outlines the background and information required for
understanding the hydrogeological conditions determining the likelihood of pollutants to
reach aquifers. Chapters 9-13 address the range of human activities potentially releasing
pollutants to the underground, i.e. agriculture, sanitation practices, industry, mining,
military sites, waste disposal and traffic. These chapters end with checklists highlighting
the type of information needed about the setting and the hmnan activities in it for
assessing health hazards potentially affecting groundwater.
Section m provides conceptual guidance on prioritizing both hazards and
management responses. Chapter 14 descnbes how information on the hydrogeological
conditions, particularly on aquifer vulnerability, can be related to hmnan activities in the
drinking-water catchment area in order to assess the potential for pollutants emitted from
these activities to reach the aquifer. Chapter 15 discusses how to prioritiz.e pollutants
according to their public health burden as well as to their likelihood of long-term
accumulation in the aquifer. It also addresses the need to consider the socio-economic
context in choosing feasible options from the range of technically appropriate
management responses for protection, control or remediation.
Section IV provides an overview of the potential management actions that may be
taken to protect drinking-water sources. These begin with their integration into a
comprehensive Water Safety Plan that covers all supply steps from catchment to
consumer (Chapter 16). Two chapters specifically cover protection of the drinking-water
source: Chapter 17 at the scale of designating and managing groundwater protection
zones in the catchment and Chapter 18 at the scale of protecting wellheads. Lastly,
Chapter 19 addresses the management of groundwater abstraction in order to avoid
impacts upon quality and quantity and thus on human health.
Section V provides an overview of control measures to prevent pollution from
human activities in the catchment. beginning with the overarching issues of
policy, land-use planning and implementation of management options for
protecting groundwater (Chapter 20). Chapters 21-25 follow with overviews of the
specific management approaches that help avoid groundwater pollution from the
range of human activities in the catchment. i.e. agriculture, sanitation practices,
industry, mining, military sites, waste disposal and traffic.
Acknowledgements
The World Health Organization wishes to express its appreciation to all those whose
efforts have made the production of this book possible. Special thanks are due to
Minist.ty of Health (Ministerium fiir Gesundheit) and the Federal Envirornnental Agency
(Umwehbundesamt), Germany, which provided financial and organiz.ational support fur
the development of this book
An international group of experts provided the material for the book and lllldertook a
process of mutual review. While authorship of individual chapters is noted below, the
quality of the volume as a whole is due in large part to the review and comments
provided by many individuals. Drafts of the text were discussed and reviewed at editorial
meetings at Bad Elster (2001 and 2002). Intellectual input and review by the following
individuals is gratefully acknowledged: Robert Bannerman, Gudnm Abbt-Braun,
Houssain Abouzaid, Richard Carter, Claudia Castell-Exner, Joseph Cotruvo, Annette
Davison, Daniel Deere, Braam de Villiers, John Fawell, Norbert Feldwisch, Richard
Franceys, Walter Giger, Alan Godfree, Andreas Grohmann, Helmut Horing, Terence
Lee, Lucy Lytton, Scott Miller, Christian Nels, Ute Ringelband, Ruprecht Schleyer, Sally
Sutton, Michael Taylor and Peter Waggitt.
Special thanks are due to Kathy Pond and Edie Campbell of the Robens Centre for
Public and Environmental Health fur editing the text; and to Monika Jllllg, Gertrud
Schlag and Penny Ward for technical and administrative support.
xiv Protecting Groundwater for Health
1be following authors are acknowledged for their major con1nbutions to the chapters
of this book:
Alistair Allen, Department of Geology, University College Cork, Cork, Ireland
Wtlliam Alley, Office of Ground Water, United States Geological Survey, San Diego,
USA
Steve Appleyard, Department ofEnvironment, Western Australia, Perth, Australia
Patricia Hotchkiss Bakir, Amman, Jordan
Mike Barrett, Department fur Environment Food and Rural Affitirs, London, United
Kingdom (formerly at the Robens Centre for Public and Environmental Health,
University of Surrey, Guildford, United Kingdom)
Jamie Bartram, Water, Sanitation and Health Programme, World Health Organization,
Geneva, Switzerland
Philip Berger, Office of Ground Water and Drinking Water, United States
Environmental Protection Agency, Washington DC, USA
Theechat Boonyakarnkul, Health Department, Minisuy of Public Health, Nonthaburi,
Thailand
Ellen Braun-Howland, New York State Department ofHealth, New York, USA
Peter Chave, Credition, United Kingdom
John Chilton, British Geological Survey, Wallingford, United Kingdom
Ingrid Chorus, Federal Environmental Agency, Berlin, Germany
Hermann H. Dieter, Federal Environmental Agency, Berlin, Germany
Jorg Drewes, Environmental Science & Engineering Division, Colorado School of
Mines, Colorado, USA
Jutta Fastner, Federal Environmental Agency, Berlin, Germany
Frank Fladerer, Bremen Overseas Research and Development Association (BORDA),
Yogyakarta, Indonesia (formerly at GTZ, Indonesian-German Government
Cooperation on Drinking Water Quality Surveillance)
Fritz Frimmel, Engler-Bunte-Institute, Department of Water Chemisuy, University of
Karlsruhe, Karlsruhe, Germany
Sam Godfrey, United Nations Children's Fund, Bhopal, India (formerly at the Water,
Engineering and Development Centre, Loughborough University, Loughborough,
United Kingdom)
Arthur Golwer, Wiesbaden, Germany
Bilqis Amin Hoque, Environment and Population Research Centre, Dhaka, Bangladesh
Guy Howard, Department for International Development, Glasgow, United Kingdom
(formerly at the Water, Engineering and Development Centre, Loughborough
University, Loughborough, United Kingdom)
Jutta Jahnel, Engler-Bunte-Institute, Department of Water Chemisuy, University of
Karlsruhe, Karlsruhe, Germany
Michael Kuperberg, Institute for International Cooperative Environmental Research,
Florida State University, Tallahassee, Florida, USA
Dennis McChesney, Groundwater Compliance Section, United States Environmental
Protection Agency, New York, USA
Broder Merkel, Geology Department, Technical University of Freiberg, Freiberg,
Germany
Hans-Martin Mulisch, Umweltbiiro Mulisch GmbH, Potsdam, Germany
Acknowledgements xv
Steve Pedley, Robens Centre for Public and Environmental Health, University of Surrey,
Guildford, United Kingdom
Brian Reed, Water, Engineering and Development Centre, Loughborough University,
Loughborough, United Kingdom
Mike Rivett, School of Geography, Earth & Environmental Sciences, University of
Birmingham, Birmingham, United Kingdom
Robert Sage, Veolia Water Partnership, Bushey, United Kingdom
Jack Schijven, Microbiological Laboratory for Health Protection, National Institute of
Public Health and the Environment., Bilthoven, The Netherlands
Wilfried Schimon, Federal Ministry of Agriculture, Forestry, Environment and Water
Management, Vienna, Austria
Oliver Schmoll, Federal Environmental Agency, Berlin, Germany
Klaus-Peter Seiler, GSF -NationaJ Research Centre for Environment and Health,
Neuherberg, Germany
Richard Taylor, Department of Geography, University College London, London, United
Kingdom
Christopher Teat; Center for Biomedical and Toxicological Research and Waste
Management., Florida State University, Tallahassee, Florida, USA
Don Wauchope, United States Department of Agriculture, Agricultural Research
Service, Tifton, Georgia, USA
Eleonora W cislo, Environmental Risk Analysis Department, Institute for the Ecology of
Industrial Areas, Katowice, Poland
Julie West, British Geological Survey, Nottingham, United Kingdom
Marylynn Yates, Department of Environmental Sciences, University of California,
Riverside, California, USA
Special thanks are also due to the following experts who provided case studies and
text boxes:
Steven Bailey (Table 9.6), Royal Society for the Protection of Birds, Bedfordshire,
United Kingdom (formerly at ADAS, Wolverhampton, United Kingdom)
Hartmut Bartel (Box 6.1 ), Federal Environmental Agency, Berlin, Germany
Rainer Hallebach (Box 5.1 ), Federal Environmental Agency, Bad Elster, Germany
Manfred Hobler (case study in Chapter 8.1.5), Federal Institute for Geosciences and
Natural Resources, Hannover, Germany
Helmut Kemdorff(Box 12.3), Federal Environmental Agency, Berlin, Germany
Klaus Kiimmerer (Boxes 12.1 and 24.2), Institute of Environmental Medicine and
Hospital Epidemiology, University Hospital ofFreiburg, Freiburg, Germany
Benjamin Mafa (Box 102), Department of Water Affairs, Gaborone, Botswana
Armin Margane ( case study in Chapter 8.1.5), Federal Institute for Geosciences and
Natural Resources, Hannover, Germany
Nicola Martin (Box 102), Federal Institute for Geosciences and Natural Resources,
Hannover, Germany
Lars Matthes (Box 11.4), Institute of Applied Geosciences, Technical University Berlin,
Berlin, Germany
Paito Obote(Box22.l), WaterAid, Uganda
Asaf Pekdeger (Box 17.1), Institute for Geological Sciences, Free University Berlin,
Berlin, Germany
xvi Protecting Groundwater for Health
Manfred Scheu (Box 243), GTZ, Eschborn, Germany
Gernard Schmidt (Box 19.1), Federal Institute for Geosciences and Natural Resomces,
Hannover, Germany
Bernd Sofuer (Box 19.1), Federal Institute for Geosciences and Natural Resources,
Hannover, Germany
Ali Subah ( case study in Chapter 8.15), Ministry of Water and Irrigation, Amman,
Jordan
Horst Vogel (Boxes 102 and 11.4), Federal Institute for Geosciences and Natural
Resomces, Hannover, Germany
Michael von Hoyer (Box 24.1 ), Federal Institute for Geosciences and Natural Resomces,
Hannover, Germany
Hans-Jiirg Weber (Box 16.1), SWL Energie AG, Lenzburg, Switz.erland
Gernard Winkelmann-Oei (Box 23.2), Federal Environmental Agency, Berlin,
Germany
Acronyms and abbreviations
ARMCANZ
ASS
BAT
BCMAFF
BGS
BMP(s)
BOD
BTEX
C&D
CCIV
cDCE
cf
OD
COD
CSOs
CTC
CVM
cw
2,4-D
DALY
2,4-DB
1,2-DCA
Agricultme and Resource Management Council of Australia mxl New
Zealmxl
acid sulphate soils
best available technology
British Colmnbia Mini.say for Agriculture, Fisheries & Forestries,
Canada
British Geological Survey
best management practice(s)
biochemical oxygen demand
benzene, toluene, ethylbenz.ene, xylene
construction mxl demolition
close circuit television
cis-dichloroethene
contamination fuctor
Creutzfeldt-Jakob disease
chemical oxygen demand
combined sewer overflows
carbon tetrachloride/tetrachloromethane
contingent valuation methodologies
chemical warfure
(2,4-dichlorophenoxy)acetic acid
Disability Affected Life Years
(2,4-dichlorophenoxy)butyric acid
l;l-dichloroethane
xviii
1,2-DCB
1,4-DCB
1,1-DCE
DCM
DDT
DFID
DNAPL
DNB
DNT
DOC
DOE
DWI
EA
EOCs
EDTA
EED
EIA(S)
EU
FS
FAO
GIS
GDWQ
HACCP
GV
Hb
HCB
HD
HIV
HMX
IARC
ICPE
ICPR
IDWSSD
ISL
illPAC
LNAPLs
LWS
MCPA
MCPP
MDG
metHb
MNA
MSW
MIBE
NA
NAPL
NCRP
NGOs
NRC
Protecting Groundwater for Health
1,2-<l.ichlorobenz.ene
1,4-dichlorobenz.ene
1,1-<l.ichloroethene
dichloromethane
dichlorodiphenyltrichloroethane
Department for International Development, UK
dense non-aqueous phase liquid
dinitrobenzene
dinitrotoluene
dissolved organic carbon
Department of the Environment
Drinking Water Inspectorate
Environment Agency
endocrine disrupting chemicals/compounds
ethylendiamine tetraacetic acid
Environmental Engineering Division
Environmental Impact A=sment (Study)
European Union
faecal streptococci
Food and Agriculture Organization
Geographical Information System
Guidelines for Drinking-water Quality, WHO
Hazard Analysis and Critical Control Points
guideline value
haemoglobin
hexachlorobenze
mustard gas
human immunodeficiency virus
High Melting Explosive cyclotetramethylenetetranitramie
International Agency for Research on Cancer
International Commissions for the Protection of the Elbe
International Commissions for the Protection of the Rhine
International Drinking Water Supply and Sanitation Decade
in situ leaching
International Union of Pure Applied Chemistry
light non-aqueous phase liquid
Lenzburg water supply
( 4-chloro-2-methylphenoxy)acetic acid
2-( 4-chloro-2-methylphenoxy)propanoic acid (mecoprop)
Millenniwn Development Goal
methaemoglobin
monitored natural attenuation
municipal solid waste
methyl tertiary-butyl ether
natural attenuation
non aqueous phase liquid
National Council on Radiation Protection Measmements, USA
non-governmental organizations
National Research Council. USA
NSW
PAH
PCB
PCE
PCP
PCPs
PCR
PRAST
POPs
PPP
RDX
REC
RNA
sm
SPA
2,4,5-T
TCA
TCE
TCM
tDCE
IDS
TeCE
1NT
TON
2,4,5-TP
TIC
UNDP
UNECE
UNEP
UNICEF
UNESCO
UNSCEAR
USEPA
USGS
VBNC
vc
VFAs
voe
WEOC
WHO
WMO
WSP
y-HOI
Acronyms and abbreviations
New South Wales, Australia
polynuclear ami;natic hydrocarbon
polychlorinated biphenyl
perchloroethylene/tetrachloroethene
pentachlorophenol
personal care products
polymerase chain reaction
participating hygiene and sanitation transfonnation
persistent organic pollutants
purchasing power parity
Royal Dutch Explosive cyclotrimethylenetetranitramine
Regional Environmental Council
nbonucleic acid
Sanitary Hazard Index
Source Protection Areas
(2,4,5-tricholorophenoxy)acetic acid
trichloroethane
trichloroethene
trichloromethane
trans-dichloroethene
total dissolved solids
tetrachloroethene
trinitrotoluene
total organic nitrogen
(2,4,5-tricholorophenoxy)propanoic acid (fenoprop)
thermotolerant colifurms
United Nations Development Programme
United Nations Economic Commission for Europe
United Nations Environment Programme
United Nations International Children's Emergency Ftmd
xix
United Nations Educational, Scientific and Cultural Organization
United Nations Scientific Committee ofEffects of Atomic Radiation
United States Environmental Protection Agency
United States Geological Survey
viable but non-culturable
vinyl chloride
volatile fatty acids
volatile organic compotmds
Water Engineering and Development Centre, University of
Loughborough, United Kingdom
World Health Organization
World Meteorological Organiz.ation
Water Safety Plan
1 a,2a,3~,4a,5a,6~-hexachlorocyclohexane (lindane)
Section I
Scientific background
1
Groundwater and public health
G. Howard, J. Bartram, S. Pedley, 0. Schmoll,
I Chorus and P. Berger
Water-related disease remains one of the major health concerns in the world Diarrhoeal
diseases, which are largely derived from poor water and sanitation, accounted for 1.8
million deaths in 2002 and contributed aroimd 62 million Disability Acljusted Life Years
per annwn (WHO, 2004a). On a global scale, this places diarrhoeal disease as the sixth
highest cause of mortality and third in the list of morbidity and it is estimated that 3. 7 per
cent of the global disease burden is derived from poor water, sanitation and hygiene
(Priiss-Ostiin et al, 2004). This health burden is primarily borne by the populations in
developing COW1tries and by children.
At 2002 estimates, roughly one-sixth of humanity (1.1 billion people) lack access to
any fonn of improved water supply within 1 kilometre of their home, and approximately
40 per cent of humanity (2.6 billion people) lack access to some fonn of improved
excreta disposal (WHO and UNICEF, 2004). These figures relate to the clear definitions
provided in the updated Global Water Supply and Sanitation Assessment Report and are
shovm in Table I.I below.
If the quality of water or sanitation were taken into accowit, these numbers of people
-without access to water supplies and sanitation would increase even further.
Endemic and epidemic disease derived fiom poor water supply affects all nations.
Outbreaks of waterborne disease continue to occur in both developed and developing
© 2006 World Health Organiz.ation. Protecting Groundwater for Health: Managing the Quality of
Drinking-water Sources. Edited by 0. Schmoll, G. Howard, J. Chilton and I. Chorus. ISBN:
1843390795. Published by 1W A Publishing, London, UK.
4 Protecting Groundwater for Health
oountries, leading to loss oflife, avoidable disease and economic costs to individuals and
oommunities. The improvement of water quality control strategies, in conjunction with
improvements in excreta disposal and personal hygiene can be expected to deliver
substantial health gains in the populatiOIL
Table 1.1. Definition of improved and unimproved water supply and sanitation facilities (WHO
and UNICEF, 2000)
Improved
Water supply
Unimproved
Household connection Unprotected well
Public standpipe
Boreholes
Protected dug well
Protected spring
Rainwater collcdicm
Unprotected spring
Vendor-provided
water
Bottled water
Tanker-truck
provided water
Improved
Sanitation
Unimproved
Connection to a Service or bucket latrines
public sewer (excreta removed
Connedion to a septic manually)
system
Pour-flush latrine
Simple pit latrine
Ventilated improved
pit latrine
Public latrines
Latrines with an open pit
1bis monograph provides information on strategies for the protection of groundwater
sources used fur drinking-water as a component of an integrated approach to drinking-
water safety management (WHO, 2004b). The importance of source protection as the
first stage of managing water quality has been an important component in both national
and international efforts. In their Guidelines for Drinking-water Quality, WHO (2004b
and previous editions) emphasiz<; the need for effective source protectioIL
The focus of this monograph is the public health aspects of groundwater protectioIL It
does not address environmental concerns, such as ecological protectioIL The control of
some pollutants, whilst of little importance for health, may be very important to
environmental protectioIL For guidance on these areas., readers should consult
appropriate texts such as Chapman (1996).
1.1 GROUNDWATER AS A SOURCE OF DRINKING-
WATER
Groundwater is the water oontained beneath the surface in rocks and soil, and is the water
that accumulates underground in aquifers. Groundwater comt:itutes 97 per cent of global
freshwater and is an important source of drinking-water in many regions of the world. In
many parts of the world groundwater sources are the single most important supply for
the production of drinking-water, particularly in areas with limited or polluted surface
water sources. For many communities it may be the only economically viable option.
This is in part because groundwater is typically of more stable quality and better
microbial quality than surface waters. Groundwaters often require little or no treatment to
be suitable for drinking whereas surface waters generally need to be treated. often
extensively. There are many examples of grotmdwater being distributed without
Groundwater and public health 5
treatment It is vital therefore that the quality of groundwater is protected if public health
is not to be compromised.
National statistics for the use of groundwater as a source of drinking-water are sparse,
but the importance of this resomce is highlighted by figures published in Europe and the
USA. The proportion of growxlwater in drinking-water supplies in some European
countries is illustrated in Table 1.2 and for the USA in Table 13. The data show that
reliance upon groundwater varies considerably between countries; for example, Norway
talces only 13 per cent of its drinking-water from groundwater sources, whereas Austria
and Denmark use groundwater resources almost exclusively for drinking-water supply.
A global estimate of one-third of the world's population depending on groundwater
supply is given by Fa1kenmark (2005).
Table 1.2. Proportion of groundwater in drinking-water supplies in seled.ed European cotmtries
(EEA, 199'); UNECE, 199'))
Coun try Pro portion Coun lTV Proportion
A~ 99% Bulgaria 60%
Denmark 98% Finland 57%
Htmgary 95% France 56%
Switterland 83% Greece 50"/o
Portugal 80"/o Swedm 49%
Slovak Republic 80"/o Czech Republic 43%
Italy 80"/o United Kingdom 28%
Germany 72% Spain 21%
Netherlands 68% Norway 13%
The data from the USA demonstrates the importance of groundwater particularly for
smaller supplies, reflecting the generally limited treatment requirements. However, this
has implications for control of public health risks as the management and maintenance of
smaller supplies is often weaker than for larger, utility operated supplies (Bartram, 1999).
Table 1.3. Proportion of grmmdwater in drinking-wata supplies in the USA by siz.e of supply (US
EPA,2004)
Population served Proportion groundwater Proportion surface water
<500 89% 11%
500-1000 78% 22%
1001-3300 70"/o 30%
3301-10000 57% 43%
IO 000-50 000 43% 57%
>50000 26% 74%
Within countries the usage of groundwater may also vary substantially, depending on
the terrain and access to alternative water sources. For instance, in the USA it ranges
from 25 per cent or less in Colorado and Kentucky to more than 95 per cent in Hawaii
6 Protecting Groundwater for Health
and Idaho. In rural areas of the US.A. 96 per cent of domestic water oomes from
groundwater. In the United Kingdom, although the national average for groundwater
usage is 28 per cent, the southern counties of Englaad depend more heavily on
groundwater than the northern cotmties, Wales and Scotland
In Latin America, many of the continent's largest cities -Mexico Uty, Mexico,
Lima, Peru, Buenos Aires, Argentina and Santiago de Chile, Onie -obtain a significant
proportion of their municipal water supply :6:om groundwater. In India, China,
Bangladesh, Thailand, Indonesia and Viet Nam more than 50 per cent of potable supplies
are provided :6:om grotmdwater. In Africa and Asia, most of the largest cities use surface
water, but many millions of people in rural areas and low-income peri-urban
oommunities are dependent on groundwater. These populations are most vulnerable to
waterborne disease. Pedley and Howard (1997) estimate that as much as 80 per cent of
the drinking-water used by these communities is abstraded :6:om groundwater sources.
Where it is available, groundwater frequently has important advantages over surface
water. It may be conveniently available close to where it is required, can be developed at
comparatively low cost and in stages to keep pace with rising demand Although small,
simple surface water supplies can be achieved relatively cheaply and pumping
groundwater :6:om deep aquifers may create significant operating costs, overall the capital
costs associated with groundwater development are usually lower than with large-scale
surface water supplies. For the latter, large, short-tenn capital investments in storage
reservoirs often produce large, step-wise increments in water availability and temporaiy
excess capacity that is gradually overtaken by the continuing rising demand for water.
An additional disadvantage in some circwnstances is that surface water reservoirs may
have multiple, sometimes conflicting functions -water supply, flood control, irrigation,
hydroelectric power and recreation -and cannot always be operated for the optimum
benefit of water supply.
Furthermore, aquifers are often well protected by layers of soil and sediment., which
effectively filter rainwater as it percolates though them, thus removing particles,
pathogenic microorganisms and many chemical constituents. Therefore it is generally
assumed to be a relatively safe drinking-water source.
However, grolindwater has been termed the 'hidden sea' -sea because of the large
amount of it., and hidden because it is not viSJble, thus pollution pathways and processes
are not readily perceived (Chapelle, 1997). This highlights a key is.sue in the use of
aquifers as drinking-water source, showing that particular attention is needed to ascertain
whether the general assumption of groundwater being safe to drink is valid in individual
settings. As discussed below, understanding the source-pathway-receptor relationship in
any particular setting is critical to determine whether pollution will occur.
Whilst there is a large volume of groundwater in this 'hidden sea', its replenishment
occurs slowly -at rates varying between locations. Over-exploitation therefore readily
occurs, bringing with it additional quality concerns.
Groundwater and public health 7
1.2 THE PUBLIC HEALTH AND SOCIOECONOMIC
CONTEXT OF GROUNDWATER PROTECTION
The use of groundwater as a source of drinking-water is often preferred because of its
generally good microbial quality in its natural state. Nevertheless, it is readily
contaminated and outbreaks of disease from contaminated groundwater sources are
reported from countries at all levels of economic development.. Some groundwaters
naturally contain constituents of health concern: fluoride and arsenic in particular.
However, tmdeIStanding the impact of groundwater on public health is often difficult and
the interpretation of health data complex. This is made more difficult as many water
supplies that use groundwater are small and outbreaks or background levels of disease
are unlikely to be detected, especially in countries with limited health surveillance.
Furthermore, in outbreaks of infectious disease, it is often not possible to identify the
cause of the outbreak: and many risk fuctors are typically involved.
Throughout the world, there is evidence of contaminated groundwater leading to
outbreaks of disease and contributing to background endemic disease in situations where
groundwater sources used for drinking have become contaminated. However, diarrhoeal
disease transmission is also commonly due to poor excreta disposal practices and the
improvement of sanitation is a key intervention to reduce disease transmission (Esrey et
al., 1991; Curtis et al., 2000). Furthermore, water that is of good quality at its source may
be re-<0ntaminated during withdrawal, transport and household storage. This may then
require subsequent treatment and safe storage of water in the home (Sobsey, 2002).
Ensuring that water sources are microbially safe is important to reduce health
burdens. However, a balance in investment must be maintained to ensure that other
interventions, also important in reducing disease, are implemented. Diverting resources
away from excreta disposal, improved hygiene practices in order to achieve very good
quality water in soura:s may be counter-productive (Esrey, 1996). Balancing investment
decisions for public health gain from water supply and sanitation investment is complex
and does not simply reflect current knowledge (or lack of) regarding health benefits, but
also the demands and priorities of the population (Briscoe, 1996).
Groundwater is generally of good microbial quality, but may become rapidly
contaminated if protective measures at the point of abstraction are not implemented and
well maintained. Further problems are caused by the creation of pathways that short-
circuit the protective measures and natural layers offering greatest attenuation, for
instance abandoned wells and leaking sewers. Pollution may also occur in areas of
recharge, with persistent and mobile pollutants representing the principal risks.
The control of the microbial qualily of drinking-water should be the first priority in all
countries, given the immediate and potentially devastating consequences of waterborne
infectious disease (WHO, 2004b). However, in some settings the control of chemical
quality of groundwater may also be a priority, particularly in response to locally
important natural constituents such as fluoride and arsenic. Furthermore, hazardous
industrial chemicals and pesticides which can accumulate over time may potentially
render a source unusable. The scale, impact and the often lack of feasible clean-up
technologies for some chemical contamination in groundwater means that they should
receive require priority for preventative and remedial strategies.
8 Protecting Groundwater for Health
Groundwater also has a socioeconomic value. It is often a lower cost option than
surfuce water as the treatment requirements are typically much lower. In many countries,
groundwater is also more widely available for use in drinking-water supply. This may
provide significant advantages to communities in obtaining affordable water supplies,
which may have benefits in terms of promoting greater volumes of water used for
hygiene and other purposes. The natural quality of groundwater also makes its use
valued in industry, and it may provide environmental benefits through recharge of
streams and rivers or for the growth of vegetation These other benefits reinforce the need
for its protection
The actions taken to protect and conserve groundwater will also create costs to
society, through lost opportunity costs for productive uses of land and increased
production costs caused by pollution containment and treatment requirements. When
developing protection plans and strategies, the cost of implementing such measures
should be taken into consideration, as well as the cost of not protecting grmmdwater, in
order for balanced decisions to be made.
1.3 GROUNDWATER QUANTITY
The interrelated issues of groundwater quality and quantity can best be addressed by
management approaches encompassing entire groundwater recharge areas or
groundwater catchments. These units are appropriate both for assessing pollution
potential and for developing management approaches for protection and remediation
Excessive groundwater abstraction in relation to recharge will lead to depletion of the
resource and competition between uses, e.g. between irrigation and drinking-water
supply. Strong hydraulic gradients ensuing from abstraction can induce the formation of
preferential flow paths, reducing the efficacy of attenuation processes, and thus lead to
elevated concentrations of contaminants in groundwater. Furthermore, changes in
groundwater levels induced by abstraction may change conditions in the subsurfu.ce
environment substantially, e.g. redox conditions, and thus induce mobiliz.ation of natural
or anthropogenic contaminants.
Groundwater quantity issues may have substantial impacts on human health. Lack of
a safe water supply affects disease incidence for instance by restricting options for
personal and household hygiene. Competing demands for groundwater, often for
irrigation and sometimes for industry, may lead to shortage of groundwater for domestic
use. In such situations it is important to ensure allocation of sufficient groundwater
reserves for potable and domestic use and health authorities often play an important role
in this. This monograph largely focuses on water quality issues, as these are of direct
relevance to the provision of safe drinking-water. Quantity issues are therefore addressed
in the context of their impact on groundwater quality.
This text is concerned with groundwater as a source of drinking-water supply.
However, in many locations other uses, for example irrigation, account for the largest
fraction of groundwater abstraction, and inter-sectoral collaboration may be needed to
develop effective groundwater allocation schemes.
Groundwater and public health 9
1.4 DISEASE DERIVED FROM GROUNDWATER USE
GroID1dwater contributes to local and global disease burdens through the transmission of
infectious disease and fiom chemical hanirds.
1.4.1 Infectious disease transmission through groundwater
The global incidence of waterborne disease is significant, though it can only be estimated
since reliable data are not sufficiently available for direct assessment of disease cases
(Priiss-Ustiin et al., 2004). The contribution of groundwater to the global incidence of
waterborne disease carmot be assessed easily, as there are many competing transmission
routes; confollllding fiom socioeconomic and behavioural factors is typically high;
definitions of outoome vary; and, exposure-risk relationships are often unclear (Esrey et
al, 1991; Payment and Hllllter, 2001; Pruss and Havelaar, 2001). Many waterborne
disease outbreaks could have been prevented by good understanding and management of
grolllldwaters for health. Pathogen contamination has often been associated with simple
deficiencies in sanitation but also with inadequate understanding of the processes of
attenuation of disease agents in the subsurface.
The most comprehensive reports of waterborne disease outbreaks come fiom two
countries, the USA and the United Kingdom, and some indications of the role of
groundwater in the infectious diarrhoeal disease burden can be estimated in these
countries (Craun, 1992; Hunter, 1997; Payment and HID1ter, 2001; Cramt et al, 2003;
2004).
Lee et al (2002) identified that of 39 outbreaks of waterborne disease in the USA
between 1999 and 2000, 17 were due to conswnption of untreated groundwater,
although approximately half of these outbreaks were reported fiom individual water
supplies, which are not operated by a utility and served less than 15 connections or less
than 25 persons. A further eight were reported in non-community supplies, which serve
facilities such as schools, factories and restaurants.
A detailed analysis of the incidence of waterborne disease in the USA was published
in the mid-l 980s by Cramt (1985), which is still relevant In his summary of data fiom
the period between 1971 and 1982, Cramt reports that untreated or inadequately treated
groundwater was responsible for 51 per cent of all waterborne disease outbreaks and 40
per cent of all waterborne illness. A recent analysis of public health data in the USA
showed little change to the epidemiology of disease outbreaks (Cramt et al, 1997).
Between 1971 and 1994, 58 per cent of all waterborne outbreaks were caused by
contaminated groundwater systems, although this is in part is due to the higher nwnber
of water supplies using groundwater than those using surface water.
Craun et al. (2003) report that for the period 1991-1998, 68 per cent of the outbreaks
in public systems were associated with groundwater, an increase from previous reports
(Craun, 1985; Cramt et al., 1997). However, this apparent increase is likely to be due in
part to the introduction of the USA Surface Water Treatment Rule in 1991, which
requires 'conventional filtration' of most surface water supplies. In general it appears that
waterborne outbreaks in the USA decreased after 1991, with the introduction of more
stringent monitoring and treatment requirements.
10 Protecting Groundwater for Health
Craun et al. (2004) provide a detailed discll$ion of waterborne outbreaks in relation
to zoonotic organisms (organisms with an animal as well as hwnan reservoir) between
1971 and 2000 in the USA They oote that 751 outbreaks were reported linked to
drinking-water supplies during this period, the majority (648) being linked to community
(year-rmmd public service) water supplies. The aetiology was either known or suspected
in 89 per cent of the outbreaks and zoonotic agents caused 118 outbreaks in community
systems representing 38 per cent of outbreaks associated with these systems and 56 per
cent of those where aetiology was identified. The data show that the majority of illnesses
and deaths were caused by zoonotic agents in the reported waterborne outbreaks.
The zoonotic agents of greatest importance were Giardia, Campylobacter,
Cryptosporidium, Salmonella, and E. cofi in outbreaks caused by contaminated drinking-
water. The majority of outbreaks caused by zoonotic bacteria (71 per cent) and
Cryptosporidium (53 per cent) were reported in grmmdwater supplies. The use of
contaminated, untreated or poorly treated groundwater was responsible for 49 per cent of
outbreaks caused by Campylobacter, Salmonella, E coli, and Y ersinia. Groundwater that
was contaminated, untreated or poorly treated contnbuted 18 per cent of all outbreaks
caused by Giardia and Cryptosporidiwn.
Kukkula et al. (1997) descnbe an outbreak of waterborne viral gastroenteritis in the
Finnish municipality ofNoormarkku that affected some 1500-3000 people, ie. between
25 and 50 per cent of the exposed population. Laboratory investigations confirmed that
adenovirus, Norwalk-like virus and group A and C rotaviruses were the principal
causative agents. The source of the outbreak was thought to be a grolllldwater well
situated on the embankment of a river polluted by sewage discharges. In 1974 an
outbreak of acute gastrointestinal illness at Richmond Heights in Florida, USA was
traced to a supply well that was continoously contaminated with sewage from a ne:arl>y
septic tank (Weissman et al., 1976). The main aetiological agent was thought to be
Shigel/a sonnei. During the outbreak approximately 1200 cases were recorded from a
population of 6500.
Outbreaks of cryptosporidiosis have also been linked to groundwater sources, despite
being usually reg.u-ded as a surfuce water problem. A large outbreak of cryptosporidiosis
occum:d in 1998 in Brush Creek, Texas, USA from the use of lllltreated grolllldwater
drawn from the Edwards Plateau karst aquifer (Bergmire-Sweat et al, 1999). There were
89 stool-<:onfirmed cases and the estimated number of cases was between 1300 and
1500. This outbreak was associated with the conswnption of water drawn from deep
wells of over 30 m located more than 400 m from Brush Creek.
In 1997, epidemiological investigations traced an outbreak of cryptosporidiosis in the
United Kingdom to water abstracted from a deep chalk borehole. Three hundred and
forty five confirmed cases were recorded by the investigation team, who claimed this to
be the largest outbreak linked to groundwater to have been reported (Willcocks et al.,
1998). This incident has particular significance because the water used in the supply was
drawn from a deep borehole and was filtered before distribution.
In the outbreak of E. coli Ol57:H7 and Campylobacter in Walkerton, Ontario in
Canada in 2000 , the original source of pathogens appears to have derived from
contaminated surfuce water entering into a surfuce water body directly linked to an
abstraction borehole (Health Canada, 2000). Although the series of events leading to the
Groundwater and public health 11
outbreak indicate a failure in subsequent treatment and management of water quality,
better protection of groundwater would have reduced the potential for such an outbreak.
An outbreak of E coli 0157:H7 occurred among attendees at the Washington Cmmtry
Fair, New Yolk, USA and was shown to be caused by consuming water from a
contaminated shallow well that had no chlorination (CDC, 1999). A total of951 people
reported having diarrhoea after attending the fair and stool cultures from 116 people
yielded E .coli 0157:H7. This outbreak resulted in hospitaliz.ation of 65 people, 11
children developed haemolytic syndrome and two people died.
In developing countries evidence of the role of groundwater in causing disease
outbreaks is more limited, although there have been numerous studies into the impact of
drinking-water, sanitation and hygiene on dianhoeal disease. In part the limited data on
groundwater related outbreaks reflects the often limited capacity of local health
surveillance systems to identify causal factors and because it is common that several
factors may be implicated in the spread of disease. However, the limited data on
outbreaks specifically linked to groundwater may also reflect that improved groundwater
sources are generally of relatively good quality. Diarrhoeal disease related directly to
drinking-water is most likely to result from consumption of poorly protected or
unimproved groundwater sources, untreated or poorly treated surfuce water,
contamination of distribution systems and recontamination of water during transport.
Pokhrel and Vmrraghavan (2004) in a review of diarrhoeal disease in Nepal in
relation to water and sanitation, cite examples from South Asia where contamination of
groundwater supplies has led to outbreaks of disease.
A study oflocal populations in Kanpur, India recorded an overall incidence rate of
waterborne disease of 80.1 per I 000 population (Trivedi et al., 1971 ). The commllllit:ies
in the study areas took water from shallow groundwater sources, analysis of which
revealed that over 70 per cent of the wells were contaminated. Of the cases of waterborne
disease investigated, the greatest proportion was of gastroenteritis, followed by
dysentery.
In addition to outbreaks, there is some evidence of contaminated groundwater
contributing to background levels of endemic diarrhoeal disease. For example,
Nasinyama et al. (2000) showed that the use of protected springs in Kampala, Uganda
which were in generally in poor condition was associated with higher rates of diarrhoea
than the use of piped water supplies. Much of this disease burden is thought to occur in
developing countries where the use of untreated water from shallow groundwater sources
is common in both rural and peri-urban settlements (Pedley and Howard, 1997).
1.4.2 Chemical hazards
The risk to health from chemicals is typically lower than that from pathogens. The health
effects of most, but not all, chemical hazards arise after prolonged exposure, and tend to
be limited to specific geographical areas or particular water source types. Much remains
to be understood about the epidemiology of diseases related to chemical hazards in water
and the scale of disease burden remains uncertain. However, some data do exist Craun et
al. (2004) report that 11 per cent of waterborne outbreaks in the USA between 1971 and
2000 were associated with acute effects following ingestion of a chemical.
12 Protecting Groundwater for Health
Ensuring that chemicals ofhealth concern do not occur at significant concentrations in
groundwaters implies understanding sources of pollution, aquifer vulnerability and
specific attenuation processes as well as recognizing the importance of naturally-
occuning chemicals of health concem In groundwater, however, there are two
contaminants in particular that represent particular hazards of concern: fluoride and
arsenic.
Fluoride affects bone development and in excess leads to dental or, in extreme form,
skeletal fluorosis. The latter is a painful debilitating disease that causes physical
impairment However, too little fluoride has also been associated with dental caries and
other dental ill-health (WHO, 2004-b). Drinking-water is the principal route of exposure
to fluoride in most settings, although burning of high fluoride coal is a significant route of
exposure in parts ofChina(Guet al, 1990).
Arsenic causes concern given the widespread occurrence in shallow groundwaters
in Bangladesh, West Bengal, India and in groundwater in several other countries. The
scale of arsenic contamination is most severe in the shallow groundwater of
Bangladesh. At present, the total population exposed to elevated arsenic concentrations
in drinking-water in Bangladesh remains uncertain, but is thought to be somewhere
between 35 and 77 million and has been described as the largest recorded poisoning in
history (Smith et al., 2000; BGS and DPHE, 2001). Problems are also noted in
countries as diverse as Mexico, Canada, Hungary and Ghana, although the source of
arsenic and control strategies available vazy. The true scale of the public health impact
of arsenic in groundwater remains uncertain and the epidemiology is not fully
understood.
In the case of Bangladesh, the lack of country-wide case-wntrolled studies makes
estimating prevalence of arsenicosis difficult In a recent evaluation of data collected by
the DPHE-Unicef arsenic mitigation programmes, Rosenboom et al. (2004) found a
prevalence rate of arsenicosis (keratosis, melanosis and de-pigmentation) of 0.78 per
1000 population exposed to elevated arsenic ( above 50 ll!ifl) in 15 heavily affected
Upazilas (an administrative unit in Bangladesh). These authors note, however, that the
data were difficult to inteipret and that exposure had been relatively short and therefore
these numbers could increase. The lack of a national cancer prevalence study makes
estimations outside small cross-section studies problematic.
Increasing numbers of countries in Asia are now identifying arsenic contamination of
groundwater (including Cambodia, China, Laos, Myanmar, Nepal, Pakistan and Viet
Nam). In India, increasing numbers of areas are being identified as arsenic affected
beyond West Bengal (School of Environmental Studies, Javadpur University, 2004).
This demonstrates that arsenic is an important contaminant ior public health and concern
is growing.
Other chemical contaminants of concern in groundwater may also lead to health
problems. These include nitrate, uranium and selenium. Of these, nitrate is of concern as
it is associated with an acute health effect (methaemoglobinaemia or infantile cyanosis).
The scale of the health burden derived from nitrate remains uncertain although it has
been suggested to cause significant health problems in some low-income countries where
levels in groundwater reach extremely high values (Melian et al., 199'1). Nitrate is also of
concern given that it is stable once in groundwater with reasonably high oxygen content,
Groundwater and public health 13
where it will not degrade. Thus it may ac.cumulate to a long-term water resource problem
that is expensive and difficult to remediate and whose effect may not Ix: noticed until
roncentrations lx:rome critical.
1.5 GROUNDWATER IN THE CONTEXT OF
INTERNATIONAL ACTMTIES TO REDUCE
WATER-RELATED DISEASE
The International Drinking Water Supply and Sanitation Decade (IDWSSD; 1980-1990)
provided a s~ focus on the need for ronc:erted eflorts to accelerate activities to
increase global access to safe water supply and to sanitation The Rio Earth Smnmit
(1992) placed water both as resource and as water supply on the priority agenda and the
World Smnmit on Sus1ainable Development in 2002 also placed safe drinking-water as a
key component of sustainable development In Septemlx:r 2000, 189 UN Memlx:r States
adopted the Millenniwn Development Goals (MDGs). Target IO of the MDGs is to
halve by 2015 the proportion of people without sus1ainable access to safe drinking-water
and basic sanitation; the baseline for this target has been set as 1990. Other important
initiatives have included a Protorol on Water and Health to the 1992 Convention on Use
ofTransboundary Watercourses and International Lakes (Box 1.1).
Box 1.1. The WHO-UNECE Protorol on Water and Health (UNECE and
WHO, 1999)
The WHO-UNECE Protocol on Water and Health to the 1992 Convention on
the Protection and Use ofTransboundary Watercourses and International Lakes
is an international legal instrwnent on the prevention, rontrol and reduction of
water-related diseases in Europe.
A major product of the Third European Ministerial Conference on Environment
and Health (1999), the Protorol was signed at the Conference by 35 rountries
and represents the first major international legal approach for rontrolling water-
related disease. It has lx:rome legally binding for the 16 countries that have
ratified it in 2005. By adopting the Protocol, the signatories agreed to take all
appropriate measures towards achieving:
-adequate supplies of wholesome drinking-water;
-adequate sanitation of a standard which sufficiently protects human health
and the environment;
-effective protection of water resources used as sources of drinking-water and
their related water ecosystems from pollution from other causes;
-adequate safeguards for hwnan health against water-related diseases;
-effective systems for monitoring and responding to outbreaks or incidents of
water-related diseases.
14 Protecting Groundwater for Health
The Global Environmental Monitoring System Water programme, launched in 1977
by UNEP in collaboration with UNESCO, WHO and WMO, has the overall objective of
observing and assessing global water quality issues in rivers, lakes and groundwater by
collecting together and interpreting data from national monitoring networks. A first
assessment of freshwater quality published in 1989 (Meybeck et al, 1989) included
discussion oflinks between water quality and health The programme was reviewed and
evaluated in 2001 with a view to enhancing its ability to contnbute to inter-agency global
programmes, including the Global International Waters Assessment and the UN-wide
World Water Assessment Programme.
Recently, the World Bank has established a groundwater management advisory team
(GW·MATE) to develop capacity and capability in groundwater resource management
and quality protection through World Bank programmes and projects and through the
activities of the Global Water Partnerships regional networks.
WHO's activities in support of safe drinking-water span across the range of its
functions as a specializ.ed agency of the UN system (Box 12).
Box 1.2. WHO activities related to safe drinking-water
Evidence and iriformation: Burden of disease estimates (at global level and
guidance on their conduct at other levels); and cost-effectiveness water
interventions (generically at global level and guidance on their conduct at other
levels).
Status and trends: Assessing coverage with access to improved sources of
drinking-water, and to safe drinking-water (with UNICEF through the Joint
Monitoring Programme).
Tools for good practice: Evidence-based guidance on effective (and ineffective)
technologies, strategies and policies for health protection through water
management
Normative guidelines: Evidence-based and health-centred norms developed to
assist development of effective national and regional regulations and standards.
Country cooperation: Intensive links to individual countries through its network
of six regional offices, regional environment centres and country offices.
Research and testing: Encouraging and orienting research; developing and
encouraging the application of protocols to increase harmonization, exchange
and use of data Publication with 1W A of the Journal of Water and Health
(http-J/www.iwapublishing.com/template.cfin?name=iwapwaterhealth).
Tools for disease reductions: Focussing especially on settings such as healthy
cities, healthy villages, healthy schools.
Groundwater and public health 15
1.6 GROUNDWATER IN THE WHO GUIDELINES FOR
DRINKING-WATER QUALITY
Since 1958 WHO has published at about ten year intervals several editi0Il5 of International
Standards for Drinking-water and subsequently Guklelines for Drinking-water Quality.
The third edition of the Guidelines, published in 2004, includes a substantial update of the
approach towards the control of microbial hazards in particular based on a preventive
management approach. In preparing the third edition of the Guidelines a series of state-of..
the-art reviews was prepared on aspects of water quality management and human health
(Box 13) of which Protecting Groundwater for Health is one.
In the overall context of the Gukfelines for Drinking-water Quality (GDWQ), this
monograph serves two purposes: it provides the backgrmmd information on potential
groundwater contamination as well as approaches to protection and remediation that
were taken into account in developing the third edition of the GDWQ. Further,
Protecting Groundwater for Health supplements it by providing comprehensive
information on: assessing the potential for contamination of groundwater resources,
prioritizing hazards and selecting management approaches appropriate to the specific
socioeconomic and institutional conditions.
Box 1.3. State of the art reviews supporting the third edition ofWHO
Gukfelines for Drinking-water Quality ( selected titles)
Water Safety Plans: Managing Drinking-water Quality from Catchment to
Consmner (Davison et al., 2005)
Safe Piped Water: Managing Microbial Water Quality in Piped Distribution
Systems (Ainsworth, 2004)
Water Treatment and Pathogen Control: ~ Efficiency in Achieving Safe
Drinking-water (LeChevallier and Au, 2004)
Assessing Microbial Safety of Drinking-water: Improving Approaches and
Methods (Dufour et al., 2003)
Quantifying Public Health Risks in the WHO Guidelines for Drinking-water
Quality: A Burden ofDisease Approach (Havelaar and Meise, 2003)
Rapid Assessment of Drinking-water Quality: A Handbook for hnplementation
(Howard et al., 2003)
Domestic Water Quantity, Service Level and Health (Howard and Bartram,
2003)
Managing Water in the Home: Accelerated Health Gains from hnproved Water
Supply (Sobsey, 2002)
Water Quality: Guidelines, Standards and Health: Assessment of Risk and Risk
Management for Water-related Infectious Disease (Fewtrell and Bartram, 200 I)
Chemical Safety of Drinking-water: Assessing Priorities for Risk Management
(in preparation)
16 Protecting Groundwater for Health
A central approach of the third edition of the GDWQ is the development of a reliable
preventive safety management approach: a Framework fur Safe Drinking-water, the
three key requirements of which are descnbed in Box 1.4. This includes the introduction
of Water Safety Plans (WSPs) as a management tool for avoidance and control of
groundwater contamination. These are descnbed in Chapter 16 of this book.
Box 1.4. lbethree key requirements ofWHO's Framework for Safe
Drinking-water (WHO, 2004b)
1. Health-based targets based on an evaluation ofhealth concerns.
2. Development of a Water Safety Plan (WSP) that includes:
• System assessment to detcrmine whether the water supply (fiom source
through treatment to the point of COINDilplion) as a whole can deliver
water of a quality that meels the health bac;ed tmge1s.
• Operatioml monitoring of the control measure; in the drinking-water
supply that are of particular impormce in securing drinking-water safety.
• Management plans docmnenting the system assessment and monitoring
plans and descnbing actions to be taken in normal operating and incident
ooooitions, including upgrading, documentation and oommunication.
3. A system of independent surveillance that verifies that the above are
operating properly.
This approach meets the need for developing an understanding of the key steps in the
supply chain at which pollution may be introduced or prevented, increased or reduced
Effective management implies identifying these, ideally through a quantitative system
assessment. The framework also includes identification of the appropriate measures to
ensure that processes are operating within the bounds necessary to ensure safety. For
drinking-water supply fiom groundwaters these oontrols may extend into the recharge
area but may also relate to more immediate source protection measures, such as well-
head protection. Some of the measures to verify safe operation of processes relevant to
gromdwater safety may be amenable to sophisticated approaches such as on-line
monitoring oflevels (e.g. oflandfill effluents). Others (such as the ongoing integrity of a
well plinth) are best approached through periodic inspection regimes.
1. 7 REFERENCES
Ainsmlrth, R (ed) (2004) Safe Piped Water: Managing Microbial Water Quality in Piped
Distribution System1i. WHO Drinking-water Quality Serie.s, IW A Publishing, LondolL
bup-//www.v.b.o.int/water sanitation healtb/dwq1924156251X/enrmdex.html (acces.sed
January 11, 2006).
Bartram, J.K. (1999) Effective monitoring of small drinking water supplies. In Providing Safe
Drinking Water in Small Systems: Technology, Operations and Economics, (eds. J.A
Cotruvo, GF. Craun and N. Hearne), pp. 353-365, Lewis Publishers, Washington OC.
Bergmire-Sweat, D., Wilson, K., Marengo, L., Lee, Y.M., MacKenzie, W.R, Morgan, J., Von Alt,
K, Bennett, T., Tsang, V.C.W. and F~ B. (1999) Cl)'Ptosporidiosis in Brush Creek:
Groundwater and public health 17
Describing the epidemiology and causes of a large outbreak in Texas, 1998. In Proc. Internal.
Corif. on Emerging lrifectious Diseases, Milwaukee, WI, A WW A, Denver, Colorado.
BGS and DPHE (2001) Arsenic Contamination of Groundwater in Banglodesh. BGS Technical
Report WC/00/19.
Briscoe, J. (1996) Financing water and sanitation services: the old and new challenges. Water
Supply, 14(3/4), 1-17.
CDC (1999) Outbreak of Escherichia coli 0157:H7 and Camplyobacter among attendees of the
Washington Cowrty Fair-New Y mk 1999. Morbidity and Mortality Weekly Report, 48(36),
803-804.
Chapelle, F.H. (1997) The Hidden Sea: ground water, springs and wells. Geoscience Press Inc.,
Ariwna
Chapman, D. ( ed.) (1996) Water Quality Assessments. E&F Spon, London.
Craun, G.F. (1985) A summary of watemome illness transmitted through contaminated
grmmdwater. J. Environ. Health, 48, 122-127.
Craun, G.F. (1992) Waterborne disease outbreaks in the United States of America: causes and
prevention. World Health Statistics Q., 45(2-3), 192-199.
Craun, G.F., Berger, P.S. and Calderon, RL. (1997) Colifurm bacteria and waterborne disease
outbreaks. JAWWA, 89, 96-104.
Craun, G.F, Calderon, RL. and Nwachuku, N. (2003) Causes ofWaterbome Outbreaks Reported
in the United States, 1991-1998. In Drinking Water and lrifectious Disease, Establishing the
Links, (eds. P. Hunter, M. WaiteandE. Rocln),pp. 105-117, CRCPress andIWAPublishing,
Boca Raton, Florida
Craun, G.F., Calderon RL. and Craun, MF. (2004) Waterborne Outbreaks Caused by z.oonotic
Pathogens in the United States. In Waterborne llJOnoses: Identification, causes and control,
(eds. JA Cotruvo, A Dufour, G. Rees, J. Bartram, R Carr, D.O. Cliver, G.F. Craun, R Fayer
and V.P J. Gannon}, pp. 120-135, WHO, IWAPublishing, London.
Curtis, V., Caimao~ S. and Yonli, R (2000) Domestic hygiene and dianboea -pinpointing the
problem. Tropical Medicine and lnternat. Health, 5(1), 22-32.
Davison, A, Howard, G., Stevens, M, Callan, P., Fewtrell, L., Deere, D. and Bartram, J. (2005)
Water Sqfety Plans: Managing Drinking-water Quality from Catchment to Consumer. World
Health Organmdion, Geneva
h llD://www.wbo.int/water sanitation health/dml{w~:p0506/en/index.html (accessed on
January 11, 2006).
Dufour, A, Snozzi, M., Koster, W., Bartram, J, Ronchi, E. and Fewtrell, L. (eds.) (2003) Assessing
Microbial Safety of Drinking-waler: Improving Approaches and Methods. WHO Drinking-
water Quality Series, IW A Publishing, London
h ://www.who.int/water sanitation health/ch\· 24154630 /en/index.html (accessed on
January 11, 2006).
EEA(1999)GroundwaterQualityandQuantityinEurope.EnvironmentalAssessmentReport,No.3.
Brey, SA, Potash, J.B, Roberts, L. and Shifl; C. (1991) Effi:cts of improved water supply and
sanitation on ascaria5is, dianboea, dracunculiasis, hoolrnorm infection, schistosomiasis, and
trachoma Bulletin of the WHO, 69(5), 609-621.
Esrey, SA (1996) Water, wastes and well-being: a multi-oountry study. Am J. Epidemiology,
143( 6), 608-623.
Gu, S.L., Ji, RD. and Cao, S.R (1990) The physical and chemical charact.tristics of particles in
indoor air where high :fluoride ooal burning takes place. Biomed Environ. Sci., 3(4), 384-390.
Falkenmark, M (2005) Water usability degradation -economist wisdom or societal madness?
Water International, 30(2), 136-146.
Fewtrell, L. and Bartram, J. (eds.) (2001) Water Quality: Guidelines, Standards and Health:
Assessment of Risk and Risk Management for Water-related lrifectious Disease. 1W A
Publishing, London.
Havelaar, AH and Meise, JM. (2003) Quantifying Public Health Risks in the WHO Guidelines for
Drinking-water Quality: A Bw-den of Disease Approach. RIVM report 734301022/2003,
18 Protecting Groundwater for Health
Billboven.
hnp://www.wbo.int/water sanitation health/dwqfquan tilvim!healthrisks/en/index.html
(accessed JanWBy 11, 2006).
Health Canada (2000) Waterbome outbreak of gastroenteritis associated with a oontaminaled
municipal water supply, Walkerton, Ontario, May-June 2000. Canada Communicable
Disease Report, 26(20), 170-173.
Howard, G. and Bartram, J. (2003) Domestic Water Quantity, Service Level and Health. World
Heallb Organi.zalion, Geneva
hl.m://1,\,ww.who.int/water sanitation health/diseases/wsh.0302/e:n/index.html (accessed
January 11, 2006).
How.ml, G., Ince, M and Smith. M. (2003) Rapid Assessment of Drinking-water Quality: A
Handbook for Implementation. Water, F.nginec:ring and Development Centre (WEDC),
Loughborough.
HIDlter, P.R (1997) Waterborne Disease: Epidemiology and Ecology, John Wiley & Sons,
Chicbe&er, New Yorlc.
Kukkula, M, ArstiJa, P., Klossner, ML~ Mai.mula, L., Bonsdorfl; CJ-I. and Jaatinen, P. (1997)
Waterbome outbreak of viral gastroenteritis. Scandinavian J. lnfectiaus Disease, 29, 415-418.
LeChevallier, MW. and Au, K.-K. (2004) Water TreatmenJ and Pathogen Control: Process
Efficiency in Achieving Safe Drinking-water. WHO Drinking-water Quality Series, 1W A
Publishing, London.
http ://1,\,ww.who.int/water sanitation health/dwq/92 41562552/e:n/index.html (am:ssal
January 11, 2006).
Lee, S.H, Levy, DA., Craun, G.F., Beach, MJ. and Calderon, RL. (2002) Surveillance for
watemome--disease outbreaks -United Staks, 1999-2000. CIX Morbidity and Mortality
Weekly Report, Sl(SS-8), v11\\-w.cdc.gov.mmwr/PDF/SS/S S5108.pdf (accessed April 29,
2005).
Melian, R, Myrlian, N., Gouriev, A, Moraru, C. and Radstakc, F. (1999) GrolDldwattT quality and
rural drinking-water supplies in lbe Republic ofMoldova Hydrogeology J. 7, 188-196.
Meybeclc, M, Chapman, D. and Helmer, R (1989) Global Freshwater QualiJy: a.first assessment.
Published fur WHO/UNEP by Blackwell Scientific, Oxfi:lrd.
Nll5inyama, G.W., McEwen, SA., Wilson, J.B., Waltner-To~ D., Gyles, C.L. and Opuda-
Astbo, J. (2000) Ri5lc mctors for ame diarrhoea among inhabitants of Kampala District,
Uganda. SA Medical J. 90(90), 891-898.
Payment, P. and Hllllter, P.R (2001) Endemic and epidemic infi:dious intestinal disease and its
relationship to drinking water. In Water Quality: guidelines, standards and health, ( eds. L.
Fe\W'ell and J. Bartram), pp. 61-88, 1W A Publishing, London.
Pedley, S. and Howard, G. (1997) The public health implications of miaobiological contamination
ofgrolDldwater. Q. J. Eng. Geo/., 30, 179-188.
Pokbrel, D. and Viraragbavan, T. (2004) Dianhoeal disease in Nepal vis-a-vis water supply and
sanitation status. .I. Water and Health, 2(2), 71-81 .
~A.and Havelaar, A. (2001) The global burden of disease study and applications in water,
sanitation and hygiene. In Water Quality: guidelines, standards and health, (eds. L. Fewtrell
andJ. Bartram), pp. 43-59, IWA Publishing, London.
Priiss-Osrun, A, Kay, D., Fewtell, L. and Bartram, J. (2004) Unsafe water, sanitation and hygiene.
In: Comparative Quantification of Health Risks: Global and Regional Burden of Disease
Attributable to Selected Major Risk Factors, (eds. Ezzati, M., Lopez, A. D., Rodgers, A and
Murray, C. J. L), Volwne 2, pp. 132 I-1352, World Health Organization, Geneva.
Rosenboom, J.W., Ahl.ned, K.M., Pfaff; A and Madajewicz, M. (2004) Arsenic in 15 Upazilas of
Bangladesh: Water supplies, health and behaviour. Report prepared for APSU, Unicef and
DPHE, Dhaka.
School of Environmental Srudies (2004). Javadpur Univeristy, Kolkatta, We& Bengal,
wv.w.soesju.org/arsenid (accessed April 29, 2005).
Groundwater and public health 19
Smith, AH., Lingas, E.O. and Rahman, M (2000) Contamination of drinking-water by arsenic in
Bangladesh: a piblic health anergency. Bulletin of the WHO, 78(9), l 093-1103.
Sobsey, MD. (2002) Managing Water in the Home: aca:lerated health gains from improved water
supply. World Health Organization, Geneva
http ://wv.w.who.int/water sanitation health/d\\tj/wsh0207/en/index.html (accessed Jammy
11,2006).
Trivedi, BK, Gandhi, H.S. and Shukla, MK (1971) Bacteriological water quality and the
incidence of waterlxnne diseases in a rural population. Indian J. Med. Sciences, 25, 795-80 l.
UNECE (1999) Invemory ofTransboundary Groundwaters. UNECE Ts Force on Monitoring
and~ Guidelines on monitoring of transbmmdary groundwaters, vol. 1, Supporting
Technical Dowments.
UNECE and WHO (1999) Protocol on Water and Health to lhe 199'1 Convention on the Protection
and use of Transboundary Wateroowses and Intemational Lakes.
hnp ://wv.w.ewo.who.int!Dorument/Peh-dlp/ProtocolWater.pdf(accessedJanuary 11, 2006).
US EPA (2004) www.epa.gov/sarewater/electronic/presentalions/sdwq/pt4/sdwa57.html (accessed
April 29, 2005).
Weissman, J.B~ Craun, G.F ~ Lawrence, DN., Pollard, RA, Suslow, M.S . and Gangarosa, E.J.
(1976) An epidemic of gastroenteritis traa:d to a mollnninaled piblic water supply. Am. J.
Epidemiology, 103, 319-398.
Willocks, L., Crampin, A, Milne, L., Seng, C., Susman, M, Gair, R, Moulsdale, M, Shafi, S.,
Wall, R, Wiggins, R and Lightfuot, N. (1998) A large outbreak of cryptospOridiosis
ll'.i.SO<--ialed with a public water supply fiom a deq> dlallc borehole. Outbreak Investigation
Team. Communicable Disease and Public Health, l, 239-243.
WHO (2004a) World Health Report 2004. WHO, Geneva
WHO (2004b) Guidelines for Drinking-Water Quality: Recommendations. 3rd edn, vol I, WHO,
Geneva
WHO and UNICEF (2000) Global Water Supply and SarriJation Assessment 2000 Report. WHO,
Geneva and UNICEF, New YOik
2
Groundwater occurrence and
hydrogeological environments
J. Chilton and K-P. Seiler
Many people are surprised to discover that groundwater is widely and heavily used
throughout the world During severe droughts in arid regions of the world, newspapers
and television cany dramatic pictures of chy wells in rural comnumities and people
walking long distances for small amounts of household water. However, groundwater
usage is important in both humid and arid regions, and it can be a revelation that many
cities are dependent on groundwater and use such large volumes of groundwater in their
public water supplies.
One reason for this general lack of awareness is that groundwater is usually a hidden
resource, out of sight and therefore out of mind. It is, nevertheless, as valuable an asset in
water supply terms as rivers, lakes and reservoirs, and deserves to be equally protected
As a consequence of this lack of awareness, the main features of grotmdwater systems
are poorly known or even misunderstood. To provide the necessary basic knowledge of
hydrogeology for the reader to fully appreciate the rest of the monograph, this chapter
aims to rectify the situation by placing place gromdwater in its appropriate context
within the wider water cycle. It then summarizes the ways in \\'hich groundwater occurs
and moves, and how it is replenished The characteristics of the main types of geological
settings are described so that the reader is able to see how different hydrogeological
environments vary in their response to the pressures of water abstraction and pollution.
© 2006 World Health Organization. Protecting Groundwater for Health: Managing the Quality of
Drinking-water Sow-r:es. Edited by 0. Schmoll, G. Howard, J. Chilton and L Chorus. ISBN:
1843390795. Published by 1W A Publishing, London, 1.JK_
22 Protecting Groundwater for Health
This knowledge will be used to help guide the information requirements outlined in
Chapter 8. In relation to the overall source-pathway-receptor approach to the assessment
of pollution this chapter is mainly focussed on the pathway through groundwater systems
to the receptor, and should be read in this context
Providing an adequate technical basis would be difficult without defining at least
some of the most important terms related to groundwater and pollutimL 1be most
important definitions are highlighted through the chapter and key concepts illustrated by
figures. A short list of suitable standard texts which can provide finther details fur the
interested reader is given at the end of the chapter, along with the refurences actually
quoted
2.1 GROUNDWATER IN THE HYDROLOGICAL
SYSTEM
2.1.1 The hydrological cycle
1be continuous movement of water between oceans, atmosphere and land is known as
the hydrological cycle (Figure 2.1 ). Considering the freshwater component of the system,
which is the part of greatest significance for this monograph, inflow is from precipitation
in the fonn of rainfall and from melting snow and ice. Outflow occurs primarily as
stream flow or runoff and as evapotranspiration, a combination of evaporation from
water surfuces and the soil and transpiration from soil moisture by plants. Precipitation
reaches streams and rivers both on the land surface as overland flow to tnbutary
channels, and also by subsurface routes as interflow and baseflow following infiltralion
to the soil. Part of the precipitation that infiltrates deeply into the ground may accumulate
above an impermeable bed and saturate the available pore spaces to form an
tmderground bcxfy of water, called an aquifer. 1be water contained in aquifers
contributes to the grotmdwater component of the cycle (Figure 2.1), from which natural
discharge reaches streams and rivers, wetlands and the oceans.
Figure 2.1 simplifies the hydrological cycle, illustrating only its natural components.
1bere are few areas of the world in which the cycle has not been interfered with and
modified by human settlement and associated activities. Large urban areas alter the
p~ of infiltration and drainage ( e.g. Lerner et al., 1990; Lerner, 1997), as do big
irrigation schemes. Negative and costly impacts of waterlogging and salinity are widely
experienced where excess infiltra1ion from irrigation with diverted surface water raises
groundwater levels beneath the irrigated land. llis is seen most dramatically in the lower
Indus Valley in Pakistan Estimates of the area affected vary, but of 16.1 million ha
irrigated, some 4.6 million ha are affected to some extent by waterlogging and salinity, of
which perhaps 2 million ha have suffered serious deterioration (Ghassemi et al., 1995).
Engineering works for flood control, irrigation, hydropower and navigation can all
change the surface water component of the cycle locally but sometimes dramatically, and
groundwater abstraction can intercept discharge to rivers, wetlands and the oceans. An
example of modification of the hydrological cycle that clearly has the potential to cause
negative health impacts is the 1.IDcontrolled discharge of 1.llltreated urban wastewater or
industrial effluents to surface water or gro1.IDdwater. Modifying the hydrological cycle by
Groundwater occurrence and hydrogeologiral environments 23
human intervention also implies changing pollutant pathways and transport mechanisms,
and these changes must be imdcrstood in developing s1n tegies for protecting the health
of water users.
swinegrwpcluzier
Figure 2.1. The manual hydrological cycle (modified from Morris et at, 2003).
DEF ■
Aquifers we layers °frock or sediments which are su�rcienity
porous to store water and permeable enough to allow water to flow
through them in economically viable quantities.
The river basin or catchment is the geo€raphical expression of the hydrological cycle,
and the spatial unit within which water resource balances can be estimated and through
which pollutants arc transported within the cycle. Figure 2.1 demonstrates that both
surface water and subsurthce prow occur. lliis means that the river basin or sub -
basin, and all of the activities within it should be the unit or basis for the management of
water rescauraes, rather than political or administrative subdivisions_ River Basin
Management Plans are, therefore, an essential feature of the European Union (ELT) Water
Framework Directive (EC, 2000), which is intended to establish the overall approach to
long-term management of water resources by EU Member States. It also follows that
catchments can contain land from more than one or indeed several countries and
transbourdlar, or multi -national authorities_ such as for the Rhine, Danube. Zambezi and
others have been established to oversee their management. Thus a key principle is that:

24
NOTE ►
Protecting Groundwater for Health
The catchment boundaries of a river basin or sulrbasin should
define the management unit for water resources, rather than
administrative or political boundaries.
2.1.2 Groundwater in the hydrological cycle
While the definition of groundwater as the water contained beneath the surface in rocks
and soil is conceptually simple and convenient, in practice the picture is a little more
complex, and confusion can arise. The water beneath the ground surface includes that
contained in the soil, that in the intermediate unsaturated zone below the soil, that
comprising the capillary :fiinge and that below the water table (Figure 22). The soil is
commonly understood to comprise the broken down and weathered rock and decaying
plant debris at the ground surface. The region between the soil and the water table is
commonly referred to as the unsaturated zone or sometimes the vadose zone.
Ground surface
r Soil water
Unsaturated I or < Vadose lntennediate
vadose l water vadose
zone zone
Water CapiUary water
table ---
!
..c: -a.
Q)
0
! Interstitial
Saturated t water Groundwater zone Subsurface
water
........... ··················
l
Water in
unconnected
pores
Water only in
chemical
combination
with rock
'
Figure 2.2. Oassificalion of subsurface water (modified fium Driscoll, 1986)
Groundwater occurrence and hydrogeological environments 25
DEF ► The unsaturuted wne contains both air and water, while in the
saturated zone all of the voids are fall of water. The water tabre
marks the boundary between the two, and is the sw:face at which
fluid pressure is exactly equal to atmospheric pressure.
Strictly speaking, therefore, groundwater refers only to water in the saturated zone
beneath the water table, and the total water ooh.mm beneath the earth's surface is usually
called subsurface water (Figure 22). In practice, of comse, the saturated and unsaturated
zones are connected, and the position of the water table fluctuates seasonally, from year
to year and with the effects of groundwater abstraction.
Appreciating this distinction is especially important in relation to protecting
groundwater from pollutants originating from activities at the surface. Such pollutants
can either be retained in the soil or they may be carried downwards by infiltrating water,
depending on the physicochemical properties of both the soil material and of the
pollutants. The soil, and the unsaturated z.one beneath it, can be considered to serve as a
reactive filter, delaying or even removing pollutants by the range of processes descnbed
in Chapters 3 and 4. The properties of the materials comprising the soil and the
unsaturated zone are, therefore, critical factors in defining the vulnerability of
groundwater to pollution, as described in Chapter 8.
In volume terms, groundwater is the most important component of the active
terrestrial hydrological cycle, as shown in Table 2.1. Excluding the 97.5 per cent of water
of high salinity contained in the oceans and seas, groundwater accounts for about one
third of the freshwater resources of the world (UNESCO, 199'J). If the water
pennanently contained in the polar ice caps and glaciers is also excluded, then
groundwater accounts for nearly all of the useable freshwater. Even if consideration is
further limited to the most active and accessible groundwater bodies, which were
estimated by Lvovitch (1972) at 4 x 106 km3
, then they still constitute 95 per cent of the
total freshwater. Lakes, swamps, reservoirs and rivers account for 3.5 per cent and soil
moisture for 1.5 per cent (Freez.e and Cheny, 1979). The dominant role of groundwater
resources is clear, their use is fi.mdamental to human lire and economic activity, and their
proper management and protection are correspondingly vital.
Table 2.1. Estimated water balana: of the world (modified fiom Nace, 1971 and UNESCO, 1999)
Parameter Surface area Volume Volume Residence time
(llr km ') (llrkm') (%)
Oceans and seas 361 l,370 97 -4000years
Groundwat.er 130 8 0.5 Weeks-100 000 years
Icecaps and glaciers 17.8 27 2 l 0-100 000 years
Lakes and reservoirs 1.55 0.13 <0.01 -IO years
Soil moisture 130 0.07 <0.01 2 weeks -several years
Atmospheric water 504 0.01 <0.01 -I0days
Sw.imps <0.1 <0.01 <0.01 1-10 years
River channels <0.1 <0.01 <0.01 -2weeks
Biospheric water <O.l <0.01 <0.01 -l week
26 Protecting Groundwater for Health
The last column of Table 2.1 provides an indication of the range of residence times of
water in the various compartments of the hydrological cycle. The great variation in
residence times in freshwater bodies is also illustrated in Figure 23, which emphasiz.es
the generally slow movement and long residence time of most groundwaters compared
to surfuce waters.
Running _ _Str_ea_ms ___ Riv_· e_rs_
waters
Shallow Deep
Standing lakes lakes
wateis Reservoirs
Karslic Alluvial Se<f1111en1ary Deep
Ground-aquifers aquifers aquifers aquifers
waters Bank
filtration
---1 I ·--
Hours Days Months Years 10 100 1,000 10,000 100,000
years years years years years
Figurel.3. Waterresidencetimeininlandfieshwaterbodies(modifiedfiomMeybedceta/., 1989)
2.2 GROUNDWATER OCCURRENCE AND
MOVEMENT
2.2.1 Groundwater occurrence and storage
Some grolllldwater occurs in most geological formations because nearly all rocks in the
uppermost part of the earth's crust, of whatever type, origin or age, possess openings
called pores or voids. Geologists traditionally subdivide rock formations into three
classes according to their origins and methods of formation:
Sedimentary rocks are formed by deposition of material, usually under water fiom
lakes, rivers and the sea, and more rarely fiom the wind In llllCOnsolidated, granular
materials such as sands and gravels, the voids are the spaces between the grains
(Figure 2.4A). These may become consolidated physically by compaction and
chemically by cementation (Figure 2.4D) to form typical sedimentary rocks such as
sandstone, limestone and shale, with much reduced voids between the grains.
Groundwater occurrence and hydrogeological environments 27
Igneous rocks have been formed from molten geological material rising from great
depth and cooling to form crystalline rocks either below the ground or at the land surfuce.
The former include rocks such as granites and many volcanic lavas such as basalts. The
latter are associated with various types of volcanic eruptions and include lavas and hot
ashes. Most igneous rocks are strongly consolidated and, being crystalline, usually have
few voids between the grains.
(A) Well-sorted, unconsolidated sedimentary
deposit having high porosity
(C) Well-sorted sedimentary deposit consis-
ting of pebbles that are themselves porous,
so the deposit as a whole has high porosity
(E) Rock with porosity increased by
solution
(B) Poorly sorted sedimentary
deposit having low porosity
(D) Sedimentary deposit whose porosity
has been diminished by the deposition of
mineral matter between the grains
(F) Rock with porosity increased by
fracturing
Figure 24. Rock texture and porosity of typical aquifer materials (based on Todd, 1980)
Metamorphic rocks have been formed by deep burial, compaction, melting and
alteration or re-crystallization of other rocks during periods of intense geological activity.
28 Protecting Groundwater for Health
Metamorphic rocks include gneisses and slates and are also nonnally consolidated. with
few void spaces in the matrix between the grains.
In the more consolidated rocks, such as lavas, gneisses and granites, the only void
spaces may be fractures resulting from cooling or stresses due to movement of the earth's
crust in the form of folding and faulting. These fractures may be completely closed or
have very small and not very extensive or interconnected opening; of relatively nanow
aperture (Figure 2.4F). Weathering and decomposition of igneous and metamorphic
rocks may significantly increase the void spares in both matrix and fractures. Fractures
may be enlarged into open fissures as a result of solution by the flowing growidwater
(Figure 2.4E). Limestone, largely made up of calcium carbonate, and evaporates
composed of gypsum and other salts, are particularly susceptible to active solution,
which can produce the caverns, swallow holes and other charact.eristic features ofkarstic
aquifers. It is worthwhile becoming aware of the main geological terms, as geological
maps are likely to be one of the main sources of information required to characterize a
catchment or area of investigation (Chapter 8), but also noting the following important
distinction:
NOTE ► A geologist's principal subdivision of rock types is according to
origin, wherefil hydrogeologists first classify aquifers fil w,consoli-
dated or consolidated and hence whether water is stored and moves
mainly between the grains of the rock matrix or through fractures.
The volume of water that can be contained in the rock depends on the proportion of
these openings or pores in a given volume of rock, and this is termed porosity of the rock.
DEF ► The porosity qf a geological material is the ratio qf the volume of the
voids to the total volume, expressed fil a decimal fraction or
percentage.
Increasing pore space results in higher porosity and greater potential to store water.
Typical porosity ranges are shown in Table 2.2 for common geological materials,
emphasizing the division between WICOnsolidated and consolidated referred to above.
Not all of the water contained in fully saturated pore spaces can be abstracted by wells
and boreholes and used. Under the influence of gravity when, fur example, the water
level fulls, some of the water drains from the pores but some remains, held by surface
tension and molecular effects. The ratio of the water that drains by gravity from an
initially saturated rock mass to its own total volume is defined as the specific yield of the
material, and typical values are also shown in Table 22.
Groundwater occurrence and hydrogeological environments 29
Table 2.2. Porosity and specific yield of geological materials (Freeze and Cheny, 1979; Driscoll,
1986; Domenico and SchW311z, 1998)
Material Porosi ty Spc:cifi c vi eld
Unconsolidated sediments
Gravel 025-0.35 0.16-0.23
Coarse sand 0.30-0.45 0.1--0.22
Fine sand 0.26--0.5 0.1--025
Silt 0.35-0.5 0.05--0.1
Gay 0.45--0.55 0.01--0.03
Sand and gravel 02--0.3 0.1--02
Glacial till 02--0.3 0.05--0.15
Consolidated sediments
Sandstone 0.05--0.3 0.03--0.15
Siltstone 02--0.4 0.05--0.1
Limestone and dolomite 0.01-025 0.005--0.1
Karstic limestone 0.05--0.35 0.02--0.15
Shale 0.01--0.1 0.005--0.05
Igneous and metamorphic rocks
Ve.5irular basalt 0.1--0.4 0.05-0.15
Fractured basalt 0.05--0.3 0.02--0.1
Tuff 0.1--0.55 0.05--02
Fresh granite and gneiss 0.0001--0.03 <0.001
Weathered granite and F55 0.05--025 0.005-0.05
Another important way of distinguishing aquifers and the way in which groundwater
occurs, when considering both its development and protection, is shown in Figure 2.5. In
the figure an unconfined aquifer is one in which the upper limit of the zone in which all
the pore spaces are fully saturated, i.e. the water table, is at atmospheric pressure. At any
depth below the water table the water pressure is greater than atmospheric. and at any
point above, the water pressure is less than atmospheric. In contrast, at greater depths. the
effective thickness of an aquifer often extends between two impermeable layers
(Figure 2.5).
DEF ► Materials thruugh which water can pass easily are said to be
permeable and those that scarcely allow water to pass or only with
dffficu/ty are described as impermeoble.
If the overlying layer bas low permeability and restricts the movement of water, then
it is known as an aquitard and causes the aquifer beneath to be partially or semi confined.
If the overlying layer bas such low permeability that it prevents water movement through
it. then the aquifer is fully confined. In these situations, at any point in the confined
aquifer, the water pressure is greater than atmospheric, because of the elevation of the
outcrop receiving recharge. If a borehole is drilled through the confining layer into the
aquifer, water rises up the borehole to a level that balances the pressure in the aquifer. An
imagiruuy surface joining the water level in boreholes in a confined aquifer is called the
potentiometric surface, which can be above or below the groundwater surface in the
30 Protecting Groundwater for Health
overlying unconfined aquifer (Figure l5 . If the pressure in a confuted aquifer is such
Mai the potentiometric surface is above ground level, then a drilled borehole will
overflow (Figure 2.5)_ For a phreatic aquifer, which is the first wiconfined aquifer to be
tinned below the surface, the potermomed7c surface and groundwater surface
correspond, and this is called the water table
From the groundwater development point of view, unconfined aquifers are often
favoured because their storage properties make them more efficient for exploitation than
confined aquifers, and they are likely to be shallower and therefore cheaper to drill into
and pump from_ On the oilier hand_
NOTE (♦ A confined aquftr which has even a modest rnerlyirgsegquence of
less permeable clay strata is likely to be much less vulnerable to
pollution than an uncovered aquifer-
- Unconfined aquifer
Outcroplrmi rge 2rea
Unstinted dipretakei
ate
m
y -+
Sabaated
Deep confined aquifer and
shallow unconfined aquifer
Wel,nlx COW
144 ,>
Walertab I -s-.•- P
surface /
tmneaa
coaling layer
Impermeade
ease
Figure 2.S. Schematic cross-section tiro rating confined and unconfined aquifers
2.2.2 Groundwater movement
Groundwater is not usually stake but moves slowly through aquifers. However, it needs a
source of energy to do so, which is provided by the hydraulic head represented by the
height of the water level in an observation well or borehole in the aquifer. The dotal
hydraulic head is made up of two components. the elevation head being the height of the

Groundwater occurrence and hydrogeological environments 31
midpoint of the section of the borehole or well that is open to the aquifer, and the
pressure head, which is the height of the column of water above this midpoint. The first
component thus reflects location and topographic position and the second reflects
conditions in the aquifer, including seasonal and longer -term changes in water levels.
Hydraulic heads are normally measured with respect to an arbitrary datum, which 'is
otter, sea level To obtain a more comprehensive description of these rather difficult
concepts, the reader should refer to standard hycirvgeological text books such as Freeze
and Cherry (1979), Price (1996) or Domenico and Schwartz (19913). For an
understanding of groundwater movement for the present purposes, however, it is
sufficient to know that groundwater moves from regions of high head to regions of low
head
The flow of groundwater through an aquifer is governed by Darcy's Law_ which
states that the rate of flow is directly proportional to the hydraulic gradient:
Q/A=q=-K(h.,—h2)II=-KAli/Al (Egn.2.1)
where Q is the rate of flow through area A under a hydraulic gradient Alt/AI which is the
difference in hydraulic heads (hi - h,) between two measuring points, and q is the
volumetric flow per unit surface area. The direction of groundwater flow in an aquifer is
at right angles to lines of equal head. A simple experimental apparatus used to
demonstrate Darcy's Law is shown in Figure 2.6, indicating also the elevation and
In essurc componerns of hydraulic head 3 canned to above. Me equation for Darcy's Law
is conventionally written with a minus sign because flow is in the direction of decreasing
hydraulic heads.
pressure
wmponent
of h,
water in
at steady
now rate
0
elevation
head
component
ofh,
gauze
4/
cross -sectional I,
area A
sand
arbitrary datum
gauze
war
out
pressure
head
component
of h,
2
elevation
head
component
❑lr h,
Figure 2.6. E'q,aimerlal apparatus to downstate Darcy's taw (modified from Price, 1996)

32 Protecting Groundwater for Health
The constant of proportionality in the equation, K, has dimensions of length/time
because the hydraulic gradient is dimensionless. This parameter is known as hydraulic
conductivity, and is a measure of the ease with which water flows through the sand
contained in the cylinder in the laboratocy experiment or through the various materials
that form aquifers and aquicludes. The similarity between Darcy's Law and other
important laws of physics governing the flow of both electricity and heat should be
noted. The ease with which water can flow through a rock mass depends on a
combination of the siz.e of the pores and the degree to which they are interconnected.
These features determine 1he overall permeability of the rock. For clean, granular
materials, hydraulic conductivity increases with grain siz.e. Typical ranges of hydraulic
conductivity for the main types of geological materials are shown in Figure 2. 7.
Hydraulic conductivity, K (m d-1)
105 104 103 102 101 10° 10-1 10·2 10-3 104 10·5 10-6 10·1 10-8
I
Relative hydraulic conductivity
Very high High Moderate Low Very low
Unconsolidated deposits
Clean fine to coarse gravels
Sarni and gravel
Coaraesands
Fine sands
Silly sands
Silts and loesses
Glacial blls
Consolidated rocks
Clays
Massive igneous and
metamorphic rocks (unfractured)
Shales
Laminated sandstones
Cemented, unjointed sandstones
Crystaftine, unjoin1ed sandstones
Volcanic tuffs
Fractured and/or weathered
granites and gneisses
Jointed sarnlstones
Dolomites
Vesicular basalts
Karst limestones
Figure 2. 7. Range of hydraulic conductivity (K) values for geological materials (based on Driscoll,
1986 and Todd, 1980)
Groundwater occurrence and hydrogeological environments 33
Darcy's Law can be written in several forms. By substituting q in the equation, it is
possible to determine the specific discharge per unit area, if the vohmetric flux: Q is
divided by the full cross-sectional area (A in Figure 2.6). However, this area includes
both solids and voids, although clearly flow can only take place through the voids or pore
spaces. A more realistic linear pore velocity, v, the volwnetric flow rate per area of
connected pore space can be calculated if the porosity is known. Thus we can define:
v=-q/n=-Ki/n (Eqn22)
Where i is conventionally used to represent the hydraulic gradient Afi/Af. To make this
calculation, it is necessary to know the effective or dynamic porosity n.,, which represents
the proportion of the total porosity that is involved in groundwater movement This is
difficult to measure, but for llllCOnfined aquifers is probably close to the specific yield
values given in Table 2.2.
DEF ► The effective porosity is defined as the proportion of the total volume
that consists of interconnected pores able to transmit fluids.
Thus most materials in which intergranular flow predominates have effective
porosities of 0.15 to 025, so in these types of aquifers the actual groundwater flow
velocity· is four to six times the specific discharge. The linear velocity will always be
greater than the specific discharge, and increases with decreasing effective porosity. This
average velocity in the direction of groundwater flow does not represent the true velocity
of water particles through the pore spaces. These microscopic velocities are generally
greater because the intergranular flow pathways are irregular and tortuous and longer
than the average linear macroscopic pathway. The average linear velocity, v, is a key
parameter in groundwater protection, as it defines the travel times for water and solutes
within aquifers. For llllCOnsolidated granular aquifers, typical natural grolllldwater flow
velocities range :fium a few mm/d fur silts and fine sands to 5-10 mid fur the clean and
ooarse gravels.
Darcy's Law provides a valid description of the flow of groundwater in most
naturally occurring hydrogeological conditions i.e. for fiactured rocks as well as granular
materials. Fractured rocks are characterized by low porosity and localiz.ed high hydraulic
conductivity, and very high flow velocities of up to several kilometres per day may result
(Orth et al., 1997; US EPA, 1997), especially where a small number of :fractures are
enlarged by solution (Figure 2.4E). Extensive development of solution in limestone areas
can result in karst terrain, which is typified by channels, sinkholes, depressions and
caves, into which all traces of surface flow may disappear. Such conditions can be very
favourable for grolllldwater supplies fi:om springs and boreholes, but aquifers of this type
are often highly vulnerable to all types of pollution (Malard et al., 1994).
Grolllldwater flow may occur through the spaces between the grains or through
fractures (Figure 2.4) or a combination of the two in, for example, a jointed sandstone or
limestone. Hydrogeologists commonly refer to these as dual-porosity aquifers, because
they have primary porosity and permeability :fium the intergranular pores and additional
secondary porosity and permeability provided by the fiacture systems. The presence of
34 Protecting Groundwater for Health
highly fractured rocks should immediately warn of the risk of rapid transport over large
distances. The occurrence of potential contaminant sources close to water supplies in
such environments should be considered to provide a very high risk of pollution.
The characteristic properties of an aquifer to store and transmit grmmdwater are
nonnally deduced from the interpretation of pwnping tests perfurmed on wells or
boreholes (Price, 19%) or by introducing inert tracers into groundwater flow systems
and observing their transport (Becker et al., 1998; Kas-5, 1998). Determinations of aquifer
parameters are often difficult and expensive, and information is usually available for at
most a few specific locations in an aquifer. However, most geological materials are far
from unifonn laterally or with depth. As an example, sediments such as river alluvium,
deltas and glacial deposits may contain alternating fine and coarse layers, clay lenses,
sand channels and many other featmes and structures which reflect the complex history
of deposition. These geological variations mean that aquifers are rarely homogeneous, in
which the properties are the same irrespective of position in the aquifer, but more often
heterogeneous, with varying properties. Obtaining or selecting aquifer parameters that
can apply to and be representative of a whole aquifur or catchment is often, therefore, a
difficult task for a hydrogeologist Describing and quantifying groundwater flow is not as
straightforward as a swnmary text such as this might suggest to the reader, especially in
aquifers with complex patterns of fracture flow. However, distinguishing whether
intergranular or fracture flow predominates for any aquifer of interest is fundamental to
understanding the hydrogeology, which is in turn the basis for developing, managing and
protecting groundwater.
OTE ► Whatever the source of pollution and type of pollutant,
understanding the wtzy groundwater occurs and moves is crucial to:
(1) setting up groundwater protection policies;
(2) establishing water quality monitoring systems;
(3) designing pollution control or aquifer remediation measures.
2.3 GROUNDWATER DISCHARGE AND RECHARGE
It is important to distinguish between infiltration of precipitation and groundwater
recharge. Thus looking back at the hydrological cycle in Figure 2.1, when rain falls,
some infiltrates into the soil. Much of this moisture is taken up by the roots of plants and
is subject to evapotranspiration from the soil zone, and some becomes interflow drainage
to streams and rivers. Only a part of the infiltration becomes recharge and moves deeper
into the subsurfuce under gravity, and in arid and semi-arid areas this may be a very
small proportion indeed. This distinction becomes very important when considering the
estimation of recharge in Chapter 8. Thus conceptually and for estimation purposes:
DEF ►
Groundwater occurrence and hydrogeological environments 35
groundwater recharge should be defined as the dawnwardj/ow of
water reaching the water table and replenishing groundwater
resources
and should be distinguished fium infiltration. The latter includes all of the water entering
the ground fium rainfall or other sources but by no means does all of this become
growidwater recharge.
In the subsoil and rock closest to the ground surfuce. the pore spaces are partly filled
with air and partly with water. This was defined as the unsaturated or vadose zone in
Figure 22, and can vary in depth fium nothing to tens of metres. In the unsaturated zone,
soil, air and water are in contact and may react with each other, which can be important
in the evolution of the hydrochemistry of the water; In the uppermost part of the
unsaturated zone, some upward movement occurs in response to seasonal
evapotranspiralion requirements. Below this, in humid areas, movement in the
unsaturated zone is dominantly vertically downwards. The most recent water arriving
fium the soil displaces downwards the whole cohnnn of water already in the unsaturated
zone, rather like the movement of a piston, so that the water at the base of the column
reaches the water table. For parts of the year, particularly when the weather is chy and no
new percolating water passes below the soil, the 'piston' moves very slowly or not at all.
In times of heavy rainfall and substantial infiltration, downward movement may be more
s1rongly established.
By sampling the unsaturated zone water to obtain vertical profiles and repeat profiles
of tracers such as bromide, nitrate and tritium, average rates of movement ofless than 5
rn/yr and often less than I m/yr have been measmed in temperate regions (Wellings,
1984; Geake and Foster, 1989; Barraclough et al., 1994). This means that it could take
20 years or more fur infiltrating water to reach a water table 20 m below the growid
surface. It is common fur the water table to be in the range IO to 50 m below ground, and
the unsatwated zone component of the pollutant pathway can therefure be substantial. In
semi-arid regions, recharge can be much less and downward displacement
correspondingly very slow (Edmunds and Gaye, 1994). In the most arid areas, the
unsaturated zone may only act as a temporary storage reservoir in which water that
percolates downwards after occasional heavy rain does not reach the water table but is
instead drawn upwards and returns to the atmosphere by evapotranspiralion fium plants.
Residence times in the unsaturated zone thus depend on the thickness and the rate of
recharge, and can vary fium almost nothing to tens or hundreds of years.
The above applies to aquifers in which downward movement of recharging water
takes place only through the intergranular matrix. In fractured and dual porosity aquifer
materials, much more rapid, preferential flow to the water table may occur, especially
after heavy rainfhll. This component of flow can carry pollutants fium the ground surface
much more quickly, allowing little or no time for attenuation, and such aquifers can be
highly vulnerable to pollution
All subsurface freshwater must have a source of recharge, even if it was long ago.
This comes either by direct infiltration of rainfall or snowmelt, or fium rivers and lakes.
Now that the hydrological cycle has been interfered with as a result of human activities,
36 Protecting Groundwater for Health
recharge can also be derived from canals, reservoirs. irrigated land, water mains and
sewerage systems in urban areas, mining waste, sewage lagoons, in fuct any artificial
water body that is in connection with the subsurfuce. Artificial recharge, which is
becoming an increasingly important resource management option, can also introduce
water of different origin and quality into aquifers. This of course means that groundwater
recharge is not always of the same good quality as infiltrating rainfall, which itself may
be contaminated by acid rain or atmospheric acid deposition
2.4 GROUNDWATER FLOW SYSTEMS
In many aquifers, the hydraulic head reflects the topographic surface of the ground, and
groundwater therefore moves from elevated regions where recharge occurs to discharge
zones at lower elevations. Thus within the context of the overall cycle shown in
Figure 2.1 and the source-pathway-receptor concept, the groundwater flow system
(Figure 28) is a useful way of descnoing the physical occurrence, movement and
hydrochemical evolution of groundwater.
DEF ► A groundwater flaw system is a discrete, closed three-dimensional
system containing.flaw paths from the point at which recharging
water enters an aquifer to the topographically lawer point at which
it leaves the aquifer.
Infiltration of rainfull on high ground occurs in a recharge area in which the hydraulic
head decreases with deplh, and net saturated flow is downwards away from the water
table and laterally towards areas of lower hydraulic head (A in Figure 28). After moving
slowly through the aquifer down the hydraulic gradient (B in Figure 2.8), groundwater
leaves the aquifer by springs, wetlands, baseflow to rivers (C in Figure 2.8) or discharge
to lakes or the oceans. These are known as groundwater discharge areas, and at C (in
Figure 2.8) the hydraulic head increases with depth and the net saturated zone flow is
upwards towards the water table. In a recharge area, the water table can be at deplh, with
a considerable thickness of unsaturated zone above it In a discharge area, the water table
is usually at, or very near to, the ground surface. Rivers, canals, lakes and reservoirs may
either discharge to or receive recharge from groundwater, and the relationship may
change seasonally or over a longer time span or along the course of a single river.
While in many cases groundwater and surface water catchments have more or less the
same boundaries, this is not always the case. Seasonally due to recharge, and in resp:mse
to heavy abstraction, groundwater catchment boundaries may deviate significantly from
surfuc.t: water catchments. In deep aquifers, or sequences of more than one layered
aquifer, groundwater recharge may come from great distances and deep groundwater
flow may have little relationship to the overlying surfuce water system. In most cases,
however, if there is no information it is a reasonable first estimate to assmne surface
water and groundwater catchments are similar and that groundwater flow patterns are
likely to be a subdued reflection of the surface topography.
Groundwater occurrence and hydrogeological environments 37
In large, deep aquifers, groundwater is likely to move slowly, at rates of a few mclies
per year, from recharge to discharge arca over tens or hundreds of kilometres. This may
take hundreds or thousands of years. and typical order -of -magnitude values from time of
recharge to point of discharge are indicated in Figure 2.8. Hydrageologists can confirm
these by isotopic dating techniques (Kendal and McDonnel, 1998; Edmunds and
Smedley, 2000). In small, shallow aquifers, recharge and discharge arras may be much
closer or even adjarer>t to each other, and residence times can be restricted to a few
months or yea-s. In and and semi -arid regions. groundwater discharge areas are often
characterized by poor quality groundwater, particukuly with high salinity. Groundwater
discharge may be from seepages or salt marshes with distinctive vegetation, known as
Salinas or playas, in which evapotranspiration at high rates for long periods of Erne has
led to a build-up in salinity_
While the flow system in Figure 2.8 is a useful general illustration, in many cases
groundwater does not flow uniformly through the entire thickness of an aquifer, but
instead flows predominantly at shallow, depths close to the water table (Seiler and
Lindner, 1995). In tuteon solidated aquifers. both hydraulic conductivity and porosity
r31rally decrease with depth due rn consolidation and compaction. In fracttued aquifers
too, the hydraulic ainductivity and porosity provided by the fracture system would be
expected to decline with depth as the fractures become less open. This general but
variable and not easily predicted decline in groundwater flow properties with depth often
restricts the flow and pollutant pathway to the most permeable. near -surface parts of the
aquifer. Further, the zone of seasonal water table fluctuation is often where the most
active solution of fractures occurs and this helps to enhance the flow dominance of the
uppermost part ofthe saturated aquifer.
Yea si
Minor
perennial
&change
area
Months;
Unsaturated
nfw
tow-panneatitity
strata
-------• Oroadwater surface
seasonal range
in unconfined aquitert
Aquilsr InlernMerit
rrrha ga discharge
area area
i�
trrwtmeable
strata
Major
perennial
a -:
f MiSertia
Low-pemneabikty
strata
Figure 2.8. Schematic grouudxaler flow system (modified from Foster er at. 2000)

38 Protecting Groundwater for Health
Given the high porosity values in the upper part of Table 22, it can be seen that most
types of aquifer, provided they are at least a few metres thick, can contain large volwnes
of water. Many aquifers are, of course, much thicker, ranging up to several hundred
metres. Even in humid areas, recharge comprises only a proportion of the total rainfall
and a simple calculation will show that, for typical annual recharge volwnes equivalent
to tens to a few hundreds of millimetres, the total volwne of groundwater in storage in
the aquifer is many times larger than the annual recharge. Aquifers are generally,
therefore, high storage, low recharge systems with substantial capacity for dilution of
incoming pollutants, except in the situations of restricted shallow flow referred to
immediately aoove. fu these cases, incoming recharge may be distributed fur from evenly
through the aquifer, and the resulting groundwater volwne available for dilution may be
much less than the total storage of the aquifer.
2.5 GEOLOGICAL ENVIRONMENTS AND AQUIFER
TYPES
As described aoove, the natural subsmface geological environment provides the
dominant control over the occurrence and movement of groundwater and hence defines
which rock types form good aquifers. However, geodiversity and the consequent
hydrogeological variability are poorly appreciated by many of those working in water
protection and management. The variability both between and within hydrogeological
environments can have a profound impact on how aquifers respond to the pressures
imposed upon them. Further, if an aquifer is to be protected and managed, it is important
to understand the groundwater flow system to be able to assess the susceptibility of the
aquifer to these external changes and the types and timescales of the likely responses.
While almost all geological materials contain some water and many different rocks
can form useful aquifers, nevertheless it is possible to develop a smrunary of the most
common aquifer types and hydrogeological environments (Table 23). This classification
is a useful overall basis for helping to identify the major potential concerns for protection
and management of groundwater. The general subdivision in Table 23 takes into
account both the rock type and the geological environment in which the rocks were
formed. While such a broad classification is useful, it inevitably involves some
simplifications of the true breadth of subsurfuce geological variation and complexity. The
classification sho\\-n in Table 2.3, or slight variations of it, proved useful as a basis for
discussion of groundwater quality monitoring (Chilton, 1996) and provided the
hydrogeological framework within which the management of groundwater in udJan
areas (Foster et al., 1998) and the development and management of groundwater in rural
areas (Foster et al, 2000) can be set Each of the seven sulxlivisions is briefly described
below.
Major alluvial and coastal plain sediments
The first sulxlivision (Table 2.3), covers a broad range of materials and lateral and
vertical scales. At one end of the scale are extensive sequences of coastal, river and
deltaic alluvium, sometimes hundreds of metres thick. These unconsolidated sedimentary
deposits form some of the most important aquifers in the world, in which very large
Groundwater occurrence and hydrogeological environments 39
volllllles of groundwater are s1Dred and from which large quantities of water are plllllped
for water supply and irrigation. Examples include the Lower Indus and Ganges-
Brahmaputra valleys, the Mekong, the Tigris-Euphrates, the north European plain and
the Nile valley. Many of the world's largest cities such as Bangkok, Beijing, Cairo,
Calcutta, Dhaka, Hanoi, Lima, Madras and Shanghai are located on such deposits and
are supplied by groundwater drawn from unconsolidated strata.
These aquifers can cover large areas and contain enormous volllllles of water. As an
example, aquifers within the unconsolidated sediments underlying the Huang-Hai-Hai
Plain of eastern China, which covers an area of 350 000 km2
, provide the potable water
requirements for nearly 160 million people and also enough to irrigate some 20 million
ha of land The sediments are of Quaternary age and are typically 200-400 m thick.
Groundwater in these sediments can be subdivided into three types: an upper unconfined
freshwater zone, a middle saline water zone and a lower confined aquifer. The total
volllllle of grom1dwater stored exceeds 2 000 000 million m3, whilst usable grom1dwater
resources have been estimated at more than 49 000 million m3/a Other unconsolidated
sedimentary aquifers may be much less extensive but can still store sufficient volllllles of
groundwater to be important sources of water supply. The coastal plain around Jakarta
and the Nile valley at Cairo are examples. Smaller but still locally-important aquifers are
provided by river valley and coastal plain sediments of more limited lateral extent and
depth, and aquifers of much more restricted siz.e and extent may occur in upland river
valleys as river terraces.
Aquifers in unconsolidated strata are rarely simple homogeneous systems but
typically consist of alternating permeable layers of productive sands and gravels
separated by less permeable aquitard layers of clay and silt, reflecting the complex
history of deposition. In such sequences, the shallowest aquifer may be the easiest and
cheapest to exploit, but is likely to be the most vulnerable to pollution. The presence of
aquitards may produce complex groundwater flow patterns, but the permeable horizons
may still have a degree of hydraulic continuity, such that plllllping from one layer will
affect the others, producing significant vertical head gradients and consequent leakage.
The high porosity of W1COnsolidated sediments, typically in the range 025 to 0.35,
and the generally low horizontal hydraulic gradients in the major alluvial plains means
that grom1dwater velocities are very low, usually in the range 0.003--0.1 m cf 1
• These low
velocities combined with the significant distances travelled (tens to hundreds of
kilometres) indicate that much of the deeper groundwater in thick alluvial sequences is
derived from recharge several hundred to several thousand years ago, and the term 'fossil'
has sometimes been used to descnbe deep, old groundwater.
Jntermontane alluvial and volcanic systems
Aquifers of this type include some volcanic lavas and pyroclastic rocks, together with
alluvial-volcanic and alluvial fan deposits. They are typically associated with rapidly
infilled and faulted troughs or basins within mom1tain regions (Table 23). Hydraulic
conductivities and porosities are generally high but variable. When combined \\'1th the
above average rainfall that is oflen found in the mountainous climatic regimes where
many of these environments are found, valuable aquifers occur and are capable of
supporting substantial borehole yields. Additional recharge to grom1dwater often occurs
40 Protecting Groundwater for Health
where surfure water flowing from the surrounding mountain slopes infiltrates into the
highly permeable valley-fill deposits, especially through the alluvial :funs and colluvial
deposits found on valley margirn. Examples of this environment include Mexioo City,
Guatemala City, San Salvador, Managua and San Jose in Central America, the
Kathmandu Valley in Nepal, Bandung and Yogyakarta in Indonesia, Davao in the
Philippines and Sana'a in Yemen. In these mountainous areas, flat land is limited and
highly valuable, and is often densely populated. Restrictions on available land for
settlement will often result in groundwater abstraction for potable supplies in the basin
occurring within densely populated areas, with significant implications for water quality.
Furthermore, the concentration of population and the consequent high water demand can
result in groundwater abstraction exceeding the safe yield of the aquifer. Long-term
decline in groundwater levels and/or contamination of groundwater can result, as for
example in Mexico City (NRC, 1995), the Kathmandu Valley (Khadka, 1991) and the
Sana'a Valley (Alderwish and Dottridge, 1999).
Consolidated sedimentary aquifers
Important aquifers occur within consolidated sedimentary strata, principally sandstone
and limestone (Table 2.3). These can be broadly subdivided into younger, Tertiary
formations and older Mesozoic or Palaeozoic formations. Globally, although their
distnbution is irregular, they are widespread and common being found both in mmmtain
belts such as the Alpine-Himalayan, Andean, Urals and North American cordilleras and
in lowlands and plateau areas such as northern Europe and central China.
Sandstones have been formed when sandy marine or continental sediments were
buried and compacted to form consolidated rocks. The degree of consolidation generally
increases with depth and age of the rocks. Thus the younger, Tertiaiy sandstones usually
retain some degree of primmy porosity between the sand grains and are typically of low
to moderate permeability. In the older, Mesoz.oic or Palaeozoic formations with more
strongly developed cementing of the grains, the primmy porosity may have become
largely eliminated. Pre-Tertiary sandstones can range from friable to highly indurated
depending on the degree of cementation, and in the latter cases it is the secondary
porosity resulting from the development of fractures which can provide adequate
permeability and storage for such rocks to form productive aquifers.
Limestones exhibiting solution enhancement of such fractures (called karst) are
widespread and can be prolific aquifers, although well yields are highly variable in time
and space. For instance, in northern China, kmst limestones occupy an area of
800 000 kni2, are typically 3~ m thick and their groundwater resouro.:s have been
estimated at 12 800 million m3 yr-1• In southern China, kmst limestones are even more
extensive where they cover an area of 1 400 000 km2, with groundwater resources
estimated at 190 000 million m3 yr-1• Similar, highly permeable limestones occur
throughout southern Europe, including along the coast of the Adriatic Sea region from
which the karst name derives, in the Middle East and in the USA
Recent cocmal calcareous formations
These fonnations fonn important local aquifers. Examples, which include Florida,
Jamaica, Cuba, Hispaniola and numerous other islands in the Caribbean, the Yucatan
Groundwater occurrence and hydrogeological environments 41
peninsula of Mexico, the Cebu limestone of the Philippines, and the Jaffua limestone in
Sri Lanka, provide important sources of potable water for 1he people living 1here and for
irrigation Their high to very high permeability derives not only from initially high
primary porosities (due to the sedimentary environment of deposition), but also from
fractures that have been enhanced by solutioIL This can produce rapid groundwater
movement with velocities :frequently in excess of 100 m a1
• The high infiltration
capacity of 1hese strata often precludes surface drainage systems and very often
groundwater is the only available source of water supply in these environments.
These characteristics have important implications for protecting groundwater quality.
Soils can be very 1hin and water movement from the soil to the water table via fissures is
often so rapid that these formations are highly vulnerable to pollutioIL In addition, being
coastal, the aquifer.; are usually underlain by seawater, often at shallow dep1hs. Excessive
abstraction of groundwater, wi1h a consequent lowering of 1he water table, may induce
saline intrusion by lateral movement of the freshwater/seawater interlace inland or local
upconing and contamination of 1he fresh groundwater body from below.
Glacial formations
Deposits of glacial and fluvioglacial origin comprise small but locally important aquifer.;
not only in temperate z.ones of1he world but also at altitude in the mountain ranges of the
Andes and Himalayas. Ice-transported sediments are commonly unsorted mixtures of all
grain siz.es from clay to boulders, typically have low permeabilities and act as aquit.ards
or aquicludes. Their geographical distnbution is often limited, as 1hey tend to occur in
regions of active erosion. In contrast, water-sorted sediments, laid down from glacial
melt-waters, include 1he sands and gravels of kames and eskers, which can form
restricted but highly productive aquifer systems. These can sometimes be more
extensive, as in the coalescing gravel outwash plains of North America, 1he eastern
Andes and 1he Himalayas/Pamir/fienshan cordilleras, or quite narrow and sinuous, as in
the glacial channels of1he North German Plain and the Great Lakes.
The environment of deposition, from melt-water streams and 1he upper reaches of
braided rivers makes fur highly variable li1hology. As a result, multiple aquifers are
typical, comprising complex systems in which lenses of highly permeable sands and
gravels are partly separated vertically and laterally from each oilier by lower-
permeability fine sands, silts and clays. The resultant 'patchy' aquifer can be very
productive, but hydraulic continuity between different lenses means that mobile
persistent contaminants are able to penetrate to significant depths.
In many glacial areas, the underlying bedrock consists of ancient, hard and
unwea1hered granites and gneisses, which are very unpromising as aquifers. In these
terrains, even the small sedimentary aquifers referred to above can provide vital but
potentially quite vulnerable water supplies to the small and scattered communities in
such regions. Sometimes these aquifer.; are used for urban supply, ei1her directly by
means of boreholes, or as prefilter.; for high volume riverbank intakes via infiltration
galleries or collector wells. Examples include Cincinnati and Lincoln (USA), Berlin and
Diisseldorf(Germany), and Vilnius (Lithuania).
42 Protecting Groundwater for Health
Loessic plateau deposits
Fine windblown deposits, called Ioess, form an important aguifur in China Although
loess is fmmd elsewhere, such as in Argentina and north of the Black Sea, thick deposits
are almost entirely restricted to north central China where they cover an area in excess of
600 000 kni2. Ofthis, some 440 000 km2 is continuously covered with a thickness of
between 100 and 300 m. The loess covets a vast plateau at elevations of between 400
and 2 400 m above sea level The loess plateau supports a population of 64-million and
7 3 million ha of cultivated land and is dependent on groundwater for domestic water and
irrigation in this semi-arid region The distinctive geomorphological features and
geological characteristics produce a complex groundwater system. The loessic plateau
aquifers are frequently cut through by gullies and ravines, so that the plateaux form a
series of independent water circulation systems. The deposits are generally of low
permeability and the presence of palaeo-soils produces a layered aquifer; the deeper
20nes being partly confined. The water table is often quite deep (30-50 m below surface).
&tensive volcanic te"ains
One of the largest and most important areas of volcanic lava flows occurs in the central
and western parts of India, where the Deccan basalts cover more than 500 000 km2•
Other extensive volcanic terrains occur in North and Central America, Central and East
Africa, and many islands are entirely or predominantly of volcanic origin, such as
Hawaii, Iceland, the Canary Islands (Spain) and some of the Canbbean islands. Older
lavas such as the Deccan basalts can often be largely impermeable in the rock mass, but
younger basalts can provide very large springs. Individual lava flows can be up to I 00 m
thick, and although the more massive flows are often impermeable, extensive jointing
allows water to infiltrate and move through them. The junctions between flows can form
highly productive aquifers, because of the cooling cracks and joints, and development of
rubble 20nes caused when the rough Slrlace of the lava is covered by the chilled base of
the next flow, weathering and soil in the period of time between successive flows.
Extensive lava tubes may be formed where lava drains fiom beneath a cooled and
congealed surface. It is the combination of these features that make the Deccan basalts
and other such volcanic rocks important and locally productive aquifers. Other materials
are thrown out as volcanic clouds, which sometimes settle as ash deposits or become
welded tufts. The mineralogy and chemistry of the volcanic rocks and their viscosity and
gas content determine the precise nature of the volcanic eruptions and resulting rocks.
Alternating sequences of ashes and lavas, in which the lavas act as conduits for
groundwater flow and the intervening ashes provide the storage, characterize the
important aquifer systems of Costa Rica, Nicaragua and El Salvador.
Weathered basement complex
These aquifers are found in ancient crystalline rocks of Precambrian or Lower Palaeo20ic
age. In sub-Saliaran Africa, such rocks cover 40 per cent of the total land area and some
220 million people live on them. Groundwater flow and storage occurs in restricted
fractures in the fresh bedrock, but usually more extensively in the superficial weathered
layers. The processes of weathering and disaggregation can enhance both porosity and
permeability (Chilton and Foster, 1995). Because these ancient rocks occupy stable,
Groundwater occurrence and hydrogeological environments 43
continental shield areas, there has been plenty of opportunity for prolonged periods of
weathering, and the zone of weathering tends to be better developed and thicker in
tropical regions where such processes are more active. As a result, the weathered zone
can be as much as 60 m thick, but more commonly in the range of 20 to 30 rn
Groundwater velocities in weathered and fractured bedrock aquifers can be very variable.
Even with the beneficial effects of weathering, the volumes of water stored within
these aquifers are generally limited, hydraulic conductivities are low, borehole yields
modest and groundwater is used mostly for providing potable water supplies for rural
communities and small towns, and for small-scale supplementary irrigation Larger cities
located on such formations, such as Kampala in Uganda, may find it difficult to abstract
the large quantities of water needed for urban supply and also to dispose of wastewater
on-site to the subsurface in a sanitary manner.
Table 2.3. Summary of characteristics of principal hydrogeological environments
Hydrogeological envlrooment Lithology Geological description and origin Groundwater flow Natuml groundwater
~:glme flow mies jm/d )
Major alluvial and coastal plain sediments Gravels, sands, silts Unconsolidated deposits of major rivers, deltas and shallow seas, high lnterb'lllI1Ular 2-IO in b'lllVels, 0.05-1 in
and clays primary porosity w,d penneability , Very extensive and thick aquifers sands, 0.001-0.1 in silts
lntennontw,e alluvial and volcanic systems Pebbles, gravels, Rapid infilling of faulted troughs wid basins in mountain regions; Interb'lllI1Ular, fiacture in 0,001-10
sands and clays, unconsolidated, primary porosity and/or penneability usually high for lavas and cemented ashes
sometimes inter-fw, sediments and ashes, but lavas wid lacustrine deposits are often
bedded with lavas poor aquifers or confining aquitards. Less extensive but can be thick
and volcanic ashes
Consolidated sedimentary aquifers Sandstones Marine or continental deposits buried, compacted and cemented to lntergmnular and fracture 0.001-0 .1
form consolidated rocks; degree of consolidation generally increases
with depth/age of deposition. Primary porosity moderate to poor but
secondary porosity introduced by fractures can be significant
Limestones Deposited ftom skeletal material (shell ftagments, reefs, reef detritus) Dominant ftacture with 0.001-0.1 in matrix, upto
in shallow seas w,d compacted to form consolidated rocks; often have variable intefb'lllI1Ular !000 in karst fissures
fractures which may be enlarged by solution processes to form component
characteristic topOb'lllPhY, cavities and tunnel systems known as karst
Recent coastal calcareous fonnations Limestones and Usually composed of com! limestones, shellbanks, chemically lntefb'lllI1ular and fracture 0.01-0.1 inmatrix,upto
calcareous sands precipitated oolites w,d calcareous oozes; often loosely cemented; 2000 in karst
porosity and permeability can be exceptionally high, especially if
fiactures are solution-enhanced
Hydrogeological environment Lithology Geological description and origin Groundwater flow Natural groundwater
n:11lme flow rates (mid !
Glacial formations Boulders, pebbles, Ice-transported sediments are commonly unsorted and have low lnterb'1'1111Ular 0.001-0.1 in tills, much
gravels, sands, silts penneability, but water-sorted sediments such as melt-water and higher in sands and
and clays outwash deposits often have high porosity and penneability. Can be b'TIIVels
thin, patchy and shallow
Loessic plateau deposits Silts, fine sands Usually well-sorted windblown deposits of silt and fine sand, with Intergranular 0,001-0.01
and sandy clays some sandy clay deposits ofsecondacy tluvial origin; low
penneability. Can be extensive and thick but divided into blocks by
deep gullies
Extensive volcanic terrains Lavas, tuffs and Extensive basaltic lava flows or ashes and luffs from more explosive Fracture with variable 0,001-10
ashes eruptions. Primacy porosity only in ashes and the less welded tuff's intergranular
and at junctions oflavas . Joints and fractures in lavas. Variable
potential , decreasing with age
Weathered basement complex Crystalline rocks, Decomposition of older igneous or metamorphic rocks can produce a Dominantly intergranular 0.001-0 .1
granites, gneisses, weathered mantle of variable thickness, moderate porosity but in weathered zone, fi'acture
schists generally low penneability, underlain by fresher rock which may be below
fractured; the combination results in a low potential but important
very wides pread but shallow uq uifer system
46 Protecting Groundwater for Health
2.6 REFERENCES
Because many of the basic concepts about groundwater occurrence and movement are
likely to be new to many of the readers of this monograph, and this introductory
discussion may not have done them justice, the following three standard texts can be
used to provide additional material if required:
Domenico, PA and Sdlwartz, F.W. (1998) Physical and Chemical Hydrogeology, 2nd edn, John
Wiley, New York
Freez:e, RA and Cheny, J.A (1979) Groundwater, Prentice Hall, Englewood Cliffs, New Jersey.
Price, M. (1996) Introducing Groundwater, 2nd edn, Chapman and Hall, London.
Additional references quoted in the chapter:
Alderwish, AM and Dottridge, J. (1999) lhban recharge and its influence on grmmdwater quality
in Sana'a, Yemen. In Groundwater in the Urban Environment: Selected city profiles, (ed. P J.
Chilton), pp. 85-90, Balkema, Roua-dam.
Barraclough, D., Gardner, CM.K., Wellings, S.R and Cooper, J.D. (1994) A tracer investigation
into lhe importance of fissure flow in lhe unsatu:raled zone of 1he British Upper Chalk J.
Hydrology, 156, 459-469.
Becker, MW., Reimus, P.W. and Vilks, P. (1998) Transport and attenuation of carboxylate-
modified latex miaospheres in fractured rock, laboratory and field tracer tests. Ground Water,
37(3), 387-395.
Chilton, P J. (1996) Grolllldwater. In Water Quality Assessments: A guide to the use of biota,
sediments and water in environmental monitoring, 2nd edn, ( ed. D. Chapman), pp. 413-5 IO, E
& FN Spon, London.
Chilton, P J. and Foster, S.S.D. (1995) Hydrogeological chm-actemation and wata supply potential
ofbasement aquifers in tropical Africa Hydrogeology J., 3, 36-49.
Driscoll, F.G. (1986) Groundwater and Wells, 2nd edn, Johnson Division, St Paul, Mirmesota.
Edmunds, W.M. and Gaye, C.B. (1994) Estimating lhe spatial variability of groundwater recharge
in lhe Sahel using dlloride. J. Hydrology, 156, 47-59.
Edmllllds, WM. and Smedley, P.L. (2000) Residmce times indicators in groundwater: lhe East
Midlands Tril!Sfilc sandstone aquifer. Appl Geochem., 15, 737-752.
European Community 2000: Directive 2000/60/EC oflhe European Parliament and oflhe Council
of 23 October 2000 csablishing a framework fur Comnnmity adion in 1he field of water
policy, Official Journal of the European Communities, 1.327, Brussels.
Foster, S.S.D., Chilton, P J., Moench, M., Cardy, F. and Schiffler, M (2000) Groundwater in Rural
Development: facing the challenges of supply and resource sustainability, World Bank
Technical Paper No. 463, Washington, DC.
Foster, S.S.D., Lawrence, AR and Morris B.L. (1998) Groundwater in Urban Development:
assessing management needs and formulating policy strategies, World Bank Technical Paper
No. 390, Washington, DC.
Geake, AK. and Foster, S.S.D. (1989) Sequential isotope and solute profiling oflhe WJSatmated
zone oflhe British Chalk Hydrological Sci. J., 34, 79-95.
Groundwater occurrence and hydrogeological environments 4 7
Gbassemi, F., Jakeman, AJ. and NIX, HA (1995) Stalini::ation of Land and Water Resources,
University ofNew Soulh Wales~ Sydney.
Kass, W. (19'J8) Tracing Techniques inGeoh;drology, Balkema, Rottmlam.
Kendal, C. and McDonnel, JJ. (19'J8) Isotope Tracers in Catchment Hydrology, Elsevier,
Amsterdam.
Kbadka, MS. (19'Jl) Nepal: Groundwater Quality. In Groundwater Quality Monitoring in Asia
and the Pacific, UN-ESCAP, Water Resources Series No 70, Bangkok
Lerner, D.N. (19'J7) Too much or too little: Recharge in mban areas. In Grmmdwater in the Urban
EnvironmenJ: Problems, processes and managemenJ, (eds. P J. Chilton et al.) pp. 41-47,
BalkCID8, Rottadam.
Lerner, D.N., lssar, A and Siimners, l (1990) Groundwater Red,arge: A guide to um:lerstanding
and estimating natural red,arge, Heisse, Hannover.
Lvovitch, M .I. (1972) World water balance: general report. In IAHS/UNESCO/ WMO Proc. of
Symposium on World Water Balance, Reading, 1970, pp. 401-415, IAHS, Wallingford.
Malaro, F., Reygrobellet, J-L., and Soulie, M (1994) Transport and reten1i!Jn offuecal bacteria at
sewage-polluted fractured rock sites. J_ Environ. Qual., 23, 1352-1363.
Meybeck, M, Chapman, D.V. and Helmer, R (eds.) (1989) Global Freshwater Quality: A first
~ent, GEMS Global F.nviromnmt Monitoring System, WHO/UNEP, Blackwell,
Oxford
Morris, B.L. Lawrwce, AR, Clriltoo, P J~ Adams, B., Calow, R and Klinck, BA (2003)
Groundwater and its Suscepnbility to Degradation: A global assessment of the problem and
options for management EadyWamingRq,ortSeries, RS 03-3, UNEP, Nairobi
Nace, RL. (ed) (1971) Scientific Frameworlc of World Water Balance, Technical Papers in
Hydrology 7, UNESCO, Pim.
NRC (19'J5) Mexioo City's Water Supply: Improving 1he outlook fur sustainability, NRC, National
Academy Press, Washington, DC.
Or1h, J.P., Nellel", R and Melkl, G. (1997) Bacterial and diemical contaminant transport tests in a
confined karst aquifer (DamJbe Valley, Swii>ian Jura, Germany). In Korst Water and
Environmental impacts, (eds. G. Gunay and AL Johnson) Ba1kema, Rottadam.
Seiler, K-P. and Lincner, W. (19'J5) Near surface and deep groundwater. J. Hydrology, 165, 33-44.
Todd, DK (1980) Groundwater Hydro/ogy,2ndedn,Jolm Wiley,NewYorlc.
UNESCO (199'J) World Water Resources at the Beginning of the 21st CenJury, CD version,
UNESCO, Paris.
US EPA (19'J7) Guidelines for Wellhead and Spring Protection in Carbonate Rocks, EPA 904-B-
97-0003, Washington, DC.
Wellings, S.R (1984) Recharge of the uppa chalk aquifer at a site in Hampshire, England J.
Hydrology, 69, 275-285.
3
Pathogens: Health relevance,
transport and attenuation ·
S. Pedley, M Yates, J.F Schijven, J. West,
G. Howard and M Barrett
This chapter will summariz.e current knowledge about the distnbution of pathogens in
groundwater and the fuctors that control their transport and attenuation The aim is to
provide a level of information and interpretation that will allow public health specialists
and water resomce managers to estimate risks to the groundwater from microbial
contaminants derived from sources descnbed in Section II of this monograph, for
example, agricultural and urban sources.
Many factors, some environmental and others linked to the properties of the
organism, control the survival and transport of microorganisms in the subsurface.
However, it is important to consider that often the factors perceived to be of importance
have been studied in isolation using controlled laboratoiy experiments and the
cooclusiom then extrapolated to predict the fate of pathogens in the environment This
process is known as tq>SCaling and is itself the sd:Jject of current research In contrast,
veiy few studies have attempted to examine the effect of multiple factors interacting in
the natural environment Current knowledge therefore offers a mnnber of guiding
principles about the transport and attenuation of pathogens in groundwater, but the
<O 2006 World Health Organmition. Protecting Groundwaler for Health: Managing the Quality of
Drinkinr,water Sources. Edited by 0. Schmoll, G. Howard, J. Chilton and I. Chorus. ISBN:
I 843390795 . Published by IW A Publishing, London, UK.
50 Protecting Groundwater for Health
complex interaction of factors controlling the fate of pathogens is poorly understood and
difficult to predict in some environments.
DEF ► Mu:roorganisms are microscopic organisms within the categories
algae, bacteria,fangi, protozoa, viruses and subviral agents
(SingletonandSainsbwy, 1999).
Pathogens are aey microorganisms which by direct interaction with
(iefection of) another organism cause disease in that organism
(Singleton and Sainsbwy, 1999). The strict definition of a pathogen
excludes those microorganisms that cause disease indirectly by the
synthesis of a toxin that may subsequently be ingested by the victim
Several microorganisms implicated in food poisoning cause disease
in this wey: Clostridium perfringens and Staphylococcus aureus are
examples of this group. Nevertheless, these microorganisms can be
pathogens in the strict sense under different conditions.
3.1 MICROBIAL PATHOGENS AND MICROBIAL
INDICATOR ORGANISMS
The ability of a pathogen to inflict damage upon the host is controlled by a combination
of factors, in particular the nature of the organism (for example its virulence) and the
susceptibility of the host Several factors combine to determine the suscept:Ibility of the
host, including age, nutritional status and immunity. Immunocompromised individuals,
for example, are highly susceptible to infection by pathogens, whereas well-nourished
young adults are typically less susceptible to infections.
DEF ► Vmdence is the capacity ef a pathogen to cause disease, defined
broadly in terms ef the severity of the symptoms. lnfectivity is the
ability of a pathogen to become established on or within the tissue of
a host.
Water can be the vehicle for the transmission of many different types of pathogenic
microorganism: some being natural aquatic organisms and some being introduced into
the water from an infected host. Overall, the pathogens in water that are the main concern
to public health originate in the faeces of humans and animals, and establish an infection
when contaminated water is consumed by a susceptible host (Boxes 3.1 and 3.2). These
are the classical waterborne pathogens (Table 3.1) that are transmitted by the faecal-oral
route of infection (Figure 3.1). Waterborne pathogens can be classified into four broad
groups according to their chemica1, physical and physiological characteristics. Listed in
Pathogens: Health relevance, transport and attenuation 51
order of increasing functional complexity the groups are viruses, bact.eria, protozoa and
helminths. In general, the transmission ofhelminths in groundwater is unlikely, although
not impossible, due to the siz.e of the organisms and their eggs. For this reason, and
because public health concerns surrotmding waterborne disea<.e transmitted through
groundwater have concentrated upon the other groups of microorganisms, helrninths will
not be discussed in this chapter. Within the other groups a large mnnber of microbial
pathogens are able to contaminate groundwater (fable 3.1 ).
Box 3.1. Health impacts of contaminated groundwater in Walkerton, Canada
(based on Howard, 2001)
In May 2000, 6 people died and over 2000 others became ill in the small town
of Walkerton, Canada, as a result of consuming groundwater contaminated by
E coli Ol57:H7 and Campylobacter. Walkerton was almost entirely deperxient
upon groundwater for domestic supply obtained fiom production wells varying
fiom 15 to >70 m deep. 1hese wells interc.ept limestones and dolomites. There
was a history of detection of E coli in a number of the wells, but this problem
was regarded as being something that could be controlled by routine
chlorination of the source water.
The first public health problems (di~ vomiting) were noted in the days
following a particularly violent storm during which 100 mm of rain fell. Initially
the problem was suspected as food poisoning and it was not until over a week
later that well water was identified as the source and a boil-water alert was
issued. E coli 0157:H7 and Campylobacter spp. were subsequently identified
as the cause of the deaths. The source of the organisms was traced to a cattle
farm close to one of the shallower production wells. It was initially suggested
that storm water runoff conveyed the contaminants to the production wells.
However, subsequent evidence indicates that the well contamination causing the
outbreak had occurred before the storm event. The precise travel paths enabling
the contaminants to enter the production wells ( even the deepest of which was
contaminated) remain a matter of speculation, although fissure flow in the
limestone aquifer is an obvious candidate. It may be significant that there were
abandoned production wells in the area that had not been properly sealed.
Improperly abandoned boreholes may facilitate the rapid vertical mixing of
contaminants entering at or near the surfuce.
It is clear that the public health impact was a result of the combination of
inadequate protection and monitoring of the groundwater resource with a failure
in treatment This is not uncommon, Bramham in the United Kingdom being a
further example (Lerner and Barrett, 19%). Clearly reliance on treatment (as a
'single barrier' approach) without other measures such as adequate groundwater
protection is a higher risk approach than that of the 'multi barrier'.
The types and numbers of the various pathogens will vary temporally and spatially
depending upon the incidence of disease in the community, the known seasonality of
human infections, and the characteristics of the aquifer systems. Furthennore, the
microbial illnesses and the severity of the disease vary marlcedly with the organism.
52
NOTE ►
Protecting Groundwater for Health
Although some enteric pathogens may circulate within a population
all year round, maey have a clearly defined seasonal distribution
For example, in temperate zones, the transmission qf rotavirus takes
place almost exclusively during cold weather.
Box 3.2. Vrrus oome outbreaks of gastroenteritis in Wyoming, USA
In Februaiy 2001, episodes of acute gastroenteritis were reported to the
Wyoming Department of Health among snowmobilers (Anderson et al, 2003).
The outbreak was believed to have been caused by noroviruses that could be
identified from 8 of 13 stool samples as well as from growxlwater samples of
one well A second outbreak of acute gastroenteritis oc.curred in Wyoming
during October 200 I among persons who dined at a tourist saloon (Parshionikar
et al, 2003). A norovirus strain (genogroup I, subtype 3) was fowxl in stool
samples from three ill persons as well as in the water from the saloon's only
well.
Although land use planning, water resource management, water treatment and
disinfection are used to control the transmission of waterborne pathogens, the safety of a
water source is frequently verified by testing for the presence of microbial parameters. A
variety of pathogens may be present., and the different methods that are required to isolate
each pathogen prolubit the direct examination of water samples for pathogens on a
routine basis. Moreover, pathogens may not be detected due to low levels, or the lade of
an appropriate detection method, but they may still be present at a density that represents
an unacceptable level of risk. Ftnthermore, currently unknown and therefore
undetectable pathogens may be present To overcome this difficulty, a separate group of
microorganisms is used as an indicator for the potential presence of pathogens. The
common descriptive term for this group of organisms is faecal indicator organisms.
Gleeson and Gray (1997) have published a thorough review of the application of faecal
indicator organisms in water quality monitoring that may be consulted for further
information. This group comprises the following:
• total coliform bacteria
• thermotolerant coliform bacteria
• E coli
• fuecal. streptococci
• bacteriophage
Pathogens: Health relevance, transport and attenuation 53
Table 3.1. Pathogenic microorganisms of concern in groundwater (adapted .from Mader and
Merkle, 2000)
Or!!anism
Viruses
Coxsackievirus
F.chovirus
Associated health effects
Fever, pharyngitis, rash, respiratmy disease, diarrhoea,
haemorrhagic conjunctivitis, myocarditis, pericarditis, aseptic
meningitis, encephalitis, reactive insulin-dependent diabetes,
hand, foot and mouth disease
Norovirus (formerly NOIWalk
virus)
Respiratory disease, aseptic meningitis, rash, fi:ver
Gastroenteritis
Hepatitis A
Hepatitis£
Rotavirus A and C
Enteric adenovirus
Calicivirus
Aslrovirus
Bacteria
&cherichia coli
Salmonella spp.
Shigella spp.
Campylobacter jejuni
Yersinia spp.
Legionella spp.
Vibrio cholerae
Fever, nausea, jaundice, liver :fuilure
Fever, nausea,jaundice, death
Gastroenteritis
Respiratory disease, haemorrhagic conjunctivitis, gastroenteritis
Gastroenteritis
Gastroenteritis
Gastroenteritis, Haemolytic Uraemic Syndrome (enterotoxic E
co/1)
Enterocolitis, endocantitis, meningitis, pericarditis, reactive
arthritis, pneumonia
Gastroenteritis, dysentay, n:active arthritis
Gastroenteritis, Guillain-Barre syndrome
Diarrhoea, n:active arthritis
Legionnaire's disease, Pontiac fi:ver
Cholera
Protama
Cryptosporidium parvum
Giardia lamblia
Dianhoea
Chronic dianhoea
NOTE ► The coliform group of bacteria consists of several genera belonging
to the family &terobacteriaceae (Gleeson and Grqy, 1997). The
relatively limited number of biochemical and physiological
attributes used to define the group (including growth at 3 7°C)
means that its members include a heterogeneous mix of bacteria.
Although the total coliform group will include bacteria of faecal
origin it also includes species that are found in unpolluted
environments. Thermotolerant coliforms are those bacteria from
within the total coliform group that grow at 44°C. E. coli is a
thermotolerant coliform.
54 Protecting Groundwater for Health
Figure3.1- Principal demons ufthe kcal -oral route ufdisease transmission (Howard el al. 2002)
An ideal microbial indicator of faecal pollution is easily detected, always present in
faecal waste. and is more durable in the environment than most enteric pathogens_ ft
should comprise a large parentage of the organisms in faecal waste, exceed the numbers
of most enteric pathogens and be roughly proportional to the degree of pollution_ Lastly,
because indicator organisms should be absent unless faecal contamination is present,
they should ideally not be present in drinking -water that is microbially safe for
consumption_ For many applications. 100 mt of water is used as lie standard volume for
analysis.
The durability of faecal indicator organisms in the subsurface is an important issue.
that has a direct bearing on the interpretation of water quality data Ideally the teal
indicator organism should be removed from the environment at a slower rate than the
most durable pathogen so that its potential for dispersal is greater. In practice, this
situation will seldom arise. For the purpose of acting as surrogates for pathogens in
groundwater tracer studies, other advantageous properties are the ease of preparing high
numbers of the indicator and the ease of enumerating the indicator. These properties
allow indicators to be used as a tool to identify and quantify removal xec ses in
laboratory and field studies_ The most important removal processes are inactivation or
die -of. adsorption to the surface of the grains of the porous medium and physical
filtration or straining when pore throats are too small to let a microorganism pass. Thus

Pathogens: Health relevance, transport and attenuation 55
robust indicalors are inactivated, adsorbed and strained not more than the pathogens they
represent The removal processes are descnbed in more detail later in this chapter.
It is acknowledged that :raecal indicator bacteria do not give absolute resolution to the
preseocelabsence of pathogenic protozoa, bacteria and viruses. These diverse groups of
microorganisms have highly variable transport and attenuation characteristics that cannot
be wholly represented by the small group of indicator bacteria However, the density of
faecal indicator bacteria does provide a measure of probability of the presence of
pathogens.
Faecal indicator bacteria are of limited use for predicting the presence or absence of
viruses as viruses often survive significantly longer in groundwater systems. It is not
practical to sample and analyse for a1I pathogenic viruses. As a result bacteriophage -
viruses that infect bacteria -fiom :raecal sources are often used to indicate the likely
presence or absence of viruses in groundwater. Several groups of bacteriophage have
been evaluated as indicator organisms a11hough the focus of attention has been upon the
coliphage group of viruses because they can be detected and quantified using relatively
simple ana1ytica1 methods.
Negatively charged bacteriophages. like MS2 and PRDl, have been found to be
useful model viruses and have been used in many field and laboratory studies on
subsurfuce transport of viruses (Schijven and Hassanizadeh, 2000). They attach poorly to
most soils, and at low temperatures they inactivate at a low rate. PRDl is relatively
insensitive to higher temperatures or extreme pH These bacteriophages have the same
size and shape as many waterborne pathogenic viruses. They are easy to prepare in high
mnnbers and are easy to detect MS2 is an F-specific RNA bacteriophage. This group of
bacteriophages may be naturally present in :raecal contaminated water in numbers 1 <i to
1 O" times higher than enteroviruses (Havelaar et al., I 993). However, F-specific
bacteriophages are less stable (more temperature sensitive) than somatic coliphages. The
latter are usually found in higher number in faecally contaminated water and are
therefore a1so useful indicators.
3.2 DISTRIBUTION OF PAfflOGENS AND FAECAL
JNDICATORS IN GROUNDWATER
Sources of faecaJ contamination in groundwater are discussed in Section Il and
potentia1Iy include leakage from on-site sanitation systems or sewers (Box 3.3), anima1
manures (Box 3.1), wastewater or sewage sludge applied in agriculture. 1be sources can
be classified according to their origin_ A point source has an identifiable source, such as a
leaking septic tank, which may result in a welHk:fined plume. More difficult to control,
and posing a greater risk to groundwater quality, are non-point sources. Non-point
sources are larger in scale and produce relatively diffuse pollution originating from either
widespread application of contaminated material or many srna1Ier sources. The aggr~e
of point sources in a leaking sewerage system may, overal1, represent a non-point source
of contamination to groundwater (usually descnbed as multi-point source of pollution).
TraditionaJly, hydrogeologists, and many public health scientists, have reg,ll'ded
groillldwater as a relatively microbia1Iy safe source of drinking-water. Unlike surface
waters, which are vulnerable to direct contamination from many sources, groundwater is
56 Protecting Groundwater for Health
often shielded fium the immediate influence of contamination by the overlying soil and
unsaturated zones as descnbed in Chapter 2. In these zones, pathogenic microorganisms
have been assumed to be attenuated by the prevailing physical. chemical and biological
conditions in the environment The risk of pathogens being transported into growidwater
and producing a threat to public health was, therefore, considered to be low.
Consequently, many groundwater sources are used for public supply with a minimum
level of treatment, normally chlorination, or with no treatment at all.
The underlying rationale for restricted dispersion in the subsurface has some merit,
but it is now known that microbial contamination of growidwater is more widespread
than previously believed. Indeed, as was found with chemical contaminant studies,
particularly in the 1980s, the more that it is looked for, the more it can be fmmd Tables
32 and 33 list some examples of studies that have demonstrated the occurrence of mecal
indicators and enteroviruses in groundwater. Although there may be a bias in some of the
studies, created by the deliberate selection of vulnerable sit.es, the data show that a
significant percentage, up to 70 per cent in some regions, of groundwater sources contain
one or more of the microbial indicators of mecal oontamination.
Table 3.2. Occum:oa: of microbial fuecal indicators in groundwater
Organism Proportion Study location Reference
of receptors
positive (%)
Colifonn bacteria IO USA: 445 public supply wells Abbaszadegan
Coliphage 21 USA: 444 public supply wells eta/., 1998
Enterococci 9 USA: 355 public supply wells
Somatic colipbage 50 USA: 30 public water supply wells Lieberman et al.,
Eroli 50 judged to be vulnaable to filecal 1994
Enterococci 70 contamination
Colifonn bacteria 40 USA, Montana Bauder eta/., 1991
Ecoli 16-24 Canada, Provina: of Ontario: Goss et al., 1998
Faecal streptococci 12-24 fimnstt:ad domestic wells
Ecoli 60 Republic ofMoldova, Ba1atina Melian et al., 1999
Faecal streptococci 50 andCaipini
Thamotolerant 10-40 Finland: rural wells Kmhonen et al.,
coliforms and filecal 1996
streplOCOCCi
Table 33 shows the OCC!B'fence of pathogenic enteroviruses fowid in public water
supply wells in the USA as defined by the Safe Drinking Water Act (US Government,
1996). Hydrogeological data fium these studies were available on aquifer type as part of
the study designs and were reviewed for accuracy by hydrogeologists. Limestone (karst),
fractured bedrock (igneous and metamorphic rocks) and gravel (formed in high energy
depositional environments with little or no sand or other fine grained materials) aquifers
are defined as sensitive aquifers under the proposed Gmwid Water Rule (US EPA,
Pathogens: Health relevance, transport and attenuation 57
2000). If a public water supply well draws water fium a sensitive aquifer, then the State
must find the well sensitive to faecal contamination unless a hydrogeological oorrier
protecl'i it. A hydrogeological barrier is defined as the physical, biological and chemical
factors, singly or in combination, that protect a well fium pathogenic organisms. In this
proposal, a confining layer is one example of a hydrogeological oorrier. If a
hydrogeological barrier is present, then the State can nullify the determination that a
system is located in a sensitive aquifer. If no suitable hydrogeological oorrier exists, then
the proposed Grmmd Water Rule requires the system to conduct fuecal indicator source
water monitoring.
Table 3.3. Occurrence of enternviruses in public water supply wells in the USA
Sensitive wells• NIIIHellSitive Samples Average Vuus type (N.) • Rdereoce
wdk per well filtered
lrlNv Pruitive N 'lNv Positive volume (L)
3/49 6% 2110 20% 200-1000 CoxsackievirusB5(1) LindseyetaL,2002
F.chovirus 13 (1)
0,9) 0% 0.0
0/0 oam 0"/4
0/92 0"/4 0/12 0"/4
11% 1% 0,9 0"/4
11% 1% 0,9 O"/o
6/12 50"/o 1/18 6%
1500
1 1500
1 200-300
2 200-300
2 200-300
12 6000
F.cbovirus 20 (1)
Poliovirus3(l)c
Roovi1Us(3)
Banksand
Bauigelli, 2002
Ro1avirus (1) • Banks et aL, 200 I
Femmer et aL. 2000
Poliovirus I (l)c Davis and Witt,
2000
Poliovirus 1 (l)c Davis and Witt,
2000
CoxsackievirusA7(1) LiebermanetaL,
Coxsackievirus Bl (4) 2002
Coxsackievirus B3 (I)
Coxsackievirus B4 (5)
Coxsackievirus BS (1)
F.chovirus 11 (2)
F.cbovirus 15 ( 4)
F.cbovirus 18 (2)
F.chovirus21 (3)
F.chovirus 24 (2)
0/31 0"/4 On9 O"lo 4 Up tol500 Dobertv etaL,1998
N'" = number of positive enteroviruses by buffillo-gn:m-monkey tis&le ru1ture; N,.. = number of
wells; a = sensitive wells are wells located in igneous or metamoJphic rock aquifers or limestone
aquifers or gravel aquifers with very low sand/fine grained content; b = serotyping results to
oonfirm cell rulture entaic virus positive samples (serological idmtification of all these samples
was determined by Dan Dahling of the US EPA); c = pos.gble laboratoJy contamination;
d = positive by Rototest assay of RD cell lysate.
Lindsey et al. (2002) fmmd enteric viruses in three sensitive aquifers and two that
were not sensitive. In Tables 32 and 3.3, the data of Lieberman et al. (2002) are most
meaningful because 12 samples were taken at monthly intervals fium each well from 30
sites in the continental USA, the Vrrgin Islands and Puerto Rico. In addition, the sample
volumes used in these studies were significantly larger than routine volumes: average
58 Protecting Groundwater for Health
6000 litres, and maximum even up to 15 000 litres. This significantly increases the
probability of detecting virus contamination of a well. Lieberman et al. (2002) found
enteric viruses primarily in sensitive aquifer.; (i.e. karst, fractured bedrock and ooarse
gravel) but only once in a very shallow sand well (5 m deep; see also Dahling, 2002).
One reason that as many as 7 of the 30 wells (24 per cent) were contaminated with
enterovirus is because almost all wells were selected for enterovirus sampling only if
they had a history of total coliform and somatic coliphage occurrence (see Table 3.2).
Echovirus 11 was possibly corning from upgradient septic tanks. Viruses found in the
karst wells must have travelled a long distance because no potential sources of
contamination were near. One of the wells was located in a populated gravel flood plain
with a nearby trailer park with septic tanks about 30 m away. fu this well water,
coxsackieviruses A7, Bl and B4, echoviruses 15, 18, 21 and 24 and reovinis were
detected. Coxsackievirus B4 was also found in the wat.er from a well in fractured basalt
with S<!pl:ic tanks nearby.
Borchardt et al. (2003) stufied the inciderx:e of viruses in WISCOnsin, USA private
household wells locat.ed near seepage land application sites or in rural subdivisions
served by septic systems. Fifty wells in seven hydrogeological areas were sampled four
times over a year, once each seasoIL Of the 50 wells, 4 (8 per cent) were found to be
positive for viruses using analytical methods that detect the presence of the viral nucleic
acid (reverse transcription PCR). Of these, three wells were positive for hepatitis A virus
and the fourth well was positive for rotavirus and norovirus in one sample and for
enterovirus in another sample. Culturable enteroviruses were not detected in any of the
wells. Virus occurrence could not be associated statistically with fuecal indicators (i.e.
total coliforms, £ coli, fuecal enterococci, F-specific RNA bacteriophages).
Clearly, the common perception that groundwater is per se a microbially safe source
of drinking-water is inaccurate. Whilst usually the microbial contamination of
groundwater is likely to be orders of magnitude lower than that of surface waters, it is
now apparent that a significant percentage of groundwater sources are contaminated by
microorganisms derived from faeces. As shown above, the known presence of infectious
pathogenic viruses in some wells, for example, represents an unequivocal message that
viruses are mobile in the subsurface, long-lived and capable of causing waterborne
illness.
fu the same way that surface waters may show rapid changes in the concentration of
pathogens, distribution and concentration of pathogens and faecal indicator bacteria in
groundwater sources is not static but also demonstrates fluctuatioIL Temporal and spatial
variations are frequently observed that may be linked to seasonal changes in land use and
changing weather patterns. For example, fluctuations in the levels of thermotolerant
coliforms have been observed in proximity to wastewater irrigation sites in Mexico and
in the United Kingdom. The distribution of enteric bacteria and viruses in urban
groundwater has been observed to vary both horizontally and vertically (Box 3.3).
Pathogens: Health relevance, transport and attenuation 59
Box 3.3. Depth and extent of microbial contamination of groundwater in British
urban sandstone aquifers (based on Powell et al., 2000; 2001a; 2001b)
There are few published data on the microbial, and particu1arly viral, quality of
United Kingdom groundwaters. This should not be taken as an indication of a
general absence of contamination. rather as a lack of detailed monitoring
studies. A nwnber of workers have identified sewage contamination of the
Triassic Sandstone aquifers in urban areas of the United Kingdom, derived fium
leaking sewers.
Powell and co-workers set out to determine the extent and penetration of
microbial contaminants in the Triassic Sandstone aquifer underlying
Birmingham and Nottingham in the United Kingdom. Five multilevel
groundwater monitoring devices were installed into the aquifer, providing a total
of some 50 depth-specific sampling points. Viral monitoring was undertaken
using a glass wool trap for the concentration of ent.eric viruses fium large
volwne groundwater samples.
The field data lead to four key findings:
-Sewer leakage-derived microbial contaminants are able to penetrate
sandstone aquifers to significant depths (>90 m).
-Hwnan enteric viruses, including pathogenic species are widespread in the
aquifer.
-The species of sewage-derived hwnan enteric viruses in groundwater are
found to vary temporally, and in parallel with their predicted prevalence in
the population. The dominant types found in March and June 200 I were
Noroviruses and Coxsackievirus B4 respectively.
-Particular horizons at depth within the sandstone aquifer were found to be
rapidly suscept.Ible to microbial contamination (i.e. contaminant distnbution
is vertically and temporally heterogeneous).
-The Triassic Sandstone aquifer ( and, by implication. other similar sandstone
aquifers around the world), the second most · important in the United
Kingdom, is far more vulnerable to microbal contamination than previously
assumed. This has public health implications where groundwater is
conswned without adequate treatment
Frequently, the microbial quality of shallow groundwater sources, including springs,
will deteriorate after heavy rainfall as surfuce contamination is washed into the source
directly and organisms in the WISaturated z.one are mobiliz.ed by water percolating
through the soil matrix (Box 3.1 ). Similarly, the relative proportions of indicator bacteria
and pathogens, and of bacteria and viruses, will fluctuate such that the dominant species
isolated at one time may be absent on subsequent sampling occasions (Box 3.4).
60 Protecting Groundwater for Health
Box 3.4. Temporal fluctuations in microbial contamination of groundwater in
Kampala, Uganda (based on Barrett et aL, 2000)
Sampling of 15 protected springs was undertaken in areas of high -density and
low -density population (peri-urban) in Kampala, Uganda an three successive
days (28-30 September 1999). The wet season was in progress, and rainfall
occurred on every day of sampling. On the night of 28-29 September, a
significantly heavier rainfall event took place. Analysis ofthe samples for faecal
indicators. including thermotolerant colifonns and faecal strept cocci, was
undertaken. As shown by Figure 3.2, there was a clear pulse reaction of
contamination in spring water within 12 hours ofthe major rainfall event
A variety of metasedimentary rucks (e.g. quartzites and phyllites) underlie
Kampala- Differential weathering has resulted in a pronounced topography with
thin weathered mantles of limited extent containing shallow (2-20 m)
groundwater flow systems that discharge to valley springs. These are fed by a
axnbination of baseflow and seasonally derived interflow. Many of these
springs are used, untreated, by lower income communities with limited access to
higher service levels of piped water supply_ Clearly the monitoring of protected
springs for microbial contaminants in these localized groundwater flow systems
must be interpreted in the context of rainfall events.
4
1231 SE 1891111u.75x151 2315676901117
Figure 3.2. Effect ofhcavy rainfall on the microbial quality of spring water in Kampala,
Uganda
3.3 TRANSPORT AND ATTENUATION OF
MICROORGANISMS IN THE UNDERGROUND
Some, perhaps many, instances of grotaidwate r receptor amtamination will occur by
rapid transport pathways accidentally introduced by human intervention and connecting
the contamination source to the groundwater abstraction point Such pathways could

Pathogens: Health relevance, transport and attenuation 61
include inadequate sanitary completion of springs, wells and boreholes, the presence of a
forgotten conduit connecting the source of contamination to the groundwater abstraction
point, or voids and fractures in soils that allow direct ingress of contaminated materials.
The implementation of management actions to reduce faecal contamination close to the
abstraction point or the rehabilitation or improvement of the well or spring is usually
sufficient to control access of pathogens to the water source (Section IV).
Rapid transport pathways cannot, however, explain all groundwater source
contamination events and it is now widely accepted that the transport of microbial
pathogens within groundwater systems is a significant mechanism for waterborne
disease transmissioIL The remainder of this chapter deals with the factors that control the
transport and attenuation of pathogens into and through groundwater.
3.3.1 Transport and attenuation of pathogens in the unsaturated
zone
Hydrogeological processes in the unsaturated z.one are complex and the behaviour of
microorganisms is often difficult to predict Nevertheless, the unsaturated :wne can play
an important role in retarding (and in some cases eliminating) pathogens and so must be
considered when assessing aquifer vulnerability, as descnbed in Chapter 8. Attenuation
of pathogens is generally most effective in the uppermost soil layers where biological
activity is greatest The presence of protoz.oa and other predatoty organisms, the rapid
changes in soil moisture and temperature, competition from the established microbial
community, and the effect of sunlight at the surface combine to reduce the level of
pathogens within this z.one. The effect of individual environmental factors will be
discussed in a later section of this chapter.
The transport of pathogens from the surface into the subsurface requires the presence
of moisture. Even during relatively dty periods, soil particles retain sufficient moisture
over their surface for pathogens to migrate downwards into the subsurface. Under these
conditions the main driving forces will be sedimentation, diffusion and bacterial motility.
Wrthin the thin film of moisture the organisms are brought into close contact with the
surface of the particle, thus increasing the opportunity for adsorption to the particle
surface and funher retarding movement If soil moisture decreases, the strength of the
association between the organism and the particle surface will increase to a point where
the organism is bound irreversibly to the surface . Passive binding to particle surfaces has
been obsetved with some strains of virus, and it is believed that the strength of the bond
can immobilize the virus and contain it at the point of interactioIL It is possible that
similar interactions occur with other groups of pathogens, but the processes are less well
defined. Bacteria, for instance, synthesiz.e extracellular substances that can enhance their
attachment to surfaces and promote binding, suggesting that the process involves both
passive and active processes. Whether alone, or in combination with the apparently
protective effect of adsorption onto surfuces, soil moisture influences the persistence of
microorganisms, in particular viruses. In laboratory experiments a soil moisture content
ofbetween 10 and 15 per cent was sho\\>n to be optimal for the survival of several strains
of enteric virus ( e.g. Bagdasaryan, 1964; Hurst et al, 1980a; 1980b ).
62 Protecting Groundwater for Health
By contrast, an increase in the moisture content of the unsaturated zone may increase
the vulnerability of the aquifer to pathogen contamination in two ways: by providing
rapid transport pathways and by mobilizing adsorbed organisms. During periods of high
recharge, for example during prolonged heavy rain, the intergranular spaces in the
unsaturated z.one become waterlogged and provide a hydraulic pathway for the rapid
transport of pathogens. Where these intergranular spaces expand into fissures the
downward migration of pathogens can be extremely rapid. For example, particles
ranging in diameter from 0.1-6.0 µm have been fmmd to move through 20 m of
unsaturated chalk in less than 3 days by passage through horizontal and vertical fis.sures
(Lawrence et al., 1996). Moreover, the rapid movement of pathogens through fissures
limits the potential for attenuation by adsorption to surfaces in the soil matrix.
In the interval between recharge events, the chemistty of the water in the unsaturated
zone will change as it equihbrates with the soil matrix. In some soil types, these changes
may fu.vour the adsorption of microorganisms to surfuces in the soil matrix. A lowering
in the ionic strength or salt content of the SlllTOunding medium, which can occur during a
rainfall event, may be sufficient to cause desorption of the organism allowing further
migration into the soil. This phenomenon has been observed in laboratory experiments
and there is evidence to suggest that it can occur in the field Furthermore, some workers
have noted that the virus particles that have desorbed from the soil surface have a
reduced capacity to resorb when the environmental conditions become :favourable. The
implication of this observation is that virus particles that have been mobiliz.ed in the
subsurface are unaffected by one of the principal methods of attenuation and are likely,
therefore, to be dispersed over a much wider area than would be anticipated.
The siz.e variability of microorganisms (Table 3.4) can, to an extent, control their
mobility in the subsurfuce. Soil and rock pore siz.es are also variable and the two ranges
are known to overlap (Figure 33). Thus in soils that are composed of fine grain particles,
typically clayey-silts, the pore space is sufficiently small (<4 µm) to physically prevent
the passage of bacterial and protozoal pathogens into the subsurfuce. This removal
process is called physical filtration or straining. Straining has been identified as the
principal mechanism for controlling the migration of Giardia and Cryptosporidium
species (Cryptosporidium oocysts: 4-6 µm; Giardia cysts: 7-14 µm) through these soil
types; indeed, experience has shown that up to 99 per cent of Cryptosporidium oocysts
are retained in the upper layers of the soil. However, the isolation of Cryptosporidium
and Giardia from a small but significant number of groundwater sources in the USA
(Hancock et al., 1998) and the United Kingdom indicates that the protective effect of the
soil layer is frequently evaded, probably by migration through preferential pathways or
bypassing; for example, from sewers that are often located below the soil z.one.
Several studies demonstrate a considerable degree of variability between the
inactivation or die-off rates of different groups of pathogens, and between inactivation
rates of the same organism in different environments. However, as a general rule, enteric
viruses persist longer in soils than bacteria Among the enteric viruses, hepatitis A virus
appears to be the most resistant to inactivation in soil (Sobsey et al, 1986) and, in
laboratory experiments, shows a lower capacity for adsorption to particle surfaces. The
oocysts of Cryptosporidium are highly resistant to environmental stress and it has been
estimated that they could be detected after 12 months in soil.
Pathogens: Health relevance, transport and attenuation 63
Table 3.4. Approximate sizes of selected micrompnisms
Bacteria
Protoz.oa
Microo~anism
Bacteriophage
Poliovirus
Bacterial spores (Bacillus, doslridia)
E.coli
Salmonella typhi
Shigella spp.
Cryptosporidium oocysts
Giardia
Enleroamoeba histolitica
Size
0.02-02 µm diameter
0.03 µm diameter
lµm
0.5 J.llil X 1.0 µmx2 .0 J.llil
0.6 µmx 0.7 µm x2.5 µm .
0.4 J.llil X 0.6 µm x2.5 J.llil
4.0-6.0 µm diameter
7.0-14.0 µm diameter
20-25 µm diameter
10-3m 10~m 10-9m
1mm 1µm 1nm 1A
I bactena . . PATHOGEN +--protozoa ..t,._. . J
• • . DIAMETER I +--Viruses-----+
I
1--fissures ----l I
apertures 1
sands
f-sandstone-i
pores I 1--limestone ---1
chalk pores
I
~
Cryptosporidium
oocyst
1-------1
silt pores
FISSURE
APERTURE/
PORE SIZES
Figure 3.3. Pathogai diame1ers compared to aquirer matrix diameters (ARGOSS, 2001; British
Geological Survey ONERC)
Some of the factors that contribute to reduced inactivation rates in the unsaturated
zone are known (lower temperatures, increased moisture, pH, reduced exposure to
sunlight, organic matter and the nature of the patmgen) but the relative contribution of
each factor at any field site is difficult to predict, and may be site specific. In these
circwnstances, the tendency of hydrogeologists and public health microbiologists is to
construct general risk assessment models based upon laboratory and field experience. At
their most sophisticated, the models comprise computer simulations of pathogen
transport (for example Yates and Yates, 1988), but also used are simple tables and
diagrams linking risk to the main observable features of the environment One such
model has been constructed by Romero (1972); a second has been developed to
accompany guidelines for assessing the risk to grmmdwater from on-site sanitation
(AR GOSS, 2001 ). However, the problem with such an approach is that only a qualitative
indication for risk levels is given \\'ithout any definition of what is meant by a high or
low risk level The actual useful infonnation from this approach is a rough indication of a
64 Protecting Groundwater for Health
relative probability for pathogenic microorganisms to reach grolilldwater. Table 3.5
shows the different classes of lithology that were defined in decreasing order of ability to
limit transport of microorganisms.
Table 3.5. Limitation of1he transport of microorganisms by 1he lithology of the unsaturated zone in
decreasing order (top to bottom) (based on ARGOSS, 2001)
lithology of the unsaturated zone
Fine sand, silt and clay
Weathered basement (soft not consolidated)
Mednnn clean sand
Coarse sand and gravels
Consolidated rock
Shallow grolilldwater (<5 m) is assumed to be at the highest probability of
contamination irrespective of the lithology of the unsaturated z.one. As the depth to the
water table increases so the capacity of the unsaturated zone to attenuate microorganisms
will also increase, although this will depend upon the composition and structure of the
unsaturated zone. For example, fine silts and clays will strongly adsorb bacteria and
viruses and also effectively filter out the larger pathogenic microorganisms. Thus the
probability of reaching groundwater at greater than five m depth is very low. By contrast,
fiacture flow through consolidated rock creates a relatively high probability of reaching
grolilldwater even at depths of over 10 m.
In smnmary, maximizing the residence times in the unsaturated zone has been
proposed as the key mechanism for eliminating bacteria and viruses (Lewis et al., 1982)
and, in general, this principle is robust However, there are exceptions, for example:
• The variability in the nature and thickness of the unsaturated zone overlying
aquifers means that the residence times may not always be adequate to attenuate
all pathogens. In particular, during periods of high recharge, an aquifer may be
vulnerable to contamination by pathogens that are transported rapidly through
the waterlogged intergranular spaces in the unsaturated zone.
• Where the flow is intergranular within the unsaturated zone there is greater
potential for contact with the soil/rock particles and hence greater potential for
retention, both sorptive and filtering. However, if excessive loading takes place
the filtering effect may lead to a blocking of the pores. The resulting reduction in
hydraulic conductivity may reduce the effectiveness of the unsaturated zone to
retard contaminants if the clogging forces recharge water into vertical fissures
where rapid downward movement can occur.
• The structure of the unsaturated zone is seldom uniform and fissures may exist
permanently or develop in any environment when the unsaturated z.one dries
out. The presence of fissures will always increase the vulnerability of the
grolilldwater to contamination from the surface, and it should be considered that
although the soil conditions may facilitate the adsorption and attenuation of
pathogens, the existence of bypass channels may offset the protective effect of
the soil.
Pathogens: Health relevance, transport and attenuation 65
3.3.2 Transport and attenuation of pathogens in the saturated
mne
On reaching the saturated z.one, microbial contaminants are subject to the same processes
of attenuation that are descnbed in Section 3.3. l but under conditions of natural or
artificially induced flow. Thus die-0ff; adsorption, filtration, predation and dilution all
contribute to the attenuation of pathogens in the saturated z.one. Due to the het.erogeneous
nature of aquifer material there may be large variations in hydraulic conductivity and this
can significantly influence the movement of microorganisms in the aquifer.
Microorganisms are transported in groundwater by advection, dispersion and diffusion,
which are defined in Chapter 4. The result is a migration and spreading of the
contaminant concentration in space and time. This may result in contamination of
increasingly large aquifer volumes as the pollutant moves downgradient Although the
transport of pathogens in some aquifer types can be both rapid and extensive, there are
several factors that may attenuate pathogens in groundwater (Table 3.6).
Table 3.6. Factors affecting transport and attenuation ofmiaoorganisms in gromulwater (adapted
fiom Westeta/., 1998)
Characteristics oftbe microorganism
Size
Shape
Density
Inactivalion rate ( die-off)
(lr)reverstble adsorption
Physical filtration
Aquifer/soil (environment) properties
GrolDldwater flow velocity
Dispersion
Pon: size (iota-granular or fracture)
Kinematic/effudive porosity
Organic carbon content (solid)
Temperature
CbemicaJ. properties of groundwater (pH, etc.)
Mineral composition of aquifi:r/soil material
Predatoiy micro flora (bacteria, fimgi, algae, etc.)
Moisture content
Pressure
From the perspective of grmmdwater management and the estimation of pathogens at
the point of abstraction (receptor), highly fractured and karstic aquifers represent a
particular problem. As discussed in Chapter 2, groundwater flow through fractured ·
systems may be very rapid, and the potential for microorganisms to be attenuated by
interaction \\'ith the aquifer matrix is much reduced, although not entirely absent.
Consequently, the inactivation rate of the pathogen and the groundwater flow rate will
primarily control dispersal in these aquifer systems . Three referenced studies will help to
illustrate the potential for rapid pathogen transport in highly fractured aquifers :
• The migration of bacteriophage in a chalk aquifer in the south of England was
investigated by Skilton and Wheeler (1988 , 1989). They injected three strains of
bacteriophage into piewmeters that intersected the water table and then
collected samples at different sites downgradient to determine the extent of
movement Very high velocities were observed at one site due to the fuct that the
majority of the water flow is through fissures , fractures, solution openings and
cavities. All three phage types were detected 355 m from the injection site
approximately 5 hours after introduction It is noteworthy that viable phage were
66 Protecting Groundwater for Health
still being recovered more than 150 days after they were injected into the
aquifer.
• Mahler et al. (2000) cite the work of Batsch and colleagues who reported the
detection of injected bacteria 14 km fium the iajection site, having been
transported at a velocity of about 250 m/h. Mahler's own studies (Mahler et al.,
2000) in a karstic aquifer located in the South ofFrance have confirmed the very
rapid transport of fuecal indicator bacteria in these systems.
• Lee (1993) investigated the contamination of a water supply well by Giardia
spp. and Cryptosporidium spp. in a karstic environment The karstic nature of
the study area provided the potential for rapid infiltration of surface waters to the
water table and subsequent transport of the organisms to the well through
:fractures mxl fissures. This connection was confirmed by the study. An analysis
of particle siz.e revealed that the full range of particle siz.es found in the surface
waters was not present in the well; there was a cut-off at both high and low
ranges. The author concluded that there had been adsorption of smaller particles
and straining of larger ones. The siz.e range of the particles that were transported
through the system included Giardia and Cryptosporidium.
These observations have significant implications for the public health risk associated
with water abstracted fium highly fractured and karstic aquifers. Not only can viral,
bacterial and protoz.oan pathogens be transported rapidly over great distances, but also
the groundwater flow pattern between the source and receptor can be very difficult to
predict due to the many interconnected :fractures in the aquifer. It is possible that well
designed tracer studies and groundwater flow models can help to define the potential
limits of pathogen dispersion in a highly fractured aquifer; however, with the current
uncertainties surrounding pathogen attenuation in groundwater it is prudent to assume
that where these aquifer types are exposed to sources of pathogens they are at high risk of
contamination over a wide area.
In other aquifer types, the radius of migration fium the source of contamination is
normally restricted to several tens of meters, or a few hundred meters, depending upon
the type of aquifer system mxl the properties of the organism (fable 3.6). Where the
groundwater flow rate is low through unconsolidated sediments the dispersal of
pathogens will naturally be limited and, in addition, this type of system offers greater
opportunity for the pathogen to interact with the aquifer matrix. Adsorption and physical
filtration may then be the major factors controlling pathogen transport.
Despite acting to limit the dispersal of pathogens in the aquifer, interaction with the
aquifer matrix may also enhance the survival of the pathogens in the environment In
several cases, adsorption to surfares in the aquifer (sediment particles and colloids, as
well as the aquifer matrix) has been shown to reduce the inactivation rate ofboth viruses
and bacteria Consequently, although the risk of contamination is contained close to the
source, the persistence of pathogens \\'ithin the zone of contamination may be increased
beyond what is predicted :from measurements of inactivation in the groundwater.
Schijven and Hassanizadeh (2000) showed that removal of virus in the subsurface
often appears to be higher near the source than further away :from the source, e.g. within
the first 8 m of aquifer passage spores of Clostridium bifermentans R5 and bacteriophage
MS2 were reduced by 5 log10 and 6 log10 respectively, while in the following 30 m MS2
concentrations were reduced only by 2 log10 and reduction of spore concentrations was
Pathogens: Health relevance, transport and attenuation 67
negligible. This may be explained by favourable attachment sites that are present in the
first meters of transport but rapidly decrea.5e with travel distance or travel time in an
exponential fashion, like sites formed by ferric oxyhychoxides.. Obviously, predictions of
virus removal over larger travel times or distances can be severely overestimated if they
are based on removal data from column or field experiments where transport was studied
over short times and distances.
Inactivation rates of bacteria and viruses in groundwater vary considerably, not only
between the bacteria and virus groups, but also between different strains within each
group and between the results of different investigations. Table 3.7 lists inactivation rate
coefficients of pathogenic viruses, bacteriophages and bacteria in groundwater. These
data are ordered according to microorganism and then according to temperature. Usually
inactivation proceeds faster at higher temperatures, although this is highly dependent on
the type of microorganism.
Often, inactivation of microorganisms can be descnbed well as a first order rate
process, especially under relatively mild conditions like temperatures from 5-20 °C and
pH values from 6-8. Under more extreme conditions, the rate of inactivation ot; for
example viruses is often found to proceed initially at a higher rate followed by a lower
rate as if two or more sub-populations exist that differ in stability (see e.g. Hurst et al.,
1992). The data given in Table 3.7 are based on the observation, or in some cases
asrumption, that inactivation or die--off proceeded as a first order process:
(Eqn. 3.1)
where C. is the remaining concentration of microorganisms after time t, Co is the initial
concentration at t=O and µ is the inactivation rate coefficient (T1). As a means of
interpretation, µ is often divided by a factor of 2.3 ( equal to the natural logarithm of 10).
The inactivation rate coefficient then reflects the number oflog10 units per time unit; e.g.
a virus decreases in number by 2 log10 (equal to a fuctoroflOO) every IO days.
From Table 3. 7 it can be seen that at common groundwater temperatures of 10-12 °C
inactivation rate coefficients for coxsackieviruses B, echoviruses 7, poliovirus 1, hepatitis
A virus, but also ofbacteriophages q>Xl 74, MS2 and PRDl are in the range from 0.oI to
0.04 day·1
• This corresponds to a decline in number or concentration of 1 log10 ( equal to a
factor of I 0) every 57 to 230 days. Some studies report an inactivation rate coefficient of
zero (e.g. Nasser et al., 1993). This is to be interpreted as no significant inactivation
within the time-scale and accuracy of the experiment and is therefore not included in
Table 3.7. Given the variation in inactivation rate between microorganisms, inactivation
rate coefficients of the more stable microorganisms need to be considered for estimating
adequate protection of groundwater wells or removal efficiency of passing
microorganisms through soil as a means of treatment
The data in Table 3.7 make clear that bacterial die--offis both highly dependent on the
type of microorganism as well as on temperature. In many cases bacterial inactivation
proceeds faster than that of viruses, implying therefore that viruses are more critical for
groundwater protection than bacteria
68 Protecting Groundwater for Health
Table 3. 7. Inactivalion rate coefficients of pathogenic viruses, bacteriophages and bacteria in
growidwater
Microorgiinlsm Temp. Olbercooditiom Jnactiwlioo rate Refermce
C'.0 codlicicot" (1/day)
Coxsadcievirus A9 10 Sterile 0.019 Matthesselal (1988)
10 O.<m
10 Deiroizm 0.031
Coxsadcievirus BI IO Sterile 0.012 Mauhess et al (1988)
IO 0.019
10 Deionized 0.040
Coxsackievirus B3 3-15 0.49 Keswiclc et al (1982)
Coxsackievirus B4 5 0.079 Schijven et al (2003)
Coxsadcievirus B5 16 12mg/102 0.12 Jansoos et al (1989a)
19.4 0.12 Jansons e1 al (1989b)
E.chovirus I 12 024 Yarese1al (1985)
13 025
17 028
18 0.35
23 0.94
F.chovirus6 22 02mgil02 025 Jansonse1al (1989a)
F.chovirus 7 10 Sterile 0.032 Manhess et al (1988)
IO 0.019
10 Deiroizm 0.038
F.chovirus 11 16 23mg/102 023 Janronsetal (1989a)
F.chovirus24 16 l.6mg/102 0.12 Jansons et al (1989a)
Hepatitis A virus IO 0.10 Nasser et al (1993)
20 0.41
23 Filtered bottled 0.038 Biziagos et al (1988)
mineral waler
25 Stenle 0.082 Sobsey et al (1986)
25 033
30 0.054 Nasser et al. (1993)
Poliovirus I 3-15 0.48 Keswick et al (1982)
4 0.016 Meschke (2001)
5 0.16 Schijvenetal (2003)
10 Sterile 0.010 Maltbess el al (1988)
IO 0.013
10 Deiroizm 0.032
IO 0.025 Nasser and Oman (1999)
12 0.18 Yaresetal (1985)
13 0.20
14 70weeks 0.16 Meschke (2001)
16 5.4mgil 02 021 Jansons et al (1989a)
16 02mgil02 0.069
17 0.19 Yat.esetal (1985)
18 0.43
20 0.038 Nasser et al (I 993)
22 0.06mg/102 0.16 Jansons et al (1989a)
22 0.10 Bittooetal. (1983)
23 0.17 Blanc and Nasser (1996)
23 12 Yat.esetal (19&5)
23 Filtered bottled 0.044 Biziagosetal (1988)
mineral waler
24 0.046 Bitton et al (1983)
25 4weeks 0.11 Meschke (2001)
30 0.12 Nassa-et al (1993)
Ro1avirus 20 0.36 Pancoroo el al 11987)
Pathogens: Health relevance, transport and attenuation 69
Microorganism Temp. Other conditions Inactivation rate Refermce
(" coeffident 11 (I/day)
Simian Rota.virus 3-15 0_83 Keswick et al(1982)
23 028 Gerba et al_ (Undated)
cl>X174 5 0_012 Schijven el al. (2002b)
F-specificRNA IO 0.025 Nasser and Oman (1999)
bacteriormges
20 0.0077 Na55eretal (1993)
30 0.031 Na5Ser et al (1993)
MS2 2-5 0.030 Scbijvenetal {1999)
4 0.037 Meschke (2001)
4 0Jl63 Yates et al (1985)
5 0.064 Scbijvenetal (1999)
5 0.082 Scbijven et al (2002b)
7 0.0058-0.10 Yahya e1 al (1993)
12 Oxic 0.10 Scbijven e1 al (2000)
12 Anoxic 0.024
12 0.16 Yates et al (I 985)
12 0.065 Yates(l992, unpublished
observati(Jll',)
13 0.22 Yatesetal (1985)
14 70weeks 0.45 Meschke (2001)
17 0.17 Yat.rsetal (1985)
18 0_19
23 0.36 Blanc and ~{1996)
23 0.58-13 Yahyaetal (1993)
23 0.73 Yat.rsetal (1985)
25 4~ 0.41 Meschke (2001)
PRDI 5 0.0004 Scbijvenetal. (1999)
5 0.044 Scbijven et al. (2002b)
7 0.010-0.10 Yah}'lletal (1993)
12 Oxic 0_054 Schijven el al (2000)
23 0.035 Blanc and~ (1996)
23 0.12-0.30 Yahya et al. (1993)
Bacillus subtilis spores 14 70weeks 0.1382 Meschke e1 al (2001)
a perftngens spores 14 70weeks O_D714 Meschke el al (2001)
Ecoli 12 0.083 Scbijven et al (2000)
14 70weeks 0.51 Meschke el al (2001)
20 0.044 Nasser and Oman (1999)
22 0-36 Bitton el al. (1983)
3-15 0.74 Keswick el al (1982)
9-13 0.84 McFeta:s et al (1974)
E coli 0157:H7 20 0.32 Rice(l992)
Faecal colifonns 12-20 0.83 Keswick et al (I 982)
Faecal SlreplOCOCci 22 0.066 Bitton et al (1983)
3-15 0.53 Keswick el al (1982)
Klebsie/Ia spp. ? 0.031 Dowd and Pillai (1997)
Salmonella SW-? 0.19 Dowd and Pillai (1997)
Salmonella typhimurium 22 0_30 Bitton et al (1983)
Salmonella typhimurium 9-13 0.50 McFeters et al. (1974)
Shigella dysen!ariae 9-13 1.7 McFeters et al. (1974)
Shigella jle:ceri 9-13 1.4 McFeters e1 al. (1974)
Shigella sonnei 9-13 1.6 McFeters et al. (I 974)
Vibrio cholerae 9-13 5.3 McFeterse1al. (1974)
70 Protecting Groundwater for Health
The low inactivation rate of Klebsiel/a spp. in growidwater provides an interesting
contrast to the other genera of enteric bacteria Klebsiel/a is an important member of the
Ent.erobacteriaceae, which are commonly, though inaccurately, referred to as 'coliform
bacteria'. Some species of Klebsiella are able to grow at 44 "C, which further defines
them as thermotolerant (formerly faecal) coliforms, the group that contains E coli.
Although K/ebsiella are fowid in the bowel and respiratory tract ofhwnans and animals,
they can be isolated also from environmental samples. The persistence of Klebsiella in
the survival experiments may therefore reflect the ability of these organisms to exist in
two very distinct environments.
One problem associated with the detection of pathogenic bacteria is that they may
become dormant in the environment (Schijven et al., 2002a). In this state, they are viable
but non-culturable, which means that the organisms show metabolizing activity even
though they cannot be grown on traditional media (Olson, 1993). As an important
example, in water the pathogen E coli 0157:H7 appears to be able to enter a viable but
non-culturable state (Wang and Doyle, 1998). The dormant state prolongs the pathogen
survival, and if its pathogenicity is unaltered, this increases the likelihood of a host
infection and illness
Some bacteria like Clostridhnn and Bacillus species can survive for extended periods
of time by producing spores. Clostridium spores are especially robust in that their
inactivation rate may be considered to be negligible. This property makes it difficult to
interpret their removal, e.g. in sand filters or river bank filtratioIL In colmnn studies by
Schijven et al. (2003) it was shown that most of the spores of Cl. perfringens attach to
the grains of sand, however followed by slow detachment:. i.e. adsorption was apparently
reversible for a large fraction of spores. It was shown that the removal is dependant on
the nwnber of spores that accumulate in the porous medium.
At the time of writing, no data are available on inactivation of oocysts of
C,yptosporidium in groundwater. C,yptosporidium oocyst in vitro viability has been
measured repeatedly U5ing a surface water matrix and in vitro excystation and/or dye
exclusion assay. For example, Heisz et al. (1997) report 0.12 day·1 (30 °C), and Medema
et al. (1997) found inactivation rates of0.023--0.056 day"1 (5-15 °C) in river water and no
differences in inactivation rates between 5 °C and 15 °C. From the data of Robertson et
al. (1992) and Chauret et al. (1995), inactivation rates of0.0051 to 0.0062 day°1 can be
calculated. Chauret et al. (1995) further found that inactivation rate was independent of
water temperature up to 20 °C.
Based on their small size and longevity in the environment viruses have the highest
potential to be transported to, and within, groundwater, and thus they have been the focus
of the majority of studies. As a comequence, the diSCUSfilon in the following chapter has
a greater emphasis toward the factors that influence the transport and survival of viruses
in the subsurface. From the data that is available, the same factors appear to affect the
survival of bacteria; however, some bacteria are able to utilize specific physiological
responses to resist environmental stress that are not available to viruses. These responses
will be discussed separately at the end of the chapter.
Pathogens: Health relevance, transport and attenuation 71
3.3.3 Summary of major factors influencing pathogen transport
and attenuation mechanisms in the underground
The mechanisms by which microbial contaminants may lilldergo transport and
attenuation in the saturated and unsaturated zones have been described in Sections 3.3.1
and 3.3.2. There now follows a description of the factors that control the degree of the
impact of these mechanisms. The potential for pathogens in manures, faeces and
wastewater to contaminate the lillderlying groundwater is dependent on a number of
factors including the physical characteristics of the site (e.g. soil texture), the hydraulic
conditions (e.g. wastewater or manure application rates, wetting/drying cycles), the
environmental conditions (e.g. rainfull, temperature) at the site and the characteristics of
the specific pathogens present in the water. The factors that influence the transport and
attenuation of pathogens in the subsurface have been the subject of a mllllber of reviews
summariz.edin Table 3.8 (Vaughn et al., 1983; Yates et al, 1985; Yates and Yates, 1988;
Bitton and Harvey, 1992; Robertson and Edberg, 1997; Schijven and Hassanizadeh,
2000). &>me of the major factors influencing pathogen transport and attenuation are
described in more detailbelow.
Temperatw-e
Temperature is probably the most important factor influencing the inactivation of
bacteria and viruses in the environment Laboratory studies have demonstrated a
negative correlation between water temperature and the survival of coliform bacteria and
enteric viruses, although the magnitude of the effect varies between different strains.
Roughly, the inactivation rate of viruses may be one order in magnitude higher at 25 °C
than at 5 °C (Table 3.7). Similarly this may be the case for enteric bacteria, such as E.
coli. The influence of temperature on the migration of bacteria and viruses is currently
unknown.
Microbial activity
There are several conflicting reports regarding the influence of indigenous populations of
microorganisms on the survival of enteric bacteria and viruses, ranging from increasing
the rate of inactivation through to having no effect to decreasing the rate of inactivation.
Overall, however, the main finding of laboratory studies is that microbial activity in the
soil and groundwater increases the inactivation rate of enteric bacteria and viruses.
Evaluating the role of microbial activity normally involves a comparison of inactivation
rates in sterile and non-sterile environments. Depending on the environmental conditions
and the experimental design. virus inactivation is either lillaffected. or accelerated in the
presence of indigenous bacteria Hmst et al. (1980a) showed that the inactivation rate of
two strains of enterovirus was more rapid in non-sterile, aerobic environments than in
sterile environments. By contrast, Matthess et al. (1988) found no significant difference
between the inactivation rates of several viruses in sterile and non-sterile groundwater.
Studies \\ith thermotolerant coliforms as a whole, and E. coli in particular, have shm,n
that the concentration of the test organism can increase rapidly in sterile environments,
but remains static, or is reduced in non-sterile environments (Gerba and McLeod, 1976).
72 Protecting Groundwater for Health
Table 3.8. Influence of major filctors on fue swvival and migration of microorganisms in fue
subsudiice (Vangbn et al., 1983; Yates et al., 1985; Yates and Yates, 1988; Bitton and Hmvey,
1992; Robertson and Edberg, 1997; Schijven and H_,..,i:mdeb, 2000)
Fador VmRS Baderia
Jnfluence 00 survival Jnflllfllre oo migration Jnffuence 00 survival Jnfluence 00 mil!flllm
Tempernture Pasislmceis Jonge.-at nknown i'elsislmceis Jonge.-at Unknown
lowte,nperallll"ff low1impelllureS
Microbial Varies: scmeviruses Unknown The II= of Unknm.w
adivity are irnctiv.mimore iixliganismia-o-
readily in the pn=n: oi,misms3R]951D
of ca1ain micro-increase the inaaiwtion
mganimls,thetJW(Ete lllleof emaic lu;taia;
may also be true, or possible~
th:re may beno eflect wilhoomepoto,m
may redure inactiwtion
llllrs
Moisture Mo& viruses survive VllllS migration IBJa!ly Most bacteria survive Bacrerial migratioo
coolf:111 longer in moist soils increasesurm longer in moist soils 1B1311y increases mm
am evm Jonge.-urm satumloo flow 1han in my soils smmded flow
saturatw croditioos; croditioos COID1ioos
1lll'3Uraloo soil may
imc1ivate viruses m the
air-w.l!frinlara:e
pH Most Wlfric viruses are LowpH typically Most eotmc l:a:1fria Low pH encwrag;:s
stableo=pH range of increases virus ~00 will survive Jonge.-at adsrxplion to soils am
3to9;hom:va-, to soils; high pH cruses near:nrutral pH thea:piirermalrix; the
smvival may be ds,qiiro~ tmdency ofulctaia to
polongedbynear fucilitaling greater bind tosudil:es may
neUlral pH values migralim redure detdnnart at
highpH
Salt species Catlin cations may Increasing iroic Unknown Increasing iroic
arxlconrm-polong survival streng1h of the strength of the
mmon dqJending upon the smmmdingnmium SID10llllding mwiwn
type of virus gmernily increases gmernllyincreases
lOJ]liro lOJ]liro
Associatiro Associatiro wilh soil V= intaa1ing wilh Adscwptionroto!did Bacta:ia intaa:ling
wilhsoil gmernily increases the soil JJ111ic1es are surfaces reduce wi1h the soil JJll1icles
smviwl, although inhibitedfiom inactivaion rares; the are inlnbited 1iom
auadnnmttoca1ain migration 1hrough the concmttalion of migration 1hrough the
mineral sudil:es may soilnwix bactaiaoosudaces soil matrix
caB: inactiv.ition may be~ on:lers
of magnitwe higher
tmn the concmttalion
in the a:µ:ous ins:
Soil Prombly relalfd to the Greaurmigration in Prooobly relalfdto the Greata-migration in
puperties degree of virus ~oo roaisetexturerl soils; degree ofumrial roaisetextured soils;
soilswilhdwgoo adsrxplion soils \>ith dwgoo
~such as clays, ~ such as clays,
!mllbviruses !mllblmfria
Pathogens: Health relevance, transport and attenuation 73
Fador VIIUSl5 Badaia
Jnfluenre m smvival Jnlluence m mi£131ioo Influeorem smvival Jnfl1H1re m mil!&llJOn
&::bm' DiffimJt virus types ~m1D51lil5is Diffimtt species of Some species of
virustype V31)' in their relaledtop!}'Sico-tumiav.nyinlheir ID:miaaremcre
~liitity1D ch:mcd diffcmx:e in ~liitityto irmi-~oflmling1D
iimivatimby sn:nlaryandtatay vatiooby~ surliK:e;; vaiatim may
~dxmicaland cap;idmilcestrudure ch:mcd and biological am oo:ur betwefn
biological lilctors andmnino ail fuclur.; strains of the same
~ IBmial!pries
Organic QganicmalttYmay SolubieOIFIBDC malttY The presence of Ogaaic mat!Ermay
Ollie" poloogsmvivalby ampelfS wi1h Wl8S ocg;micmallfrmayac:t cmditioo solid surla:;es
competitively lmling furadsotpliorlmm asa soun:e of llllllieJl!s am Jmloole lmaial
atair-w.ila"inlErnl:e; pal1icles mlich may forhldl:ria,pmx,ting adupim
\\meina:tiwlirocan result in increaserl virus grIM1h and exremed
oocur migralim smvival
Hydraulic Unknmw VIIUS~ Unkm\m Ba:lmalmigrdlicn
aJOditims genaallyiocreasmat grnerally iocreasm al
higher hydraulic loads higher hydraulic loads
and flowrates aodflowl3e:5
Soil moisture content
Although some investigators have observed no difference between the inactivation rates
of viruses in dried and saturated soils (Lefler and Kott, 1974) the majority ofreports have
shown that soil moisture content influences the survival of viruses in the subsurfuce. For
example, Hurst et al. (1980a) found that moisture content affected the survival of
poliovirus in loamy sand. The inactivation rate of poliovirus decreased as the moisture
content increased from 5 to 15 per cent. However, further increases in soil moisture
content increased the inactivation rate of the virus. It was noted that the inactivation rate
peaked near the saturation moisture content of the soil (15-25 per cent), and was slowest
at the lowest moisture contents (5-15 per cent). Soil moisture has been reported to
influence the fate of bacterial contaminants (Robertson and Edberg, 1997), but the
magnitude of the effect, and the value of any correlation has not been described
pH
The effect of pH on the survival of pathogens in the environment has not been studied
extensively and the impact can only be inferred from laboratory investigations of the
physiological characteristics of the bacteria and the effect on the structural integrity of
viruses. In general, every species of bacteria has a narrow pH range that is optimum for
growth. Depending on the normal environment of the organism, the pH requirements can
range from highly acidic to highly alkaline: for many human pathogenic bacteria the
optimum pH is close to neutral. Despite having a preference for a narrow pH range, most
species of bacteria can tolerate a short exposure to a much broader range of pH. Outside
these limits, the organisms are rapidly killed. It is likely that pH affects the survival of
viruses by altering the structure of the capsid proteins and viral nucleic acid.
Some authors have suggested that pH indirectly influences pathogen survival by
controlling adsorption to soil particles and the aquifer matrix. It is the adsorption to
surfaces that ultimately reduces the inactivation rate of the pathogens. Generally, bacteria
74 Protecting Groundwater for Health
and viruses have negative surface charges generated by the level of ionization of the
carboxyl and amine groups that are a major comp:ment of surfuce proteins. As the pH of
the meditm1 changes, the ionization of the two groups will change, causing a shift in the
net strength and polarity of the surface charge. At a specific pH, which is determined by
the molecular structure of the protein, the net charge will be z.ero; this is termed the
isoelectric point of the molecule. The isoelectric point has been determined for many
different proteins and for a number of virus strains. At pH values below the isoelectric
point a virus will have a net positive charge, whereas the charge will be negative at pH
values above the isoelectric point
Wrthin the pH range of most unpolluted groundwater both the matrix surfaces and the
surfaces of the microorganisms carry a net negative charge. Under these conditions the
microorganisms will be repelled by most mineral grain surfaces. At low pH values the
surface charge on the microorganisms will shift to being net positive, which will favour
their adsorption to soils and the aquifer matrix by electrostatic attractioIL This hypothesis
has been confirmed by several groups of workers for both bacteria and viruses (Sobsey,
1983; Gema and Bitton, 1984; Bales et al, 1991, 1993; Bitton and Harvey, 1992; Grant
et al, 1993; Loveland et al, 1996; Pemod et al., 1996; Redman et al, 1997; Ryan et al,
1999).
There are many complicating factors that can interfere with the mechanism discussed
above. One is that a given virus may have more than one isoelectric point and the factors
responsible for passage -from one form to another are unknown at this time. Other factors,
such as cations and humic and fulvic acids, may also influence the net surfuce charge of
the organism.
Whilst changes in pH may affect the mobility of microor~ in the subsurface,
the significance of this factor in any particular aquifer is uncertain. Robertson and Edberg
(1997) have noted that the pH of most unpolluted groundwater is generally very stable,
and in their experience falls within the near-neutral range of 6.5 to 8.5. There are
exceptions, and in those African countries underlain by acidic basement gneisses and
granites the pH is likely to be much lower, frequently in the range 5.5 to 6.5. In addition,
geological materials comprising most aquifers have a significant buffering capacity that
helps to maintain a relatively constant pH Robertson and Edberg conclude that it is
unlikely that significant changes in microbe mobility will occur due to these minor pH
changes. This assumption may be valid for many stable groundwater systems but in
groundwater that is exposed to contamination fiom a variety of sources, which may be of
unknown and variable quality, for example sewage, pH may emerge as a dominant factor
in the mobility of pathogens.
Salt species
The adsorption of microorganisms onto surfaces in the groundwater system has been
shown to have two counteracting effects: It reduces the dispersal of the organism in the
subsurfuce, but reduces the inactivation rate of the organism in the affected area. If the
prevailing geochemical conditions in the groundwater system create opposing charges on
the surfuce of the organism and the aquifer matrix, adsorption will occur by electrostatic
attractioIL Frequently, however, these conditions do not exist and the organism and the
aquifer matrix each have a negative charge.
Pathogens: Health relevance, transport and attenuation 75
The types and concentrations of salts in the environment can have a profound
influence on the extent of pathogen transport in the subsmface. Cations (positively
charged inorganic species), in particular multivalent cations such as Magnesium (Mt')
and Calcium (Ca2"'), can form a bridge between the solid surface and the organism and
significantly enhance adsorption. Clearly, the concentration of the salt is also important.,
as this will influence the number of sites that are available for binding as well as the
number of bridges that can be formed between the two surfaces. Thus the capacity for
binding and the strength of the bonds will be affected by the salt concentration. Several
studies of virus and bacterial transport through simulated groundwater systems have
confirmed this hypothesis (Taylor et al., 1981; Sobsey, 1983; Bitton and Harvey, 1992;
Simoni et al., 2000).
A decrease in the salt concentration or ionic strength of the soil water, such as would
occur during a rainfall event., can cause desorption of viruses and bacteria from soil
particles (Gema and Bitton, 1984). This phenomenon has been observed in both
laboratory and field experiments (Landry et al., 1980; Wellings et al., 1980).
Furthermore, there is evidence t.o suggest that only small changes in the salt
concentration can dramatically affect the mobilization of some organisms in groundwater
systems (Redman et al., 1999). The implication of this discussion is that salt
concentration in the groundwater system may be of greater significance t.o pathogen
transport than pH, although it is important t.o consider that neither factor will act in
isolation.
Organic matter
There are conflicting reports about the influence of organic matter on the survival and
1ransport of microorganisms in the subsmface, with different responses being noted for
bacteria and viruses, and for different species and strains within each group. The
influence of organic matter on virus survival has not been firmly defined. In some studies
it has been found that proteinaceous material present in wastewater may have a
protective effect on viruses; however, in other studies no effect has been observed.
Whilst similar observations have been made of bacterial survival in the presence of
organic matter, there remains an additional concern that enteric bacteria, in particular the
major pathogens and faecal indicator organisms, may be able t.o undergo a certain level
of growth in the environment if the conditions are suitable. There is some support for this
hypothesis, a few reports have been published demonstrating regrowth of fuecal indicator
bacteria in organically rich tropical surface waters, but the evidence is still insufficient t.o
confirm that regrowth is a significant issue for most enteric bacteria in groundwater.
Dissolved organic matter has generally been found t.o decrease virus adsorption by
competing for binding sites on soil particles and the aquifer matrix. The consequence of
this observation is that organic matter will increase the mobility of viruses in the
subsurface (Powelson et al., 1991 ). However, at relatively low concentrations of organic
matter the effect may be reversed, causing increased virus adsorption and significantly
reduced mobility in the subsurface (Robertson and Edberg, 1997).
Overall, bacteria may respond differently. Binding t.o surfaces is a characteristic of the
growth cycle of most., if not all species of bacteria Unlike the passive processes that
characteriz.e the attachment of viruses t.o surfaces, bacterial attachment involves active
76 Protecting Groundwater for Health
p~ including the synthesis of extracellular appendages specifically required to
stabiliz.e the bacteria-surface interactioIL The initiation of this binding is favoured by the
formation of a conditioning film of organic molecules deposited on the solid surface
(Bitton and Harvey, 1992; WIIDpenny, 1996). Thus the presence of organic matter may
restrict the dispersal of bacteria in the subsurfuce but reduce the inactivation rate at the
site of attachment.
3.4 REFERENCES
Abbaszadegan, M, Stewart, P., Le Chevallie:r, M, Rosen, J.S. and Gerba, C.P. (1998) Occurrence
of Viruses in Groundwater in the United States, American Water Worlcs Service Company,
Voomees, New Jersey, Interim Report
Anderson, AD., Heryford, AG., Sarisky, J.P., Higgins, C., Monroe, S.S? Beard, RS., Newport,
CM., Cashdollar, C.L., Fout, G.S., Robbins, D.E., Seys, S.A, Musgrave, IU., Medus, C.,
Vmje, J., Bresee, J.S., Mainzer, HM. and Glass, RI. (2003) A wateibome outbreak of
Nmwalk like virus among snowmobilc:rs -Wyoming, 2001. J lrrfectious Diseases, 187, 303-
306.
ARGOSS (2001) Assessing the risk to groundwater from on-site sanitation, BGS Commissioned
ReseardJ. Report, w allingfurd.
Bagdasaiyan, G. (1964) Smvival of viruses of the Enterovirus group (Poliomyelitis, Echo,
Coxsackie) in soil and on vegetables. J. Hyg. Fpidemiol Microbiol. andlmmunol, 8,497.
Bales, RC., Hinkle, SR, Kroeger, T.W. and Stocking, K. (1991) Bacteriophage adsorption during
transport through porous media: Chemical pertmbalions and reversibility. Environ. Sci.
Technol., 25, 2088-2095.
Bales, RC., Li, S., Maguire, K.M, Yahya, M.T. and Gaba, C.P. (1993) MS-2 and poliovirus
transport in porous media: hydrophobic effects and chemical pertmbalions. Water Resow-.
Res., 2, 957-963.
Banks, W.SL. and Battigelli, D.A (2002) Occurrence and Distribution of Microbiological
Contamination and Enteric Viruses in Shallow Grmmd Water in Baltimore and Harford
Counties, Maryland. Water-Resources Investigations Report 01-4216, Reston, Virginia
Banks, W.S.L., Klohe, C.A and Battigelli, D.A (2001) Occurrence and Distribution of Enteric
Viruses in Shallow Grmmd Water and Factors Affecting Well Vulnerability to Microbiological
Contamination in Worcester and Wicomico Cuunties, Maryland. USGS, Water-Resources
Investigations Report 0 1-414 7, Reston, Virginia
Barrett, M.H? Johal, K., Howard, G., Pedley, S. and Nalubega, M. (2000) Sources of fuecal
contamination of shallow groundwater in Kampala Jn GroundwaJer: Past achievements and
fature challenges (eds. O.TM. Sililo, S. Appleyard, M. Barrett) pp. 691-696, Balkema,
Rotterdam_
Bauder, J.W., White, B.A and lnskeql, W.P. (1991) Montana extension initiative fuaises on
private well quality. J. Soil and Waler Conservation, 46, 69-74.
Bitton, G. and Harvey, R W. (1992) Transport of palhogens tlrrougb soils and aquifers. 1n
Environmental Microbiology, (ed. R Mitchell), pp. 103-124, Wiley-Lis.s, Inc.
Bitton, G., Farrah, S.~ Ruskin, RH., Butner, J. and Chou, Y J. (1983) Smvival of pathogenic and
indicator organisms in groundwater. Grmmd Water, 21, 405-410.
Biziagos, E., Passagot, J., Crance, J. and Deloince, R (1988) Long-term survival of Hepatitis A
virus and Poliovirus Type 1 in mineral water. Appl. Environ. Microbial., 54, 2705-2710.
Blanc, R and Nasser, A (1996) Effect of effluent quality and temperatme on the pe:rsistmce of
viruses in soil. Water Sci Technol., 33, 237-242.
Borchardt, M.A. Be:rtz, P.D., Spencer, S.K. and Batigelli, D.A (2003) Incidence of enteric viruses
in groundwater fiom household wells in Wisronsin. Appl. Environ. Microbiol., 69, 1172-
1180.
Pathogens: Health relevance, transport and attenuation 77
Chauret, C., Chen, P., Springlhorpe, S. and Sattar, S. (1995) Effect of environmental stressors on 1he
survival ofCtyptosporidium oocysts. In Proc. Water Quality Technology Coriference, New
Orleans, Louisiana.pp. 1567-1585. AWWA, Denver, Colorado.
Dahling, D. (2002) An improved filter elution and cell culture as a procedure fur evaluating public
groundwater systems for rultnrable enteroviruses. Water Environ. Res., 74, 654-568.
Davis, J. and Witt, E.C. (2000) Microbiological and Chemical Quality of Ground Water used as a
Source of Public Supply in SouJhem Missouri-Phase I, May 1997-March 1998. USGS
Wat.er-Resources Investigations Report 00-4038. Reston, Virginia
Doherty, K. (1998) Status of1he New England ground water viral study. In Proc. AWWA Annual
Meeting Dallas Texas, June 23, 1998, A WW A, Denver.
Dowd, S.E. and Pillai, S.D. (1997) Survival and transport of selected bacterial pathogens and
indicator viruses under sandy aquifer conditions. J Environ. Sci & Health Part A, 32, 2245-
2258.
Femmer, S.R (2000) Microbiological and Chemical Quality of Ground Water Used as a Source of
Public Supply in SouJhem Missouri-Phase II, April-July, 1998. USGS Wat.er-Resources
Investigations Report 00-4260, USGS, Denver, Colorado.
Gerba, C.P. and Bitton, G. (1984) Microbial pollutants: Their survival and transport pattern to
groundwater. In Grotmdwater Pollution Microbiology, (ed,. G. Bitton and C.P. Gaba), pp.
65-88, Wiley, New York
Getba, C.P. and McLeod, J.S. (1976) Effects of sediments on 1he survival of Escherichia roli in
marine wat.ers. Appl. Environ. Microbiol., 32(1), 114-120.
Getba, C.P ~ Kutz, S.M., Bradford, A.W. and DeLeon, R (Undated) Rotavirus Survival and
Transport in the Subswface. Cooperative Agreement Final Report to S1eve Hutcbson, US
EPA, Ada, Oklahoma, CR-813566-0101.
Gleeson, C. and Gray, N. (1997) The Coliform Index and Waterborne Disease, 1st edn, E&FN
Spon, London.
Goss, MJ., Barry, D.AJ. and Rudolph, D.L. (1998) Contamination of Ontario finmstead domestic
wells and its association wi1h agrirulture: I. Results from drinking water wells. J. Contam.
Hydro!., 32, 267-293.
Grant, S.B., List, EJ. and Lidstrom, M.E. (1993) Kinetic analysis of virus adsorption and
inactivation in batch experiments. W~ Resour. Res., 29, 2067-2085.
Hancock, C.M., Rose, J.B. and Callahan, M. (1998) Crypto and Giardia in US groundwater.
JA WWA, 90(3), 58-61.
Havelaar, AH., van Olphen, M. and Drost, Y.C. (1993) F--specific RNA bacteriophages are
adequate model organisms for ellteric viruses in fresh water. Appl. Environ. Microbiol., 59,
2956-2962.
Heisz, M., Chauret, C., Chen, P., Springthorpe, S. and Sattar, SA (1997) In vitro survival of
Cryptosporidium oocysts in nab.Jral waters. In Proc. International Symposium on Waterborne
Cryptosporidium, March 2-5, 1997, Newport Beach, California, (eds. C. Fricker, J.L. Clancy
and PA Rochelle),pp. 71-175, AWWA, Denver, Colorado,.
Howard, K W.F. (2001) Polluted groundwater -deadly lessons from Walkerton, Ontario, Canada.
In New Approaches to Characterising Groundwater F1uw, (eds. K.P. Seiler and S. Wobnlich),
pp. 521-525, Swets andZeitlinger, Balkema, Rotterdam.
Howard, G., Bogh, C., Goldstein, G., Morgan, J., Priiss, A, Shaw, R and Teuton, J. (2002).
Healthy villages: A guide for rommunities and community health workers. WHO, Geneva.
Hurst, CJ., Getba, C.P. and Cech, I. (1980a) Effects of environmental variables and soil
characteristics on virus survival in soil. AJPI. Environ. Microbiol., 40, 1067-1079.
Hurst, CJ., Gerba, C.P. Lance, J.C. and Rice, RC. (1980b) Survival of enteroviruses in
rapid-infiltration basins during 1he land application of wastewater. Appl. Environ. Microbiol.,
40,192.
Hurst, CJ., Wild, DK and Clark, RM. (1992) Comparing 1he accuracy of equation formats for
modelling microbial population decay rates. In Modelling the Metabolic and Physiologic
78 Protecting Groundwater for Health
Activities of Micro-organisms, (ed. CJ. Hurst), pp. 149-175, Jolm Wtley & Sons. Inc., New
York
Jansons, J, Edmonds, L. W., Speight, B. and Buceos, MR. {1989a) Movement of viruses after
artificial recharge. Water Res., 23, 293-299.
Jansons, J., Edmonds, L.W., Speight, B. and Buceos, M.R.{1989b) Survival of viruses in
grmmdwater. Water Res, 23, 301-306.
Keswick, B.~ Gerba, C.P., Secor, S.L, Cech, L (1982) Survival ofenteric viruses and indicator
bacteria in groundwater. J. Environ Sci. Health, Al 7, 903-SU.
Kolhoneo, L.K, Niskanen, M, HeinonenTanski, Ji, Martikaineo, P J, Saloneo, L. and Taipalineo,
L (1996) Groundwata quality in wells in central rural Finland: A microbiological and
radiochemical survey. Ambio, 25, 343-349.
Landry, E.F., Vaughn, JM. and Peaello, W.P. (1980) Poliovirus n:tention in 75-cm soil cores after
sewage and rainwater application. Appl Environ Microbial., 40, 1032.
Lawrence, A.R., Williams, AT., Brewerton, L.T, Barker, JA, Bird, Mand Ward, RS. (1996)
Pathogen transport in the Chalk aquifer: An assessment of the risk to chalk groundwater
resources. BGS Technical Report No. WD/96/48, Wallingfurd. ·
Lee, J.V. (1993) Contamination ofa well by the direct influence ofsurfilce water. In Proc. of the
Anmml Coriference of the American Waterworks Association, San Antonio, Texas, A WW A,
pp. 215-231.
Lefler, E. and Kott, Y. (1974) Virus Retention and Survival in Sand, Centre fur Research in Water
Resources, Austin, Teiw.
Lerner, D.N. and Barrett, MH. (1996) Urban groundwater issues in the Unired Kingdom.
Hydrogeology J., 4, 80-89.
Lewis, W J., Foster, S.S.D. and Drasar, B.S. (1982) Toe risk of groundwata pollution by on-site
sanitation in developing countries: A 1itera1un: review. Jntemat. Ref Centre for Wastes
Disposal, 01, 82.
Lieberman, R, Shadiz, L.C., Newport, B.S, Frebis, C.P., Saffi:rman, RS., Stetler, RE, Lye, D.,
Fout, G.S. and Dahling, D. (2002) Microbial Monitoring of Vulnerable Public Ground Water
Supplies. A WW ARF, Denver, Colorado.
Lieberman, RJ., Shadix, L.C, Newport, B.S., Crout, S.R., Buescher, S.~ Saffi:rman, R.S., Stetler,
R.E., Lye, D., Fout, GK and Dahling, D.R. (1994) Source Water Miaobial Quality of Some
Vulnerable Public Groundwater Supplies. A WW A, Denver, Colorado.
Lindsey, B.D., Raslxny, J.S. and Zimmerman, T.M (2002) Microbiological Quality of Water from
Noncommunity Supply Wells in Carbonate and Crystalline Aquifers of Pennsylvania. USGS,
Water-Resources InvestigationsReport0I-4268, Reston, Virginia
Loveland, J.P., Ryan,· J.N., Amy, G.L. and Harvey, R W. (1996) Toe reversibility of virus
attachment to mineral surfilces. Colloids and Surfaces. A: Physicochemical and Engineering
Aspects, 107, 205-221.
Macler, B.A. and Merkle, J.C. (2000) Current knowledge on groundwater microbial pathogens and
their control Hydrogeology J., 8, 29-40.
Mattler, BJ., Pasonne, J.C., Lods, G.F. and Drogue, C. (2000) Transport offtee and particulate-
a-;sociated bacteria in karst. J. Hydrology, 238, 179-193.
Matthess, G.A., Pekdeger, A and Schroeter, J. (1988) Pasistence and transport of bacteria and
viruses in grmmdwater-a conceptual evaluation. J. Contam. Hydro!., 2, 171-188.
McFeters, G.A, Bissonnette, GK, Jezeski, J., ThOIDSOn, C.A. and Stuart, D.G. (1974)
Comparative surviwl of indicator bacteria and enteric pathones in well water. Appl.
Microbial., 27, 823-829.
Medema, GJ., Bahar, M. and Schets, F.M. (1997) Survival of Cryptosporidium parvum,
Escherichia coli, fiiecal enterococci and Clostridium perfringens in river water. Influence of
temperature andautochthonous microorganisms. Water Sci. Technol., 35(11-12), 249-252.
Melian, R., Myrlian, N., Gouriev, A., Moraru, C., and Radstake, F. (1999) Groundwater quality and
rural drinking-water supplies in the Republic ofMoldova Hydrogeology J., 7, 188-196.
Pathogens: Health relevance, transport and attenuation 79
Meschke, J.S . (2001) Comparative Adsoiption. Persistence, and Mobility of Norwalk virus,
Poliovirus type 1, and F+RNA Coliphages in Soil and Grouoowater. PhD. thesis, School of
Public Health, University ofNol1h Carolina, Chapel Hill, Nol1h Carolina
Nasser, A, Tchorch, Y. and Fattal, B. (1993) Comparative survival ofE. coli, F+bacteriopbages,
HAVandpoliovirus 1 inwastewaterandgroundwater. Waier&i. Technol, 27,401-407.
Nasser, AM and Oman, S.D. (1999) QuantitatiVP. assessment of the inactivation of pathogenic and
indic:mrvirusesinnatural w.m-sources. Waler Res.,33, 1748-1752.
Oson, B.R (1993) Pathogen occmrence in source waters: fuclors affi:cting survival and growth. In
Safety of Water Disinfection' Balancing Chemical and Miavbial Risks, (ed. G.F. Crmm), ILSI
Press, WashingtonOC.
Pancomo, o.c., Evanshen, B.G., Campbell, W.F., Lambert, S., Curtis, S.K. and Woolley, T.W.
(1987) Infectivity and antigenicity reduction rates of human Rotavirus strain Wa in Fresh
Waters. Appl Environ Miavbiol., 13, 1803-1811.
Parshionikar, S.U., Willian-True, S., Fout, G.S., Robbins, D.E ., Syes, S.A, Cassady, J.D. and
Harris,.R (2003) WateJbome oulbrealc ofgaslmenteritis as!Deiated with a Norovirus. Appl
F.nviron. Miavbiol, 69(9), 5263-5268.
Penrod, S.L, Olson, T.M and Grant, S.B. (1996) Deposition kinetics ofnw viruses in packed beds
of quartz granular media umgmuir, 12, 5576-5587.
Powell, K.L., Barrett, M.H ., Pedley, S., Tdlam, JR, Stagg, KA, Gm.well, RB. and Rivett, MO.,
(2000) Enteric virus detection in groundw.lrer using a ~I trap. In Groundwater: Past
Achievements and Fulure Challenges, (ed. 0. T.M. Sililo), pp. 813-816, Balkema, Rotterdam.
Powell, K.L., Cronin, AA, Bam:tt, MR, Sell\\OOd, J. and Pedley, S. (2001a) Distribution of
Miavbiological Contaminants in Trias.sic Sandstone Urban Aquifers. Environment Agency
R&D Technical Report P2-255/IR, Bristol.
Powell, K.L., Cronin, AA, Pedley, S., Bam:tt, MR (2001) Microbiological quality of
groundwater in UK urban aquifers: do we know enough? In Proc. 3rd lntemalional
conference on Groundwater Quality, 'Groundwater Quality 2001', (eds. S. Thornton and S.
Oswald), Univasity ofSheflield, June 2001, pp. 236-238.
Powelson, D.K., Simpson, J.R and Gerba, C.P. (1991) Effects of organic matta on virus transport
in llllS3lmaed flow. Appl Environ. Miavbiol., 51, 2192-2196.
Redman, J.A, Grant, S.B~ Olson, TM., Amkins, J.M., Jackson, L~ Castillo, MS. and Ylllko,
W .A (1999) Physiochemical tnedlanisms responsible fur the filtration and mobilisalion of
filamentous bactaiophage in quartz sand. Water Res., 33(1), 43-52.
Redman, J.A, Grant, S.B., Olson, T.M., Hardy, M.E. and Estes, M.K. (1997) Filtration of
recombinant Norwalk virus particles and bacteriophage MS2 in quartz sand: Importance of
electrostatic interactions. F.nviron. Sci. Technol, 31, 3378-3383.
Rice, E.W., Johnson, C.R, Wild, D.K. and Reasoner, D.J. (1992) Survival of Escherichia coli
O157:H7 in drinking-water IIS.50Ciated with a waterborne disease oulbrealc of hemonbagic
colitis. Le«ers in Appl Microbiol., 13, 38-40.
Robertson, J.B . and Edberg, S.C. (1997) Natural protection of spring and wcll drinking water
against surfilre mia:obial contamination. I. Hydmgeological Parametas. Crit. Rev. in
Microbial., '13 , 143-178.
Robertson, L.J~ Campbell, AT. and Smith, H.V. (1992) Survival of Cryptosporidium parvum
oocysts under various environmental pres.sures. Appl. Environ. Microbiol, 58, 3494-3500.
Romero, J.C. (1972) The movement of bacteria and viruses through porous media In Water
Quality in a Stressed F.nvironment, (ed. W .A Pettyjohn), pp. 200-224, Btrrgess Publishing
Company,Minnesota.
Ryan, J.N ., Elimelech, M, Ard, RA, Harvey, R W. and Johnson, P.R (1999) Bacteriophage PRDl
and silica colloid transport and recovery in an iron oxide-coated sand aquifer. Environ. Sci.
Technol, 33, 63-73.
Sd:rijvai., J.F . and Hassanizadeb, S.M. (2000) Removal of viruses by soil passage: Overview of
modelling, processes and parameters. Crit. Rev. Environ. &i Technol., 30(1), 49-127 .
80 Protecting Groundwater for Health
Schijven, J.F., Hoogenboezem, W., Jfassaoizadd:t, S.M and Peters, J.H. (1999) Modelling removal
ofbacteriopbages MS2 and PRDI by dune infiltration a1 Castricum, 1he Netherlands. Wa1er
Resour. Res.,'35, 1101-llll.
Schijven, J.F., Medema, G~ Vogelaar, AJ. and l-lassanizaddJ, S.M (2000) Removal of
miaoorganisms by deep well injedion. J Contam. Hydro/., 44, 301-327.
Schijven JF, Berger P, Miettinen I. (2002a) Removal of pathogens, smmgates, indicators and toxins
using bank filtration. In Riverbank Filtration: Improving source water quality, ( eds. R
Cbittaranjan, G. Mlin and RB. Linsky), Klimer Academic, University ofHawaii.
Schijven, J.f., J'fassanindeb, S.M., de Bruin, HAM. (2002b) Two-site kinetic modeling of
bacteriopbages transport through columns of saturated dune sand. J Contam. Hydrol, 57, 259-
279.
Schijven, J.F., de Bruin, HAM., HassaoizaddJ, S.M~ de Roda Husman, AM (2003)
Bacteriophages and dostridium spores as indicator organisms fur removal of pathogens by
passage through saturated dune sand Water Res., 37, 2186-2194.
Simoni, S.F., Bosma, T.N.P., Hanns, H. and Zehnder, AJ.B. (2000) Bivalent cations increase bo1h
the subpopulation of adhering bacteria and their adhesion efficiency in sand cohmms. Environ.
Sci Technol., 34(6), 1011-1017.
Singleton. P . and Sainsbury, D. (1999) Dictionary of Microbiology and Mo/eadar Biology, 2nd
edn, John Wiley & Sons Ltd, New York.
Skilton, H. and Wheeler, D. (1988) Bacteriophage tracer experiments in gromdwater. J. Appl.
Bacteriol., 65(5), 387-395.
Skilton, H. and Wheeler, D. (1989) The applicalion of bacteriophage as 1racels in chalk aquirer
systems. J. Appl Bacterial., 66, 549-557.
Sobsey, M.D. (1983) Transport and :rate of viruses in soils. In Microbial Health Considerations of
Soil Disposal of Domestic Wartewaters, US EPA, Cincinnati, Ohio.
Sobsey, MD., Shields, PA, Haucbmao, F.H., Hazard, RL~ and Canton, L.W. (1986) Smvival and
transport of hepatitis A virus in soils, grmmdwater and wastewater. Water Sci. Technol., 18,
97-106.
Taylor, D.H., Moore, RS. and Sturman, LS. (1981) Influence of pH and elee1rolyte composition
on ad<ioiption of poliovirus by soils and minerals. Appl Environ. Miavbiol~ 42, 976.
US EPA (2000) National Primary Drinking Wmer Regulations: Ground Watec Rule. Proposed
Rule. Federal Register, 10, 30194-30274.
US Government (1996) Safe Drinking Watec Act, Public Law 104-182.
USGS (1998) Miavbiological Quality of Public Water Supplies in the {>-_ark Plateaus Aquifer
System, Missouri. Washington DC, USGS, RefType: Pamphlet
Vaughn, J.M., Landry, E.F. and Thomas, MZ. (1983) Entrainment of viruses from septic tank leach
fields through a shallow, sandy soil aquirer. Appl. Environ. Microbial., 45, 14 74-1480.
Wang, G. and Doyle, M.P. (1998) Smvival of enterbemonhagic Escherichia coli 0157:H7 in
water. J. Food Protection, 61, 662-667.
Wellings, M., Lewis, A.C., Mountain, C.W. and Pierce, L.V. (1975) Demonstration of viruses in
gromdwater after effluent discharge onto soil. Appl. Miuobiol., 29, 751.
West, J.M~ Pedley, S., Baker, SJ., Bmrott, L., Morris, B7 Storey, A, Ward, RS. and Barrett, M.
(1998) A Review of the Impact of Microbiological Contaminants in Groundwater, UK
Environment Agency, Rand D Technical Report Pl39, Bristol
Wimpenny, J.W.T. (1996) Ecological determinants ofbiofilm formation. Biofouling, 10(1-3), 43-
64.
Yab.ya, M.T., Galsomies, L., Gerba, C.P. and Bales, RC. (1993) Smvival ofbacteriophages MS2
andPRDl ingroWidwater. WaterSci Tech.,27,409-412.
Yates, M.V. IBld Yates, S.R (1988) Modelling miaobial :rate in the subsurface environment Crit.
Rev. Environ. Control, 17, 307-344.
Yates, M.V., Gerba, C.P. and Kelley, L.M (1985) Virus persistence in gromdwater. Appl Environ.
Mia-obiol., 49(4), 778-781.
4
Chemicals: Health relevance, transport
and attenuation
M Rivett, J. Drewes, M Barrett, J. Chilton,
S. Appleyard, HH Dieter, D. Wauchope and
J. Fastner
The presence of substances in grmmdwater may be affected by naturally occurring
proces.5t:S as well as by actions directly associated with human activities. Naturally
occurring processes such as decomposition of organic material in soils or leaching of
mineral deposits can result in increased concentrations of several substances. 1bose of
health concern include flooride, arsenic, nitrate, selenium, uranium, metals, and
radionuclides such as radon Problems of aesthetic quality and acceptance may be caused
by iron, manganese, sulphate, chloride and organic matter.
Sources of groundwater contamination associated with human activities are
widespread and include diffi.Jse as well as point source pollution like land application of
animal wastes and agrochemicals in agriculture; disposal practices of human excreta and
wastes such as leaking sewers or sanitation systems, leakage of waste disposal sites,
landfills, underground storage tanks and pipelines; and pollution due to both poor
practices and accidental spills in mining, industry, traffic, health care facilities and
military sites.
© 2006 World Health Organimion. Protecting Groundwater for Health: Managing the Quality of
Drinking-water Sources. Edited by 0. Schmoll, G. Howanl, J. Chilton and I. Chorus. ISBN:
1843390795. Published by IW A Publishing, London, UK.
82 Protecting Groundwater for Health
The exploitation of petroleum pnxiucts and the development of industrial chemistry
has given rise to a large number of organic chemicals, many of which can be fotmd in the
environment. Many organic chemicals are known to have potential hmnan health
impacts, and some of these may occur in drinking-water in heallh-relevant
coocentrations. In consequence, the list of those for which guideline values and national
quality standards have been developed has been continually added to and revised as data
on the occurrence of chemicals in water and new toxicological data emerge. Organic
chemicals commonly used by industry with known or suspected human health impacts
that are often encountered in groundwaters include, for example, aromatic hydrocarbons
such as benzene, toluene, ethylbenzene and xylenes (BTEX) as well as volatile
chlorinated hydrocarbons such as tetrachloroethene (PCE) and trichloroethene (fCE). A
diverse range of pesticides is also found in groundwaters. These are primarily, but not
exclmively, ascnbed to agricultural activities. Typically pesticide concentrations
encountered are low. In some cases they have exceeded regulatoiy limits for drinking-
water supplies, although often the regulatory levels exceeded were often much lower
than those based on public health considerations.
This chapter concentrates on the groups of chemical substances that are toxic to
humans and have reasonable potential to contaminate drinking-water abstracted fiom
grolilldwater. It provides folilldational knowledge of natural groundwater constituents
and anthropogenic groundwater contaminants and discus.ses their relevance to human
health, origin, and transport and attenuation in groundwater systems. The chapter is sub-
divided as follows: Section 4.1 provides introductory theory on the transport and
attenuation of chemicals in the subsurfuce; Sections 42 to 4.4 focus upon inorganic
chemicals -natural inorganic constituents, nitrogen species and memls respectively;
Sections 4.5 to 4.8 focus upon organic chemicals including an introductory section on
concepwal contaminant models and transport and attenuation theory specific to organic
contaminants followed by sections on some organic chemical groups of key concern -
aromatic hydrocarbons, chlorinated hydrocarbons and pesticides respectively; finally, the
chapter closes with a brief consideration of currently emerging issues (Section 4.9).
Further information on the individual chemicals disc~ in this chapter is available in
the WHO Guidelines for Drinking-water Quality, Volume I (WHO, 2004a), as well as
in detailed background documents on WHO's Water, Sanitation and Health website
(http://www.who.int/water _sanitation_ health/dwq/chemicals/enfmdex.html).
4.1 SUBSURFACE TRANSPORT AND ATTENUATION
OF CHEMICALS
Understanding of the transport and attenuation of chemicals in the subsurface is
fimdamental to effective management of risks posed by chemicals and their possible
impact on groundwater resources. A risk assessment approach to groundwater protection
incorporates the three-stage combination of source, pathway and receptor. All three must
be considered and understood to arrive at a balanced view of the risks to health of
groundwater users. Informed consideration of the pathway, which in the context of this
monograph means transport through the groundwater system, is vital. This not only
includes consideration of the general and local hydrogeologic characteristics covered in
Chemicals: Health relevance, transport and attenuation 83
Oiapters 2 and 8, but also the transport and attenuation of chemicals within that pathway.
The latter depend upon the properties of the chemical itself: particularly those properties
that control interactions of the chemical with the subsurface regime, a regime that
includes not only the host rock and groundwater, but other natural and anthropogenic
chemical oonstituents present as well as microbial life.
Within the overall transport process, attenuation processes may cause movement of
the chemical to differ from that of the bulk flowing grotmdwater, for example dispersion,
sorption and chemical or biological degradation of the chemical. Such attenuation
processes potentially act to mitigate the impact of chemicals and are a fimction of both
the specific chemical and geologic domain. Indeed, attenuation may vary significantly
between individual chemicals and within different geological settings. In recent years
natural attenuation of organic contaminants has been increasingly recognized to play an
important role in many aquifer systems leading to monitored natural attenuation
becoming a recognized remedial strategy to manage risks to groundwater at some
contaminated sites (EA, 2000).
This section provides an overview of the key processes that control the transport and
attenuation of chemicals in groundwater. Elaboration of some of the more specific
attenuation processes is also included in later sections. Further details may be found in
the following texts and references therein: Freez.e and Cherry (1979), Appelo and Postma
(1993), Stumm and Morgan (1996), Domenioo and Schwartz (1998), Bedient et al.
(1999), Fetter (1999) and Schwartz and Zhang (2003).
4.1.1 Natural hydrochemical conditions
It is important to understand at the outset the natural hydrochemical conditions that exist
in a given aquifer system, as these provide the baseline from which quality changes
caused by human impacts can be determined. The natural hydrochemical oonditions may
also affect the behaviour of some pollliants. Because groundwater movement is typically
slow and residence times long, there is potential for int.eraction between the water and the
rock material through which it passes. The properties of both the water and the material
are therefore important, and natural groundwater quality will ·vary from one rock type to
another and within aquifers along groundwater flow paths. Water is essentially a highly
polar liquid solvent that will readily dissolve ionic chemical species. Rock material is
predominantly inorganic in nature and contact of flowing groundwater with the rock may
dissolve inorganic ions into that water, i.e. dissolution of the rock occurs. 'Major ions '
present are the anions nitrate, sulphate, chloride and bicarbonate and the cations sodium,
potassium, magnesium and calcium. Ions typically present at lower concenlration,
'minor ions ', include anions such as fluoride and bromide and a wide variety of metal
ions that are predominantly cations. Combined, the total inorganic concentration within
the water is referred to as the total dissolved solids (IDS).
Natural grotmdwater quality changes start in the soi~ where infiltrating rainfull
dissolves carbon dioxide fiom biological activity in the soil to produce weak carbonic
acid that may assist removal of soluble minerals fiom the underlying rocks, e.g. calcite
cements. At the same time, soil organisms consume some of the oxygen that was
dissolved in the rainfall. In temperate and humid climates \\-1th significant recharge,
84 Protecting Groundwater for Health
groundwater moves relatively quickly through the aquifer. Contact time with the rock
matrix is short and only madly soluble minerals will be involved in reunions.
Groundwater in the outcrop areas of aquifers is likely to be low in overall chemical
content, Le. have low major ion contents and low ThS, with igneous rocks usually
having Ions dissolved constituents than sedimentary rocks (Hcm, 1989). In coastal
regions, sodium and chloride may exceed calcium, magnesium and bicarbonate and the
presence of soluble cement between the grains may allow major ion concentrations to be
increased. Grotmdwaters in carbonate rocks have p1-1 above 7, and mineral contents
usually dominated by bicarbonate and calcium.
In many small and shallow aquifers the hydmchemisrry does not evolve (other_
However, the baseline natural quality of groundwater may vary spatially within the same
aquifer if the mineral assemblages vary, and also evolves with time as the water moves
along groundwater flow lines. Han aquifer dips bekaw a confining layer (Figure 2.5), a
sequence of hydrochemical processes occurs with progressive distance downgradient
away from the outcrop. including precipitation of some solids when relevant ion
concentrations reach saturation levels far a solid mineral phase. These processes have
been clearly observed in the United Kingdom, where the geological history is such that
all three of the major aquifers eichibit the sequence shown in Figure 4.1, which has been
characterized by sampling transacts of abstraction boreltoles across the aquifers
(Edmunds et at,1987).
sw,rearb7,5 ---•`
�o
= tr ca>auntbi E,rmr. _ _ . Surface w r
sod omaior4amle nat.-z m�s rodec5m j��
Xy hkn
Sodiar-chloride hour
Figure 4.1. Scticaudic mpritseniallori of downgradient bydrocheinical changes
In the recharge area oxidizing conditions occur and dissolution of calcium and
bicarbonate dominates. As the water continues to move downgradient. further
modifications are at fast limited, By observing the redox potential (F.c) of abstracted
groundwater. a sharp redox barrier is detected beyond the edge of the confining layer,
corresponding to the complete exhaustion of dissolved oxygen. Bicarbonate increases
and the pH rises until buffering occurs al about Si. Sulphate concentrations remain
stable in the oxidizing water. but decrease suddenly just beyond the redox boundary due
to sulphate reduction_ Groundwater becomes steadily more reducing as it flows
dnwrtgradient, as demonstrated by the presence of sulphide. increase in the solubility of
iron and manganese and denitrification of nitrate_ After some farther kilometres, sodium
begins to increase by ion exchange at the expense of calcium, producing a natural
softening of the water. Eventually, the available calcium in the water is exhausted._ but
sodium continues to increase to a level greater than could be achieved purely by cation
exchange. As chloride also begins to increase. this marks the paint at which recharging

Chemicals: Health relevance, transport and attenuation 85
water moving slowly down 1hrough the aquifer mixes with much older saline water
present in the sediments (Figure 4.1). The observed hydrochemical changes can thus be
interpreted in terms of oxidation/reduction, ion exchange and mixing processes.
In arid and semi-arid regions, evapotranspiration rates are much higher, recharge is
less, flow paths longer and residence times much greater and hence much higher levels
of natural mineralization, oflen dominated by sodium and chloride, can be eocountered.
Thus the major ion contents and IDS are oflen high. In some desert regions, even if
groundwater can be found it may be so salty (extremely high IDS) as to be undrinkable,
and the difficulty of meeting even the most basic domestic requirements can have serious
impacts on health and livelihood.
Natural variations in pH and oxygen status are also important and are not restricted to
deep environments. Many groundwaters in tropical regions in weathered basement
aquifers and alluvial sequences have low pH, and the reducing conditions which prevail
can promote the mobilization of metals and other parameters of health significance such
as arsenic. Thus prevailing hydrochemical conditions of the groundwater that are
naturally present and develop need to be taken into accomt when: (i) developing
schemes for groundwater abstraction for various uses and in protecting groundwater; and
(ii) considering the transport and attenuation of additional chemicals entering
groundwaters due to human activity.
4.1.2 Conceptual models and attenuation processes
Effective prediction of transport of chemical pollutants through a subsurface
groundwater system and associated assessments of risk requires a valid conceptual
model of the contaminant migration scenario. 1he c~ical contaminant conceptual
model is one of a near-surfuce leachable source z.one where chemical contaminant is
leached, i.e. dissolved/solubilized, into water infiltrating through the source (Figure 42).
A dissolved-phase chemical solute plume subsequently emerges in water draining fiom
the base of the contaminant source z.one and moves vertically downward through any
unsaturated z.one . present 1he dissolved solute plume ultimately penetrates below the
water table to subsequently migrate laterally in the flowing groundwater. Many sources,
e.g. a landfill, chemical waste lagoon, contaminated industrial site soils, pesticide
residues in field soils, may have sufficient chemical mass to enable them to act as long-
term generators of dissolved-phase contaminant phnnes; potentially such sources can last
decades. This will lead to continuous dissolved-phase plumes extending fiom these
sources through the groundwater pathway that grow with time and may ultimately reach
distant receptors unless attenuation processes operate. This near-surface leachable source
-dissolved-plume conceptual model is the model most frequently invoked and the one to
which groundwater vulnerability and protection concepts and groundwater risk-
assessment models are most easily applied. It is important to note, however, that the
above conceptualization may be too simplified and alternative conceptual models need to
be invoked in some cases, most notably for non aqueous phase liquids (NAPLs) as
discussed in Section 4.5.
86 Protecting Groundwater for Health
Contaminated
soli
SOURCE
Cantarniuted Y YRol
land
Pore war
Dissolved
4 .•
• PATHWAY
GrolverNater sone
RECEPTOR
Drini]ng.waler
abstraction
4
Unsat/riled
tone
Lone
iAlternator
zone
It
sates 1�
Aquifer base
Figure 4.2. classical contaminant conceptual rnodcl
Attenuation processes operative in the groundwater pathway, both for unsaturated and
sated zones, are summarized below and briefly described in the text that follows.
Further dctaiis may be found in the texts referenced in other sections ofthis chapter.
DEF ■ AdvecOion and asEspenion
Advection Ls the trmarport of dixcolvedsolute mars present in
groundwater due to the bulk flow (movement) of this groundwater.
Advec Lon alone (with no dispersion or reactive processes °curing}
would cause a non -reactive solute to advert (move) at die mean
groundwater pore veloeily. All solutes undergo advection, Jv wever,
reacirve solutes are subject to influences by other processes detailed
below.
Molecular di Thsion is the movement ofsolute ions in the direction
of the concentration gradient front high towards low concentrations.
It affects all solutes.
Mechanical dispersion r.on sPs spreading ofsolute and hence
dilution of concentrations, it arises from the tortuosity of the pore
channels in a gratudor agterferand ofthefractures in a consolidated
aquifer and the different speeds ofgroundwrtter withinflow
ow
channels of varying width It affects all solutes.

Advection
Chemicals: Health relevance, transport and attenuation 87
Relardation
Sorption is a process by which chemicals or organisms become
al/ached to soils and/or the geologic rock material (aquifer solids)
and removed from the water. Often the sorption process is
reversible and solutes desorb and hence dissolved-solute plwnes are
retarded, rather than solutes being permanently retained by the
solids.
Cation exchange is the inJerchange between cations in solution and
cations on the swfaces of clay particles or organic colloids.
Filtration is a process that affects particulate contaminants (e.g.
organidinorganic colloids or microbes) rather than dissolved
solutes. Particles larger than pore throat diameters or fracture
apertures are prevented from moving by advection and are
therefore attenuated within the soil or rock.
Reaelions and transformations of chemicals
Chemical reactions (abiotic reactions) are classical chemical
reactions that are not mediated by bacteria. They may include
reaction processes such as precipitation, hydrolysis, complex:ation,
elimination, substitution, etc., that tran.eform chemicals into other
chemicals and potentially alter their phase/state (solid, liquid, gas,
dissolved).
Precipitation is the removal of ions from solution by the formation of
insoluble compounds, ie. a solid-phase precipitate.
Hydrolysis is a process of chemical reaction by the addition of
water.
Complexation is the reaction process by which compounds are
formed in which molecules or ions form coordinate bonds to a metal
atom orion.
Biodegradation (biotic reactions) is a reaction process that is
facilitated by microbial activity, e.g. by bacteria present in the
subsurface. Typically molecules are degraded (broken down) to
molecules of a simpler structure that often have lower taxicity.
As descnbed in Chapter 2, groundwater moves due to the presence of a hydraulic
gradient and may be characterized by the Darcy velocity q (alternatively named the
specific discharge). The Darcy velocity may be calculated via Darcy's law and is the
product of the geologic media hydraulic conductivity K and the gro1IDdwater hydraulic
gradient i. The actual mean groundwater pore (linear) velocity of groundwater,
henceforth referred to as the 'grolilldwater velocity' v differs from the Darcy velocity as
flow can only occur through the effective porosity n,, of the fonnation. The gro1IDdwater
velocity may be quantified by modifying the Darcy equation:
V =-Ki In,, (Eqn. 4.1)
88 Protecting Groundwater for Health
Advection is The transport of dissolved solutes in groundwater due to the bulk
movement of groundwater. The mean advecdve velocity of non -reactive solutes is equal
to the groundwater velocity, v (Eqn. 4.3) and is normally estimated by knowledge of the
Equation 4.1 hydrogeologic l paraenelers. Occasionally v may be estimated from the
mean position of a solute plume, typically within a groundwater trader test. (Mackay el
aL, 1986). Reactive solutes also advect with the flowing groundwater, however, their
velocities are modified due to co -occurrence of attenuation processes.
Dispersion
All reactive and non -reactive solutes will undergo spreading due to dispersion, causing
dissolved -phase plumes to broaden both along and perpendicular to the groundwater
flow direction (Figure 43). Dispersion is most easily observed for 'conservative' non-
readive solutes, such as chloride, as these only undergo advection and dispersion_
Dispersion causes mixing of the dissolved -solute plume with umcontaminated water and
hence concentration dilution as welt as plume spreading. Longitudinal dispasion.
spreading in the direcon of predominant groundwater flow, is greatest causing solutes to
move at greater or less than the mean advativc velocity v. Solute spreading is due to
mechanical elusion that can arise at the pore -scale due to (Fetter, 1999): (i) fluids
moving faster at pore centres due to less friction.; (i} larger pores allowing faster fluid
movement; (iii) routes of varying tortuosity around grains_ At a larger scale, macro -
dispersion is controlled by the distribution of hydraulic conductivities in the geologic
domain; greater geological heterogeneity resulting in greater plume spreading. The above
processes cause increasing dispersion with plume travel distance, i.e. dispersion is scale
dependent (Gelhar, 1986: Fetter, 1999).
x
CO 0]
f \c(t1)
f
x2 =
Flow
Direction
(Velocity V)
c ltiz} Longitudinal
F Spreading
x
Transverse
c(12) Spreading
y
Figure 43. Dispassion ofa pulse release of dissolved -solute plume
Plume dispersion in other directions is much reduced. Transverse horizontal
spreading may arise from fowpath tortuosity and molecular diffusion due to plume

Chemicals: Health relevance, transport and attenuation 89
chemical-concentration gradients. Transverse vertical spreading occurs for similar
reasons, but is generally more restricted due to predominantly near-horiznntal layering of
geologic strata. Overall, a hydrodynamic dispersion coefficient D is defined for each
direction (longitudinal. transverse horiznntal, transverse vertical):
D=av+D* (F.qn42)
which is seen to depend upon D*, the solute's effective diffusion coefficient and a the
geologic media dispersivity. Dispersion parameters are most reliably obtained from
tracer tests or, less reliably, at the larger (>250 m) scale, by model fitting to existing
plumes. Collated values have yielded simple empirical relationships to estimate
dispersion, e.g. the longitudinal dispersivity is often approximated to be 0.1 (10 per cent)
of the mean plume travel distance (Gelhar, 1986). However, such relationships are very
approximate.
Retardation
The processes that cause retardation (slowing down) of dissolved-solute plume migration
include filtration, sorption and cation exchange. Filtration is a proces.s that affects
particulate contaminants (e.g. organicfmorganic colloids or microbes) rather than
dissolved solutes, the key focus here. Sorption is a proces.s by which chemicals or
organisms become attached to soils and/or the geologic rock material (aquifer solids) and
are removed from fue water. Often fue sorption process is reversible and solutes desorb
back into fue water phase and hence dissolved-solute plumes are retarded, rather than
solutes being permanently retained by the solids. Preferred sorption sites depend upon
the chemical solute properties, in general clay strata or organic matter within the geologic
solid media are key sorption sites. Such sites may, however, be limited and sorption to
other mineral phases, e.g. iron oxyhydroxides, may become important in some cases.
Sorption processes normally lead to a Retardation Factor, R;, being defined that is the
ratio of the mean advective velocity (conservative solute velocity) (v) to fue mean
velocity offue retarded sorbing solute plume (v;):
(Eqn 4.3)
Typically R.; is not estimated from Equation 4.3, rather various methods may be used to
estimate R.; relating to the specific chemical nature of the sorption interaction and a
relevant sorption coefficient (e.g. see Section 4.52). Sorption-related processes can be
sensitive to the environmental conditions. For example, relatively small pH changes may
cause significant changes to the mobilization of metals or perhaps organic contaminants
that are themselves acids or bases, e.g. phenols or amines.
Reactions and transformations of chemicals
Many chemicals undergo reaction or transformation in the subsurface environment In
contrast to retardation, contaminants may be removed rather than simply slowed down.
Reactions of harmful chemicals to yield benign products prior to arrival at a receptor are
the ideal, e.g. many toxic hydrocarbons have potential to biodegrade to simple organic
acids (of low health concern and themselves potentially degradable), carbon dioxide
(bicarbonate) and water. Transformation often causes a deactivation (lowering) of
90 Protecting Groundwater for Health
toxicity. Reactions and/or transformations incorporate processes such as chemical
precipitation, complexation, hydrolysis, biodegradation (biotic reactions) and chemical
reactions (abiotic reactions).
Chemical precipitation and complexation are primarily important for the inorganic
species. The formation of coordination complexes is typical behaviour of transition
metals, which provide the cation or central atom. Ligands include common inorganic
anions such as er, F, Br-, so/-, PO/-and CO/-as well as organic molecules such as
amino acids. Such complexation may facilitate lhe transport of metals.
Biodegradation is a reaction process mediated by microbial activity (a biotic
reaction). Naturally present bacteria may transform the organic molecule to a simpler
product, e.g. anolher organic molecule or even COi. Biodegradation has wide
applicability to many organic chemicals in a diverse range of subsurface environments.
Rates ofbiodegradation vmy widely, some compounds may only degrade very slowly,
e.g. high molecular weight polynuclear aromatic hydrocarbons (P Alls) 1hat are relatively
recalcitrant (unreactive). Rates are also very dependent upon environmental conditions,
including redox, microbial populations present and lheir activity towards contaminants
present
Abiotic reactions, classic chemical reactions not mediated by bacteria, have been
found to be of fairly limited importance in groundwater relative to biodegradatioIL For
example, a few organics, e.g. 1,1,1-trichloroethane (1,1,1-TCA) and some pesticides,
may readily undergo reaction wilh water (hydrolysis), olhers such as the aromatic
hydrocarbon benz.ene are essentially unreactive to water and a range of olher potential
chemical reactions.
Potential for attenuation
Potential for attenuation processes to occur varies wilhin the various subsurface z.ones,
i.e. soil unsaturated and saturated z.one. Attenuation processes can be more effective in
1he soil rather 1han aquifers due to higher clay contents, organic carbon, microbial
populations and replenishable oxygeIL This makes 1he soil a very important first line of
defence against groundwater pollution, often termed 'protective layer'. Consideration of
1he soil and its attenuation properties is a key factor in assessing the vulnerability of
groundwater to pollution (Chapter 8). This also means 1hat where lhe soil is 1hin or
absent lhe risk of groundwater pollution may be greatly increased. Many human
activities that give rise to pollution by-pass lhe soil completely and introduce pollutants
directly into 1he unsaturated or even saturated z.ones of aquifers. Examples include
landfills, leaking sewers, pit latrines, transportation routes in excavated areas and
highway drainage.
4.2 NATURAL INORGANIC CONSTITUENTS
The occurrence of natural constituents in groundwater varies greatly depending on lhe
nature of lhe aquifer. In general, aquifers in magmatites and metamorphic rocks show
lower dissolved contents 1han in carbonate or sedimentary rocks. The mobility and thus
lhe concentration of nearly all natural groundwater constituents can be significantly
Chemicals: Health relevance, transport and attenuation 91
influenced by changes of physical and chemical conditions in groundwater through
hlllil3Il activities.
Fluoride and arsenic are now recogniz.ed as the most serious inorganic contaminants
in drinking-water on a worldwide basis. Fmther natural constituents that can cause a
public health risk addressed in this chapter (in alphabetical order) are selenium, radon
and uranium. Although nitrate has occasionally been fmmd naturally in health-relevant
concentrations, in most cases these are caused anthropogenically, and therefore nitrate is
addressed in Section 43.
NOTE ►
4.2.1 Arsenic
Health aspects
Arsenic, fluoride, seleniwn. radon and uraniwn are examples of
health-relevant naturally occwring groundwater constituents. Their
concentrations in groundwater are strongly_ dependant on
hydrogeologica/ conditions. In some settings, nitrate may naturally
occur in health-relevant concentrations.
The International Agency for Research on Cancer (IARC) has classified arsenic (As) as a
Group 1 human carcinogen (IARC, 2001). The health effects of arsenic in drinking-
water include skin cancer, internal cancers (lung, bladder, kidney) and peripheral
vascular disease (blackfoot disease). Evidence of chronic arsenic poisoning includes
melanosis (abnormal black-brown pigmentation of the skin), hyperlceratosis (thickening
of the soles of the feet), gangrene and skin and bladder cancer (WHO, 2003a). Arsenic
toxicity may not be apparent for some time but the time to appearance of symptoms and
the severity of effects will depend on the concentration in the drinking-water, other
sources of exposure, dietary habits that may increase arsenic concentrations in staple
dishes and a variety of other possible nutritional factors. •
While earlier maximum allowable concentrations recommended by WHO for arsenic
in drinking-water were higher, in 1993 the provisional WHO guideline value for arsenic
in drinking-water was reduced to 10 µg/1 based on concerns regarding its carcinogenicity
in humans (WHO, 2004a). Regulations in some countries, e.g. the Emopean Union
(EU), Japan and the USA follow this guideline value and Australia has established a
drinking-water standard for arsenic of7 µg/1. A number of countries operate at present at
a 50 µg/1 standard, which corresponds to the provisional WHO guideline value before
1993. Some national authorities are currently seeking to reduce their own limits in line
with the WHO guideline value. It is important to realize that the WHO Guidelines
emphasize the need for adaptation of national standards to local health priorities, social,
cultural, environmental and economic conditions and also advocate progressive
improvement that may include interim standards. Fmthennore, the WHO Guidelines
emphasises the scientific uncertainty of the dose-response curves at low intakes and thus
in deriving the guideline value of 10 µg/1. For improving public health benefits, other
issues may therefore take priority over upgrading the sensitivity of analytical facilities for
92 Protecting Groundwater for Health
detecting lower concentrations or investing in upgrading drinking-water supplies to
reduce arsenic concentrations 1D levels below 50 µwJ.
In recent years both the WHO guideline value and CUITeDt national standards for
arsenic have been found 1D be frequently exa:eded in drinking-water sources. The scale
of the arsenic problem in terms of population exposed 1D high arsenic concentrations is
greatest in Bangladesh with between 35 and 77 million people at risk (Smith et al.,
2000). However, many other countries are also faced with elevated arsenic
concentrations in grmmdwater, such as Hllllgary, Chile, Mexico, nonheast Canada, the
western USA and many countries in South Asia More detailed information on
occurrence and health significance of arsenic can be found in 'Arsenic in Drinking-
water' (WHO, InPress).
Occurrence
Arsenic is an ubiquitoll'! element folllld in soils and rocks, natural waters and organisms.
It occurs naturally in a number of geological environment<;, but is particularly common in
regions of active volcanism where it is present in geothermal fluids and also occurs in
sulphide minerals (principally arsenopyrite) precipitated from hydrothermal fluids in
metamorphic environments (Hem. 1989). Arsenic may also accumulate in sedimentary
environments by being co-precipitated with hydrous iron oxides or as sulphide minerals
in anaerobic environments. It is mobilized in the environment through a combination of
natural processes such as weathering reactions, biological activity and igneous activity as
well as through a range of anthropogenic activities. Of the various routes of exposure 1D
arsenic in the environment, drinking-water probably poses the greatest threat to human
health.
Background concentrations of arsenic in groundwater in most countries are less than
IO µwJ. However, surveys performed in arsenic-rich areas showed a very large range,
from <0.5 to 5000 µwJ (Smedley and Kinruburgh, 2001). Cases of large scale naturally
occurring arsenic in groundwater are mainly restricted 1D hydrogeological environments
characterized by young sediment deposits (often alluvilDil), and low-lying flat conditions
with slow-moving groundwater such as the deltaic areas forming much of Bangladesh.
Investigations by WHO in Bangladesh indicate that 20 per cent of 25 000 boreholes
tested in that colllltry have arsenic concentrations that exa:ed 50 µwJ. High
concentrations of arsenic in groundwater also occur in regions where oxidation of
sulphide minerals (such as arsenopyrite) has occwred (Alaerts et al., 2001 ).
Arsenic concentration in German groundwater downstream of abandoned waste
disposal sit.es was found to have a mean concentration of61 µwJ (n = 253 sit.es) due 1D
arsenic leaching from domestic coal ashes deposited with household wastes. In contrast,
the mean arsenic concentration in uncontaminated aquifers is 0.5 µwJ (n = 472 sites)
(Kerndorff et al., l 992).
Transport and attenuation
The concentration of arsenic in natural waters is normally controlled by solid-solution
interactions, particularly in groundwater where the solid/solution ratio is large. In most
soils and aquifers, mineral arsenic interactions are likely to dominate over organic
matter-arsenic interactions, although organic matter may interact to some extent through
its reactions with the surfaces of minerals (Smedley and Kinrnburgh, 2001 ). One of the
Chemicals: Health relevance, transport and attenuation 93
best correlations between the concentration of arsenic in sediments and other elements is
with iron. These interactions have also been the basis for the use of iron, almninium and
manganese salts in water treatment for arsenic removal.
Arsenic shows a high sensitivicy to mobilization at the pH values typically found in
groundwater (pH 6.5-8.5) and under both oxidizing and reducing oonditions. Arsenic can
occur in the environment in several oxidation states (-3, 0, +3 and +5) but in natural
waters is mostly found in inorganic oxyanion forms as trivalent arsenite (As(lll)) or
pentavalent arsenate (As(V)). Redox potential (F,i,) and pH are the most important factors
controlling arsenic speciation. Relative to the other oxyanion-fonning elements, arsenic
is among the most problematic in the environment because of its mobility over a wide
range of redox conditions (Smedley and Kinniburgh, 2001 ). Under oxidizing conditions,
~Aso4• is dominant at low pH (less than -pH 6.9), while at higher pH, HAsO/
becomes dominant (H.0S()4 and AsO/· may be present in extremely acidic and alkaline
conditions, respectively). Under reducing conditions at less than-pH 92, the uncharged
arsenat.e(III)-species (H.0S()3) will predominate.
Transport is largely controlled by the aquifer conditions, particularly by adsorption on
ferric oxohydroxides, humic substances and clays. Arsenic adsorption is most likely to be
non-linear, with the rate of adsorption disproportionally decreasing with increasing
concentrations in groundwater. This leads to reduced retardation at high concentrations.
Since different arsenic species exlubit different remrdation behaviour, arsenate (V) and
arsenate (Ill) should travel through an aquifa with different amounts of interactions
resulting in different velocities and increased separation along a flow path. This was
demonstrated by Gulens et al. (in Smedley and Kinniburgh, 2001) using controlled soil-
column experiments and various groundwaters. They showed that: (i) As(III) moved five
to six times faster than As(V) under oxidizing conditions ( at pH 5. 7); (ii) with a 'neutral'
groundwater (pH 6.9) under oxidizing conditions, As(V) moved much faster than under
(i) but was still slower than As(III); (iii) under reducing conditions (at pH 8.3), both
As(III) and As(V) moved rapidly through the column; (iv) when the amount of arsenic
injected was substantially reduced, the mobility of the As(IIl) and As(V) was greatly
reduced.
There is no process in the subsurface that alters arsenic species beside precipitation
and adsorption. 1f groundwater with elevated arsenic levels is used for drinking-water
supply, then treatment should be applied. There has been increasing research into this
area and a mnnber oflow-cost household treatment technologies are available. Data from
studies in Bangladesh suggest that low-cost technologies can remove arsenic to below
0.05 mg/I and sometimes lower (Ahmed et al, 2001). Technologies are also available for
system treatment including activated alumina, chemical precipitation and reverse
osmosis (for arsenate). However, in some situations, source substitution or mixing is
preferable to arsenic removal (Alaerts et al., 2001).
4.2.2 Fluoride
Health rupects
Because fluoride is widely dispersed in the environment., all living organisms are widely
exposed to it and tolerate modest amounts. 1n humans, fluoride has an affinity for
94 Protecting Groundwater for Health
accumulating in mineralizing tissues in the body, in ymmg people in bone and teeth, in
older people in bone, and incorporation of fluoride into the matrix of teeth during their
formation is protective against dental caries.
Health problems =iated with the oondition known as fluorosis may occur when
fluoride ooncentrations in groundwater exceed 1.5 mg'l: staining of the tooth enamel
may beoome apparent ( dental fluorosis) and, with oontinued exposure, teeth may beoome
extremely brittle. The incidence and severity of dental fluorosis, and the much more
serious skeletal fluorosis, depend on a range of factors including the quantity of water
dnmk and exposure to fluoride from other sources, such as from high fluoride ooal in
China. Nutritional status may also be important. Estimates based on studies from China
and India indicate that for a total intake of 14 mg/day there is a clear excess risk of
skeletal adverse effe.cts, and there is suggestive evidence of an increased risk of effects on
the skeleton at total fluoride intakes above about 6 mg/day (WHO, 2004b). In its most
severe form, this disease is characteriz.ed by irregular bone deposits that may cause
arthritis and crippling when occurring at joints.
The WHO guideline value for fluoride is 1.5 mg'l since 1984 (WHO, 2004a). The EU
maximum admissible ooncentration for fluoride in drinking-water is 1.5 mg'l. The US
Environmental Protection Agency (US EPA) set an enforceable primary maximum
oontaminant level of 4 mg'l in water systems to prevent crippling skeletal fluorosis. A
secondary contaminant level of2 mg'l was reoommended by US EPA to protect against
ol!jectionable dental fluorosis. In setting national standards for fluoride, it is particularly
important to consider volumes of water intake (which are affected by climatic
conditions) and intake of fluoride from other sources (e.g. food. air). Where higher
fluoride ooncentrations occur in groundwater used as drinking-water source, treatment
and/or change or mixing with other waste sources oontaining lower fluoride levels is
necessary in order to meet drinking-water standards. In areas with high natural fluoride
levels, the public health benefits of investments in the treatment neces.saiy to meet the
WHO guideline value may need to be balanced against other priorities for optimising
public health benefits.
More detailed information on occurrence and health significance of fluoride can be
found in 'Fluorides in Drinking Water' (Bailey et al., In Press).
Occurrence
Fluoride (F) naturally oc.curs in rocks in many geological environments (Hem, 1989) but
fluoride concentrations in groundwater are particularly high in groundwater associated
with acid volcanic rocks, e.g. in Sudan, Ethiopia, Uganda, Kenya and Tanzania (Bailey
et al., In Press). High concentrations of fluoride also occur in some metamorphic and
sedimentary rocks that contain significant amounts of fluoride-bearing minerals such as
fluorite and apatite. Fluoride in water supply based on groundwater is a problem in a
number of countries and over 70 million people worldwide are believed to be at risk of
adverse health effects from consumption of water oontaining high levels of fluoride.
India and China have particular problems and estimates suggest up to 60 million are
affected in these two countries alone.
Exposure to fluoride from drinking-water depends greatly on natural circumstances.
Levels in raw water are normally below 1.5 mg'l, but groundwater has been found to
Chemicals: Health relevance, transport and attenuation 95
oontain >50 mgll in some areas rich in fiuoride-oontaining minerals. For example, in
Kenya, 61 per cent of groundwater samples oollected nationally from drinking-water
wells exceeded I mgll (Bailey et al, In Press). In general high fluoride concentrations in
groundwater show a strong positive oorrelation with dissolved solids, sodium, and
alkalinity, and a strong negative oorrelation with hardness.
Transport and attenuation
The concentration of fluoride ions in grolllldwater is driven by the presence of calcium
ions and the solubility product offluorite (CaF2}. In equilibrium, a calcium ooncentration
of 40 mgll equates to a ooncentration of 32 mgll fluoride. In groundwater with a high
ooncentration of calcium ions, fluoride ooncentrations rarely exceed I mgll. Substantially
higher fluoride concentrations in grolllldwater are usually caused by a lack of calcium.
During high percolation rates, Fliihler et al. (1985) observed increased fluoride
ooncentration in the leachate of fluoride-enriched soils due to a limited additional
delivery of calcium.
In groundwater with a high pH (>8) and dominated by sodium ions and carbonate
species, fluoride ooncentrations commonly exceed 1 mg/I. and concentrations in excess
of 50 mgll have been recorded in grolllldwater in South Afi-i.ca, and in Arizona in the
USA (Hem, 1989). Moreover, the fluoride-ion (F) can interact with mineral surfuces, but
is substituted by hydroxyl-ions at high pH values. Hem (1989) observed a fluoride
concentration of 22 mgll in a caustic thermal grolllldwater (pH 9.2, 50 °C) in Owyhee
Collllty, Idaho. Fluoride ions form strong oomplexes especially with aluminium,
beryllium and iron (Ill).
4.2.3 Selenium
Health aspects
Selenium is an essential trace element with a physiologically required intake of about
I µg per kg body weight and day for adults. Deficiencies of selenium in diets may cause
a number of health effects, although few reports of clinical signs of deficiency are
available. However, the range of concentrations of this element in food and water that
provide health benefits appears to be very narrow. When ingested in excess of nutritional
requirements in food and drinking-water, selenium can cause a number of acute and
chronic health effects including damage to or loss of hair and fingernails, finger
deformities, skin lesions, tooth decay and neurological disorders (WHO, 2003b ).
Although drinking-water generally acoollllts for less than 1 per cent of the typical
dietary intake of selenium, in some circumstances naturally-occurring ooncentrations of
selenium in grolllldwater may be sufficiently high to cause health problems. The WHO
guideline value for selenium in drinking-water is 0.01 mgll (WHO, 2004-a).
Occurrence
Selenium has similar chemical properties and behaviour to sulphur (Hem, 1989), and is
oommonly associated with metal sulphide minerals in mineral deposits in a wide range
of igneous rocks and with sulphur-rich ooal. Sedimentary rocks and overlying soil in
some regions may have high backgrolllld concentrations of selenium. In the western part
of the USA, these are associated with uranium and vanadium mineralization in shales
96 Protecting Groundwater for Health
and sandstones. In some semi-arid areas in China and India, selenium reaches high
concentrations in soil and accumulat.es in plant tissue. Runoff from irrigated agriculture
on seleniferous soil may contain dissolved selenium concentrations of up to l mg')
(Hem, 1989), and groundwater in these areas also typically contains high concentrations
of leached selenium (Barceloux, 1999). Groundwater concentrations of selenium rarely
exceed l µg/1 (Hem, 1989), but up to 6000 µgll have been reported (WHO, 2003b), and
high concentrations (tens to hundreds of micrograms per litre) may occur in surface
water and groundwater near metal-sulphide mine sites.
Selenium concentrations are often particularly high in surface waters and
groundwater in coal mining areas where solid wastes and wastewater from coal power
stations are disposed to the environment (Barceloux, 1999; US EPA. 2000).
Transport and attenuation
Selenium can exist in nature in four oxidation states: 0 (elemental selenium), -2
(selenide), +4 (selenite) and +6 (selenate). Under oxidizing conditions, the selenium
oa:urs predominantly as selenite (SeO/) and selenate (SeO/) ion<i in natural waters.
These ion<i have a very high solubility, and can reach very high concentrations in
conditions when water is being subjected to high rat.es of evapotranspiration such as in
regions with semi-arid or arid climates. Selenate and selenite minerals can accumulate
with sulphates in soils in regions with semi-arid or arid climates.
High concentrations of selenium may also occur in groundwater benealh areas where
intense irrigated agriculture :flushes selenium compounds through the soil profile, and if
groundwater pumping rates are high, the concentration of selenium may be progressively
increased by the recycling of salts by the process of pumping, evaporation and recharge
of pumped effluent. Consequently, selenium concentrations in shal1ow groundwater and
in drainage from irrigated agriculture on seleniferous soils are often highly toxic to
wildlife that ingests the water, as in the widely studied case of the Kesterson National
WIidlife Refuge in the San Joaquin Valley of California (NRC, 1989). This water is also
potentially toxic to humans who might use shallow groundwater as a drinking-water
source, although water contaminated with high selenium concentrations is often too
saline for potable use.
Under reducing conditions in groundwater or in marshes, selenium can also be
removed from water through co-precipitation with sulphide minerals such as pyrite
(FeSi) or the precipitation of ferroselite (FeSei); through volatiJization as dimethyl
selenide or hydrogen selenide, or through the uptake of orgaro-seleniwn compounds by
plants. Consequently, anaerobic bioreactors or artificial wetlands are being used for
selenium removal from water, predominantly to protect receiving environments from the
discharge of wastewater contaminated by selenium.
Selenium can be removed from water by adsorption onto iron oxyhydroxide minerals
( especially ferrihydrite) and this is one of the preferred water treatment methods.
Selenium can also be removed from drinking-water by reverse osmosis and through the
use of anion~change resins.
Chemicals: Health relevance, transport and attenuation 97
4.2.4 Radon
Health aspects
Radon is a radio~ve gas emitted from radium, a daughter product of uranium that
occlD'S na1urally in rocks and soil. The main health effect of radon is to cause lung
cancer. Radon, together with its decay products, emits alpha particles that can damage
lung tissue. Although most radon is exhaled before it can do significant damage, its
decay products can remain trapped in the respiratory system attached to dust, smoke and
other fine particles from the air.
The global average human exposure to radiation from natural sources is 2.4 mSv per
year with an average dose from inhalation of radon of 1.2 mSv per year. There are large
local variations in this exposure depending on a number of factors, such as height above
sea level. the amount and type of radionuclides in the soil. and the amount taken into the
body in air, food, and water (WHO, 2004a). Unlike most other naturally occurring
grourxlwater contaminants, most of the health effects of radon in grourxlwater are
considered to be due to its contn'bution to indoor air quality rather than due to effects
caused by direct ingestion of water. UNSCEAR has calculated the average doses from
radon in drinking-water as low as 0.025 mSv/year via inhalation and 0.002 mSv/year
from ingestion as compared to the inhalation dose from radon in the air of 1.1 mSv/year
(UNSCEAR, 2000). The WHO has recommended a reference level of committed
effective dose ofO.l mSv from I year's consumption of drinking-water (WHO, 2004a).
Stining and transferring water from one container to another will liberate dissolved
radon. Water that has been left to stand will have reduced radon activity, and boiling will
remove radon completely. As a result, it is important that the fonn of water consumed is
taken into account in assessing the dose from ingestion. Moreover, the use of water
supplies for other domestic pmposes will increase the levels of radon in the air, thus
increasing the dose from inhalation. This dose depends markedly on the form of
domestic usage and housing construction (NCRP, 1989). The form of water imake, the
domestic use of water and the construction of houses vary widely throughout the world.
It is therefore not possible to derive an activity concentration for radon in drinking-water
that is universally applicable.
WHO recommends implementing controls if the radon concentration of drinking-
water exceeds 100 Bq/litre (WHO, 2004a), and the EU likewise recommends assessing
the need for protective measures at concentrations above this level (Euratom 2001/928;
CEC,2001).
Occurrence
Radon (Rn) is a naturally occurring, colourless, odourless gaseous element that is soluble
in water. It OCCI.D'S naturally only as a product of the radioactive de.cay of radium, itself a
radioactive decay product of uranium. As is the case for uranium, concentrations of
radon are directly related to the local geology, and are particularly high in granitic rocks
and pegmatites and sediments with phosphate nodules or heavy mineral sand deposits.
Radon-222 is a :frequently encountered radioactive constituent in natural waters and
typically exceeds the concentration of other radionuclides, including uranium, thorium
and radium, by orders of magnitude. High radon emanation, especially along fiacture
surfaces, contn'butes significantly to radon concentrations in groundwater. Data from
98 Protecting Groundwater for Health
sampling campaigns indicate that there is a great degree of variability in the radon-222
concentration of samples drawn :from any given rock type. 1he United States Geological
Smvery (USGS) conducted a study on occmrence of dissolved radon in grmmdwater in
Pennsylvania (Senior, 1998). Findings of this study indicated that rock types with the
highest median radon concentrations in groundwater include schist and phyllite (2400
pCi/1) as well as quartzite (2150 pCi/1). 1he geohydrologic groups with lowest median
radon concentrations in ground water include carbonate rocks (540 pCi/1) and other rocks
(360 pCi/1). Water :from wells in gneiss bad a median radon concentration of I 000 pCi/1,
and water from wells in Triassic-age sedimentary rocks had a median radon
concentration of 1300 pCi/1. Radon concentrations generally do not correlate with well
characteristics, the pH of water or concentrations of dissolved major ions and other
chemical constituents in the water samples.
Transport and attenuation
The rate of radon's radioactive decay is defined by its half-life, which is the time required
for one half of the amount of radon present to break down to form other elements. The
half.life of radon is 3.8 days. Several factors probably control the concentration of radon-
222 in a water supply. The flux of radon-222 within the ground may be controlled by the
radium-226 concentration in the surrounding rocks, the emanation fraction for the radon-
222 :from the rock matrix and the permeability of the rock to radon-222 movement For a
given flux, the concentration of radon-222 in a water supply would then also be
controlled by the ratio of aquifer surfuce area to volume.
4.2.5 Uranium
Health aspects
Uranium is a heavy metal of toxicological rather than radiological relevance in drinking-
water. In particular, it is of concern because of its impact on kidney function following
long-term exposure. Bearuse of uncertainties regarding the toxicity of uranium for
human beings the WHO has proposed a provisional drinking-water guid~line value of
15 µg/1 (WHO, 2004a; 2005a). The US EPA maximum contaminant level for uranium in
drinking-water is 30 µgll.
Occurrence
Uranium (U) is widely distributed in the geological environment, but concentrations in
groundwater are particularly high in granitic rocks and pegmatites, and locally in some
sedimentary rocks like sandstones. Uranium often occurs in oxidizing and sulphate-rich
groundwater. There are three naturally occurring isotopes of uranium: 234U (<0.01 per
cent), 23\J (0.72 per cent), and 238U (9927 per cent). All three isotopes are equally toxic.
Concentrations of uranium in natural waters usually range between 0.1 and l O µg/1
(Hem, 1989), but are often up to 100 µgll in groundwater in areas underlain by granitic
rocks, and may exceed I mg/1 near uranium mineral deposits.
Transport and attenuation
The transport of uranium in groundwater varies widely according to the aquifer
conditions. In anoxic conditions, uranium is reduced to U(IV) which is relatively
Chemicals: Health relevance, transport and attenuation 99
insoluble and precipitates. In oxidizing environments, uranium exists mainly as UOzX2-
(= uranyl}-compmmds with U(VI) which is considerably more soluble. Even with the
higher solubility ofU(VI), transport ofU(VI) can be limited as it sorbs strongly to solid
surfuces at circwn-neutral pH Very low and very high pH conditions limit soiption as
does the presence of certain complexing ligands such as natural organic matter, organic
chelating agents and carbonate, all of which can significantly enhance the transport of
uraniUIJL
4.3 NITROGEN SPECIES
Ammonia, nitrate and nitrogen containing organic compolDlds of humic type are the
dominating nitrogen compoWlds in groundwater. Though nitrite is highly toxic, it usually
occms only in very low concentrations in gro1D1dwater and these are not relevant to
human heallh. However, nitrite can become relevant from conversion of ammonia or
nitrogen in the drinking-water supply system or human body.
NOTE ►
Health aspects
Though nitrogen may occur naturally in groundwater, the main
sources of groundwater pollution are human activities such as
agriculture and sanitation (see Chapters 9 and JO).
Ammonia in chinking-water is not of direct health relevance, and therefore WHO have
not set a health-based guideline value. However, ammonia can compromise disinfection
efficiency, can cause the firilure of filters for the removal of manganese. and can cause
taste and odour problems. Also in distnbution systems it can lead to nitrite formation
which is ofheahh relevance.
1h: toxicity of nitrate to hmnans is mainly attnbutable to its reduction to nitrite.
Nitrite, or nitrate converted to nitrite in the body, causes a chemical reaction that can lead
to the induction of methaemoglobinaemia, especially in bottle-fed infants.
Methaemoglobin (metHb), normally present at 1-3 per cent in the blood, is the oxidi7..ed
form ofhaemoglobin (Hb) and cannot act as an oxygen carrier in the blood. The reduced
oxygen transport becomes clinically manifest when the proportion of metHb
concentration reaches 5-10 per cent or more of normal Hb values (WHO, 1996a). Nitrate
is enzymatically reduced in saliva forming nitrite. Additionally, in infunts under one year
of age the relatively low acidity in the stomach allows bacteria to form nitrite. Up to 100
per cent of nitrate is reduced to nitrite in infunts, as compared to IO per cent in adults and
children over one year of age. When the proportion of metHb reaches 5-l 0 per cent, the
symptoms can include lethargy, shortness of breath and a bluish skin colour ('blue baby
syndrome'). Anoxia and death can occur at very high uptakes of nitrite and nitrate from
drinking-water.
Methaemoglobinaemia is observed in populations where fixxl. for infant formula in
prepared with water containing nitrate in excess of around 50 mg/I, but other factors are
also involved in disease causation. The risk is enhanced by sewage contamination. This
100 Protecting Groundwater for Health
contributes nitrate and renders chemical conditions in the water to be reducing, thus
supporting the presence of nitrate reducing bacteria Moreover, ingestion of microbially
contaminated water causes gastroenteritis infuction which would also predispose the
infant to a nitrate reducing conditions and thereby more nitrite exposure (WHO, 2004a).
A review ofmnnerous case studies of water-related infunt methaemoglobinaemia in the
1980s indicated high correlation with microbial contamination of the water (US EPA,
1990; WHO,2005b).
The weight of evidence is strongly against an association between nitrite and nitrate
exposure in humans and the risk of cancer (WHO, 2004a). Studies in laboratory animals
demonstrate increased tumour incidence only after exposure to extremely high levels of
nitrite in the order of 1000 mg/I in drinking-water and simultaneously high levels of
nitrosatable precursors (WHO, 1996b). At lower nitrite levels, tumour incidence
resembled those of control groups treated with the nitrosatable compound only. On the
basis of adequately performed and reported studies, it may be concluded that nitrite itself
is not carcinogenic to animals (WHO, 1996a).
Based on methaemoglobinaemia in infunts (an acute effect), the WHO has established
a guideline value for nitrate ion of 50 mg/I as NO3• and a provisional guideline value for
nitrite of 3 mg/I as NOi (WHO, 2004a). Because of the possibility of simultaneous
ocunrence of nitrite and nitrate in drinking-water, the sum of the ratios of the
concentrations (¼irate or ½tritJ of each to its guideline value (GV .-or GV runire) should
not to exceed one.
Sources and occurrence
Nitrogen is present in human and animal waste in organic form, which· may then
subsequently be mineralized to inorganic forms. Ammonia (ioniz.ed as NH/, non-
ioniz.ed as NH3) as well as urea (NHz)2CO is a major component of the metabolism of
mammals. Ammonia in the ·environment mainly results from animal feed lots and the use
of manures in agriculture (Chapter 9), or from on-site sanitation or leaking sewers
(Chapter IO). Thus ammonia in water is often an indicator of sewage pollution. The
nitrite ion (Nffi) contains nitrogen in a relatively unstable oxidation state. Nitrite does
not typically occur in natural waters at significant levels, except temporarily under
reducing conditions. Chemical and biological processes can further reduce nitrite to
various compounds or oxidize it to nitrate. The nitrate ion (N0-3) is the stable form of
combined nitrogen for oxygenated systems. Nitrate is one of the major anions in natural
waters, but as for ammonia, concentrations can be greatly elevated due to agricultural
activities (Chapter 9), and sanitation practices (Chapter IO).
Natural levels of ammonia in ground and surfuce waters are usually below 0.2 mg/I,
and nitrate concentrations in groundwater and surface water typically range between 0-
18 mg/I as N~--Although elevated concentrations of nitrate in groundwater are mostly
caused by agricultural activity or sanitation practices, natural nitrate concentrations can
also exceed 100mg/I as NO3" as observed in some arid parts of the world such as the
Sahel and north Aftica (Edmunds and Gaye, 1994) and the arid interior of Australia
(Box4.l).
Chemicals: Health relevance, transport and attenuation IO I
Box 4.1. Naturally-occurring high nitrate in Australia
High groundwater nitrate concentrations have been observed in the arid interior
of Australia, commonly exceeding 45 mg/I, and often exceeding 100 mg] in
groundwater which otherwise meets national and international drinking-water
guidelines (Lawrence, 1983; Barnes et al., 1992). The nitrate in this region is
partially derived :from nitrogen fixing by native vegetation, and by
cyanobacteria crusts on soils. Termite mounds appear to be a significant
contributory source of the nitrate (Barnes et al., 1992), possibly due to the
presence of nitrogen fixing bacteria in many termite species, and the nitrogen-
rich secretions used to build the walls of the mounds. Nitrate is leached to the
water table in arid Australia after periodic heavy rainfall events, particularly
after bush fires that allow soluble nitrate salts to accumuJate in soils .
Denitrification in these soils appears to be inlnbited by low carbon levels.
Transport and attenuation
Ammonium (NH/) shows a high tendency for adsorption to · clay minerals, which limits
its mobility in the subsurfuce (saturated and unsaturated :zones). In contrast, interactions
between minerals and nitrate or nitrite are usually negligible and both ions are mobile in
the subsurfuce.
Under aerobic conditions in the substrrface oxidation of ammonium through nitrite to
nitrate by microorganisms is 1he only process where nitrate is formed in natural systems.
DEF ► Nllriftcalion is the biological conversion of ammonium through
nitrite to nitrate. Denilrijica.ion is the biological process of
reducing nitrate to ammmria and nitrogen gas.
Despite the natural high concentrations of nitrate in groundwater in much of inland
Australia, there have been no verified cases ofMetHb in Aboriginal people (Heam et al.,
1993), who are the main users of groundwater in this part of the country. Because
potable quality groundwater is scarce in the interior of Australia, and because 1he use of
water is vital for maintaining hygiene in the region, the National Heal1h and Medical
Research Council revised the national water quality guidelines in 1990. The revised
guidelines allow the use of groundwater wi1h concentrations of nitrate exceeding 100
mg/I tor all non-potable needs, up to I 00 mg] for potable use except for infants under 3
months old, and up to 50 mg] for infimts under 3 months old Although technologies
exist to remove nitrate :from drinking-water using microbial denitrification, the
equipment is difficult to maintain in remote aboriginal settlements, and it was considered
in this case that changing guideline concentrations would produce better health
· outcomes. These changes were incorporated into the Australian drinking-water
guidelines in I 996. The autotrophic conversion of ammonia to nitrite and nitrate
(nitrification) requires oxygen. The discharge of ammonia nitrogen into groundwater and
its subsequent oxidation can thus seriously reduce the dissolved oxygen content in
102 Protecting Groundwater for Health
shallow groundwater, especially where high ammonia loads are applied and re-aeration
of the soil is limited.
In the absence of dissolved oxygen (such as in some deep or confined groundwat.ers),
denitrification can occur, driven by denitrifying bacteria. Under fully anaerobic
conditions, in an aquifer where predominantly sulphides serve as reduction agents, the
microbial oxidation of sulphides into sulphate and simultaneous reduction of nitrate to
nitrogen gas can occur which also reduces the nitrate content
As microbial processes, both nitrification and denitrification are affected by many
factors that are of importance to microbial activity. Nitrification and denitrification are
optimal at about 25°C and are inlnbited at 10°C or less. Other regulating factors are pH
and all factors affecting the diffusion of oxygen such as soil density, grain structure,
porosity and soil moisture. Warm, moist and well aerated soils provide ideal conditions
for nitrification. Denitrification occurs only under anoxic or almost anoxic conditions.
Beside the presence of nitrate, the denitrifying bacteria require a carbon source. A soil
moisture of more than 80 per cent has been found to be essential for denitrificatioa Thus
in many settings natural attenuation can substantially reduce nitrate concentrations in
groundwater over time, but rates of attenuation strongly depend on conditions in the
aquifer.
4.4 METALS
The following focuses on those metals which are toxic to humans and which have
:frequently been observed as groundwater contaminants in connection with human
activities and/or have physical and chemical properties which make them potential
groundwater contaminants, i.e. cadmium (Cd), lead (Pb), nickel (Ni), clromium (Cr),
and copper (Cu).
Health aspects
Cadmium has a high renal toxicity, which is not only due to its mode of action but also to
its irreversible accumulation in the kidney. The health based guideline value for
cadmium in drinking-water is 3 µg/1 (WHO, 2004a).
Lead is a strong newutoxin in the unborn, newborn and young children. It crosses the
placenta easily and is toxic to both the central and the peripheral nervous system, thus
causing cognitive and behavioural effects (WHO, 2004a). The threshold of
nemotoxicological concern, defined as a group based mean blood lead level, has
decreased continually during the last 10 to 20 years, and epidemiological evidence
indicates lead levels above 30 µg of lead per litre of blood to be associated with
intelligence quotient deficits in children (WHO, 2004a). The use oflead in antiknock and
lubricating agents in petrol is being phased out in many countries, thus decreasing this
source of contaminatioa However, a major main source of expostu'e to lead through
water is household plumbing systems, i.e. pipes, fittings, solder and connections from the
mains to homes. Dissolution from such materials strongly depends on chemical
properties of the drinking-water, with soft, acidic water dissolving the largest amount
The WHO guideline value for lead in drinking-water is 10 µgll (WHO, 2003c; 2004a).
Chemicals: Health relevance, transport and attenuation 103
The significance of Nickel from the health point of view is mainly due to its high
allergenic potential. The WHO drinking-water guideline value for the protection of
sensitive persons is 20 µg/l (WHO, 2004a).
Chromium can be found in the environment in two valency states, Cr(III) and Cr(VI).
The former predominates in soils, whereas the latter occurs exclusively as chromate
(Cr04
2) from anthropogenic sources. Cr(VI) is the form which is · of toxicological
significance because of its easy uptake into cells together with SO/" and PO/'. Within
cells and during its reduction to CI(IIl), the chromate ion represents a considerable
geootoxic and clastogenic potential (O,sta, 2002). However, since even very high doses
of Cr(VI) are subjected to rapid chemical reduction in the upper gastrointestinal tract
(Kerger et al., 1997), only negligi"ble amounts of Cr(VI) should reach the blood
compartment and other body fluids and organs. The health based guideline value for
chromiwn in drinking-water is 50 µg/l (WHO, 2004a), and while higher concentrations
have been reported from some drinking-water supplies, most studies indicate the
concentration of chromium in groundwater to be low (WHO, 2003d). Cr(III) in drinking-
water may eventually be oxidized to Cr(VI) during its ozonation.
Copper is an essential trace element with an optinlal daily oral intake of 1-2 mg per
person. Naturally occurring copper concentrations in groundwater are without any health
significance and scatter mostly around 20 µgll. · If drinking-water drawn from
groundwater contains elevated levels, in most situations corrosion of copper pipes is the
primacy source. Mean concentrations of more than 2 mg/I could lead to liver cirrhosis in
babies if their formula is repeatedly prepared using such water (Zietz et al., 2003). The
prevalent endpoint of acute copper toxicity by time, concentration and dose is nausea
(Araya et al., 2003). The health based guideline value for copper in drinking-water is 2
mgll (WHO, 2004a; 2004c).
Sources and occwrence
Metals from activities such as mining, manufacturing industries, metal finishing,
wastewater, waste disposal, agriculture and the burning of fossil fuels can reach
concentrations in groundwater which are hazardous to hwnan health. Chapter 11 lists
industry types together with the metals they commonly emit (see Table 11.2.) Metals are
natural constituents in groundwaters, having their origin in weathering and solution of
numerous minerals. However, natural concentrations of metals in groundwaters are
generally low. Typical concentrations in natural groundwaters are <10 µg/l (copper,
nickel), <5 µg/l (lead) or <I µgll (cadmium, chromiwn). Even so, the concentrations can
locally increase naturally up to levels which are of toxicological relevance and can
exceed drinking-water guidelines, e.g. in aquifers containing high amom1ts of heavy
metal bearing minerals (ore). Metal concentrations in groundwater may be of particular
concern where it is directly affected by manufacturing and mining as well as downstream
of abandoned waste disposal sites. Another anthropogenic cause of elevated metal
concentrations in groundwaters is the acidification of rain and soils by air pollution and
the mobilization of metals at lower pH values. This problem predominantly appears in
furested areas, because the deposition rates of the acidifying anions sulphur and nitrate
from the atmosphere are evidently higher in forests due to the large surface of needles
104 Protecting Groundwater for Health
and leafs, and because soils in forests are generally poor in nutrients and have a low
neutralization capacity agaimt acids.
Transport and attenuation
Most of the metals of concern occur in groundwater mainly as cations ( e.g. Pb2+, Cu2+,
Ni2+, of"') which generally become more insoluble as pH increases. At a nearly neutral
pH typical for most groundwaters, the solubility of most metal cations is severely limited
by precipitation as an oxide, hydroxide, carbonate or phosphate mineral, or more likely
by their strong adsoiption to hydrous metal oxides, clay or organic matter in the aquifer
matrix. The adsorption decreases with decreasing pH. As a consequence, in naturally or
anthropogenical.ly acidified groundwaters metals are mobile and can travel long
distances. Furthennore, as simple cations there is no microbial or other degradation.
In a soil solution containing a variety of heavy metal cations that tend to adsorb to
particle surfaces, there is · competition between metals for the available sites. Of several
factors that determine this selectivity, ionic potential, which is equal to the charge of an
ion over its ionic radius, has a significant effect Cations with a lower ionic potential tend
to release their solvating water molecules more readily so that inner sphere surface
complexes can be formed Selectivity sequences are arranged in order of decreasing ionic
radius, which results in increasing ionic potential and decreasing affinity or selectivity for
adsorption. As an example the following selectivity sequence of transition elements
belonging to group 11b has been determined (Sposito, 1989):
H~ > of+> m2+
As a consequence, mercucy is the most strongly adsorbed; this being the probable reason
for its generally very low concentration occurrence in groundwater.
Metals within the transition group differ in that electron configmation becomes more
important than ionic radius in determining selectivity. The relative affinity of some
metals belonging to different transition groups is given by:
Clf+ > Ni2+ > Co2+ > Fe2+ > Mn2+
However, this sequence can be more or less changed in groundwater by naturally
occurring complexing agents like fulvic acids which is especially true for copper
(Schnitzer and Khan, 1972).
In addition, most oxyanions tend to become less strongly sorbed as the pH increases
(Sposito, 1989). Therefore, the oxyanion-forming metals such as chromium are some of
the more common trace contaminants in groundwater. Chromium is mobile as stable
Cr(V1) oxyanion species under oxidizing conditions, but fonns cationic CI(III) species in
reducing environments and hence behaves relatively immobile under these conditions.
For example, in conlaminated groundwater at industrial and waste disposal sites
Chromium occurs as c?+ and CrO/ species, with CrOl being much more toxic but less
common than cr3+_ In most aquifers chromium is not very mobile because of
precipitation of hydrous chromiurn(III)oxide. In sulphur-rich, reducing environments,
many of the trace metals also form insoluble sulphides (Smedley and Kinniburgh, 2001 ).
Chemicals: Health relevance, transport and attenuation 105
4.5 ORGANIC COMPOUNDS
Organic compounds in groundwater commonly derive from breakdown and leaching of
naturally occwring organic material, e.g. from organic-rich soil horm>ns and organic
matter associated with other geologic strata, or hmnan activity, e.g. domestic,
agricultural, commercial and industrial activities.
Natural sources will always contnbute some organic compounds to groundwater,
often at low levels. Natural organic matter comprises water-soluble compounds of a
rather complex nature having a broad range of chemical and physical properties.
Typically, natural organic matter in groundwater is composed of hmnic substances
(mostly fulvic acids) and non-hmnic materials, e.g. proteins, carl:Johydrates, and
hydrocarbons (Thmman, 1985; Stevenson, 1994). While natural organic matter is a
complex, heterogeneous mixture, it can be characteriz.ed according to size, stnx:ture,
fimctionality, and reactivity. Natural organic matter can originate from terrestrial sources
(allochthonous natural organic matter) and/or algal and bacterial sources within the water
(autochthonous natural organic matter). Dissolved organic carl:Jon (DOC) is considered
to be a suitable parameter for quantifying organic matter present in groundwater;
however, DOC is a bulk organic quality parameter and does not provide specific
identification data and may also incoipOrate organic compotmds arising from human
activity. Natural organic matter, although considered benign, may still indirectly
influence groundwater quality. For example, contaminants may bind to organic-matter
colloids allowing their fucilitated transport within grotmdwater, a process proposed (but
not proven) to be of most significance for the more highly soming organic compotmds.
Also, routine chlorination of water supplies containing natural organic matter may form
disinfection by-products such as trihalomethanes. However, became of their low direct
health relevance, natural organic substances are not addressed further herein.
Human activity has released a vast range of anthropogenic , organic chemicals,
commonly termed 'micro-pollutants', to the environment, some of which may
detrimentally impact groundwater quality. This chapter focuses on commercially and
industrially derived chemicals which (i) have a high toxicity, (Ii) have physical and
chemical properties fucilitating their occurrence in groundwater and (iii) have been
ohrerved to occur frequently as groundwater contaminants. Chapter 11 lists industry
types together with substances that may potentially be released to the subsurface from
their respective industrial activities. The occurrence of organic pollutants in groundwater
is controlled not only by their use intensity and release potential, but also by their
physical and chemical properties which influence subsurface transport and attenuation.
Disc~ion of this aspect specific to organic chemicals follows and extends the general
concepts covered in Section 4.1.
4.5.1 Conceptual transport models for non aqueous phase liquids
A correct conceptual model of contaminant behaviour is essential for assessing
subsurface organic contaminant migration 1be classical near-surfuce leachable source
z.one -dissolved plmne model presented earlier (Section 4.1.2, Figure 4.2) is not
applicable for all organic substances. Of key importance is the recognition that organic
chemicals have very different affinities for water, ranging from organic compounds that
106 Protecting Groundwater for Health
are hydrophilic ("love" water) to organics that are hydrophobic ("fear" water). Such
concepts are used below to develop appropriate contaminant conceptual models followed
by discussion of specific transport p~ applicable within the models developed.
Water is a highly polar solvent, so polar in fact that it develops a hydrogen-bonded
struclllre and will easily dissolve and solvate ionic species . The vast majority of organic
compounds are covalent IOOlecules, rather than ionic species, and most have a limited
tendency to partition or dissolve into water. Further, many organic compounds found in
groundwater are used as liquids, e.g. hydrocarnon fuels or industcy solvents. A focus
upon organic liquids is hence relevant Organic compounds that most easily partition or
dissolve into water tend to be small molecules, have a polar structure and may hydrogen-
bond with water. Typically they have only a few carbon atoms and often contain oxygen.
Examples include methanol (CH30H) and other short-chain alcohols, e.g. ethanol and
propanols that may be used as de-icers, and ketones such as methyl-ethyl-ketone and
ethers such . as dioxane that are used as . industrial solvents. Some compotmds are so
hydrophilic that they fonn a single fluid phase with the water and are said to be miscible
with the water, e.g. methanol. acetone, dioxane.
Most organic compounds are, however, relatively hydrophobic as they are
comparatively large molecules of limited polarity with low hydrogen-bonding potential.
Most organic liquids are so hydrophobic that they fonn a separate organic phase to the
water (aqueous) phase. They are immiscible with water and a phase botmdary exists
between the organic phase and the aqueous phase, with the organic phase generally being
referred to as the non aqueous phase liquid (NAPL). When a separate organic NAPL
exists it is important to consider the density of the NAPL relative to water as this controls
whether the NAPL will be upper or lower phase relative to the water phase. Most
hydrocarbon-based organic liquids have a density <I (g/ml), e.g. benz.ene is 0.88 and
pentane is 0.63 and when in contact with water will be the upper phase and "float"' upon
the water phase of density I. Such "light"' organic compounds are generally referred to as
being LNAPLs.
In contrast, other hydrophobic organics have a relatively high density due to
incorporation of dense chlorine (or other halogen) atoms in their structure and for
example chlorinated solvents such as trichloroethene (TCE) and 1,1,1-trichloroethane
(1,1,1-TCA) and polychlorinated biphenyl (PCB) mixtures have densities in the 1.1 to
1.7 range. Due to their density such organic phases will be the lower phase and 'sink'
below the water phase. Such "dense" organic compounds are generally referred to as
DNAPLs.
Although hydrophobic, LNAPL and DNAPL organics still have potential for some of
their organic molecules to dissolve into the adjacent aqueous phase. The organics are
'sparingly soluble' and will have a finite solubility value in water leading to dissolved
concentrations in the water phase. Solubility values achieved by individual organic
compounds in water are highly variable between organics and controlled by their relative
hydrophobicity. For example, small and/or polar organics have the greatest solubility
with for example dichloromethane (DCM) (CH2Cli) being one of the most soluble with a
solubility of ca -20,000 mg/I, which contrasts with e.g. DDT, a large pesticide molecule
that is not easily accommodated in the polar water structure and has a solubility of just
about 0.1 mg/I. Similarly benz.ene, as single aromatic ring hydrocarbon, has a solubility
Chemicals: Health relevance, transport and attenuation 107
ca 1,800 mg/I that is much greater than benz.o[a]pyrene, a PAH of solubility ca 0.004
mg/I that is composed of five adjacent aromatic rin~
The above provides fimdamental understanding for conceptual models of organic
contaminant transport in the subsurface and why specific organic compotmds have a
tendency to occur or not occur in grotmdwater. Hydrophilic miscible organics behave
similarly to the classical leachable source model (Figure 42). In essence, a spill of e.g. a
de-icer fluid at surface would migrate as a concentrated organic-aqueous fluid through
the unsaturated z.one and then migrate laterally in the groundwater as a concentrated
dissolved-phase plwne. Importantly, hydrophobic immiscible organics, i.e. NAPLs,
exhibit very different behaviom. Conceptual models for LNAPL releases and DNAPL
releases (Mackay and Cheny, 1989) are shown in Figures 4.4 and 4.5.
NAPLs may migrate as a separate NAPL phase and displace air and water from the
pores they invade if they have sufficient head (pressure) to overcome the entry pressure
to the pores or fractures. This head is controlled by aspects such as the spill volume and
rate and the vertical column of continuous NAPL developed in the subsurface. NAPL
migration is also controlled by its density and viscosity. Petrol fuel and chlorinated
solvents have viscosities lower than water and more easily migrate in the subsurface; in
contrast, PCB oils or coal tar (PAR-based) hydrocarbons may be very viscous and
perhaps take years for the NAPL to come to a resting position in the subsurface.
Chlorinated solvents such as PCE have high densities and may penetrate to significant
depths through aquifer systems in very short time periods. Whereas dissolved pesticides
may take years to decades to migrate through a 30 m unsaturated z.one, DNAPL may
migrate through such a z.one in the order of hours to days (Pankow and Chroy, 19%).
DNAPLs may penetrate discrete sand horizons and hairline fractures in clays and com-
promise clay units that are normally an effective barrier to dissolved plume migration
At the water table, LNAPLs, being less dense than water, will form a floating layer of
LNAPL on the water table often slightly elongated in the direction of the water table
hydraulic gradient. DNAPL, in contrast may penetrate as a separate immiscible DNAPL
below the water table. Predominant movement will be vertically downward due to its
density, but some lateral spreading will occur as it encotmters lower permeability strata
If spilt in sufficient volume and with sufficient driving head, the DNAPL may penetrate
the full aquifer depth to the underlying aquitard/bedrock (Kueper et al., 1993). This
should not be assumed to occur in all cases. Migrating NAPL leaves a trail of immobile
residual NAPL droplets behind its migration pathway held by capillwy forces causing
NAPL to spread across an aquifer thickness. DNAPL accumulating on low permeability
features, often referred to as pools, is potentially mobile. It may ultimately penetrate that
formation due to changes in pressure arising from continued DNAPL spillage, pumping
or remediation attempts or via drilling (for boreholes, piling etc) through that layer.
108 Protecting Groundwater for Health
Figure 4.4. Conceptual model ofa light non riquoous phase liquid (LNAPL) rdeasz
DNAPL
Residual
DNAPL
Vapours
Vapours
1
Dissolved -phase
DNAPL pool . , • .
Aquifer base
Figure 45. Conceptual model ofa dense non aqueous phase liquid (DNAPL) nlease

Chemicals: Health relevance, transport and attenuation I 09
Often NAPL will remain relatively local to a site, possible exceptions being the
migration of LNAPL to a local surfuce water and pemaps huge NAPL spills, e.g. at a
poorly operated oil refinery/distnbution facility. Risks posed to groundwater resources
and supplies are most often concerned with the migration of the di~lved-phase plwne
formed by the contact of flowing groundwater with the spilt NAPL. Although the
presence of NAPL may impede the flow of groundwater, e.g. in DNAPL pools and
central LNAPL body, areas where NAPL residual saturations are lower will still be
permeable to water and NAPL ~lution will occur. Often the mass ofNAPL is so
large and the dissolution (solubilization) of NAPL into water so slow that the entire
NAPL body post spill should be regarded as a largely immobile source zone able to
continuously generate a ~lved-phase solute phnne of organics downgradient for years
to decades, even centuries for low-solubility NAPLs. Thus historic spill sites may still
constitute major sources ofNAPL in the deep subsurface and cause very large di~lved-
phase plwnes, particularly where ~lved-phase plume attenuation is limited. In
general, DNAPLs tend to pose the greatest groundwater threat as they reside deep in
groundwater systems and many, being chlorinated, are less suscepllble to attenuation. In
contrast, LNAPLs are restricted to shallower groundwater-table depths, and are more
susceptible to attenuation via biodegradation.
The above provides a basic introduction to NAPLs in grotmdwater. Much research
and field experience has been gained since the pioneering NAPLs research of Schwille
(1988) and the reader is referred to Mercer and Cohen (1990) and Pankow and Cheny
(1996) and references therein fur further details.
4.5.2 General aspects of transport and attenuation of organics
Some of the transport and attenuation proces.5eS introduced earlier require specific
discussion for organic contaminants. Several physiochemical properties/parameters exert
a key corirol over subsurface organic contaminant migratioIL A selection of parameters
is listed in Table 4.1 fur a range of organic chemicals of groundwater-health coricem.
Values for a specific parameter generally vaiy over orders of magnitude across the listed
chemicals and infer substantial variations in transport and attenuation between organic
contaminants. Table 4.1 is not exhaustive: there are many more organic chemicals;
values of individual chemical parameters may show significant variability across the
literature; and other parameters exist., most ootably half-life, that due to their dependency
on site conditions display significant variability (see Section 4.5.4 for references to some
half-life literature). For more detailed databases and their supporting literature see e.g.
US EPA (1996; 1999) and Montgomery (1996).
llO Protecting Groundwater for Health
Table 4.1. Selected physiochemical parameter values fur important organic groundwater
rontamirumts at 20-25"C (Merrer and Cohen, 1990; US EPA, 1996; 1999) (see Section 4.5.3 fur an
e:xplanalionoftheabbreviations)
Chemical Density Absolute Aqueous Vapour Henry's Koc Kaw
(Jdml) vucosity solubility pressure constant (ml/g)
{cP ) {mr.l!I {atm.} {atm. m3/mol )
Aromatic Hydrocarbons (single aromatic ring)
Benzene 0.87 0.60 1750 0.13 0.0056 62 130
Toluene 0.86 0.55 535 0.037 0.0064 140 540
Ethylbenzme 0.87 0.68 152 0.0092 0.0064 200 1400
o-Xylene 0.88 0.81 175 0.0087 0.0051 240 890
Chlorinated Hydrocarbons
DCM 1.34 0.45 20000 0.48 0.0020 8.8 20
TCM 1.50 0.60 8200 020 0.0029 53 93
ere 1.58 0.97 757 0.12 0.024 152 440
TCE · 1.47 0.57 1100 0.076 0.0091 94 240
PCE 1.63 1.93 150 . 0.024 0.026 265 400
vc Gas Gas 2760 3.7 0.027 18 14
1,2-0CA 126 0.89 8520 0.084 0.00098 38 30
1,1-0CE 122 036 2250 0.79 0.034 65 69
cOCE 127 0.44 3500 027 0.0076 49 5.0
tOCE 126 0.40 6300 0.43 0.0066 38 3.0
1,2-0CB 1.30 132 100 0.0018 0.0021 379 2790
1,4-0CB 128 1.04 73 0.0014 0.0028 616 2580
Others
Naphthalene l.16 solid 31 0.00012 0.00048 1190 2290
Anthracene 124 solid 0.043 3.6xl0.s 0.00007 23500 35500
Benro(a) 1.35 solid 0.0016 6.4x10·12 0.000001 969000 1260000
pyrene
PCB-1248 1.44 212 0.054 6.6xur7 0.0035 437000 562000
V o/atilization
Although other processes may be enhanced in the unsaturated zone relative to the
saturated zone, e.g. biodegradarion through the ready availability of oxygen,
volatilization is a key process that only occurs in the unsaturated zone. Organic
compounds with high vapour pressures (P) (>0.008 atm., i.e. xylene in Table 4.1) are
termed (volatile organic compounds (VOCs). The vapour concentration adjacent to a
NAPL or organic solid is dictated by its vapour pressure. Although volatilization of
subsurfuce organic contaminants, e.g. NAPL sources, may occur and organic vapours
potentially lost to the above ground atmosphere, VOCs are nevertheless very widely
enoountered in groundwaters; possible reasons include many VOCs are NAPLs of
relatively high solubility (S) and low sorbing potential (K..,). Vapours migrate due to
diffusion and advection within the air phase and may migrate due to pressure
(barometric) and temperature fluctuations, water infiltration and preferential conduit
routes (Mendoza et al., 1996). In relation to vapour-phase diffusion, it is emphasized
diffusion coefficients in the air phase are ---4 orders of magnitude greater that the water
phase. This allows much greater opportunity for lateral (radial) migration of vapour
plwnes and, due to vapour contact, contamination of underlying groundwaters over a
wide area (Rivett, 1995).
Chemicals: Health relevance, transport and attenuation 111
Partitioning of dissolved organic solutes between a contnninat.ed water phase and an
adjacent air-phase is controlled by the Heruy's Law partition coefficient:
H=CA/Cw (Eqn.4.4)
where H is the Heruy's Law comtant, Cw is the concentration of an organic compolDld
in water and CA is its vapour concentration in the gaseous phase. It should be noted that
similar concentration units will yield a dimensionless (i.e. unitless) H value, however, the
air concentration is often expressed in terms of pressure and hence H values may be
quoted with units of the type atmos m3 mor1, e.g. fur benz.ene H (dimensionless) is 0.24
and with units His 5.5xHT3 atoms m3 mor' (fable 4.1).
So/ubili7Dlion
As indicated in Section 4.5.1, the differing hydrophobic nature of organic compowids
meam their solubility in water varies over orders of magnitude (fable 4.1). Solubility
values represent maxinunn concentrations that may be achieved in a dissolved-phase
plume. VOC contaminants, e.g. benzene and TCE tend to be small molecules that are
moderately soluble, amenable to analysis and hence often detected in growidwater.
Although solubilities are relatively low compared to inorganic ions, they may
nevertheless achieve concentrations 4-5 orders of magnitude greater than drinking-water
standards or guideline values. Larger molecular weight organics will have lower
solubilities, hence concentrations e.g. of DDT in groundwater may only reach about 0.1
mg/I (its solubility).Thus DDT at solubility only exceeds the WHO guideline value of 1
µgll by a factor of approx. 50, hence allowing for dilution and some attenuation, the
prospects of exceeding the DDT standard in growidwater are low except in close
proximity to a DDT source. DDT's high hydrophobicity and hence high sorption (see
below) tends to cause DDT (and other organic chemicals of similw: properties, e.g. high
molecular weight PCBs and PAHs) to perhaps be more of a soil rather than a
groundwater problem.
Sorption
Soiption exerts a key control over the transport of anthropogenic organic contaminants.
Organic sorption is a complex topic, a detailed review is provided by Allen-King et al.
(2002). Sorption is a fi.mction of the properties of both the organic solute and aquifer
solid. Hydrophobic non-ionic organic contaminants prefi:rentially sorb to the low-
polarity components of geosolids, e.g. any organic material present Sorption is inversely
related to organic compound solubility; the more hydrophobic and less soluble an
organic solute, the greater its intrinsic potential fur sorption to any organic material
present in the aquifer solids. Hence hydrophilic organics have negligible soiption, and
mild to moderately hydrophobic organics such as the VOCs show limited soiption. ln
contrast, hydrophobic, high molecular weight, large organics such as P AHs and PCBs of
low solubility exhibit high sorption (fable 4.1).
An additional measure of organic compound hydrophobicity often used in soiption
research is the octanol-water partition coefficient CK-) (fable 4.1), that is simply an
equihbrium partitioning of the organic solute between the organic octanol phase and
112 Protecting Groundwater for Health
water phase. The higher the K.,..., value, the more hydrophobic, less soluble and more
sorptive the organic compound.
The degree ofsorption is also controlled by the sorption potential of the sorbate, i.e.
the aquifer material that dissolved concentrations in groundwater contact. Frequently
sorption is assumed to be at equilibrium and linear with organic solw: concentrations,
the magnitude of sorption being expressed by the sorption partition coefficient K.i:
K.i = Cs I Cw (Eqn. 4.5)
where Cs is the sotbed concentration. The main sorbing phase for organic solutes is any
organic material present in the rock phase originating for example from organic detritus,
e.g. humic material. deposited at the time of rock depositioIL This organic material is
refurred to as the fraction of oig.mic carbon (t;,J within the geologic or soil matrix.
Although t;,. values may be on the order of one per cent or more in organic-rich soil
horizons, many aquifers comprise geologic strata with low t;,. values, e.g. an t;,.-0.02 per
cent is recorded for the Borden glaciolacustrine sands, Canada (Rivett and Allen-King,
2003). The foe, even at such low concentrations, may still be the dominant sorption phase
l'liher than poorly sorbing mineral surfaces. Simultaneous laboratory mt:afillrements oft;,.
and K.i have shown that they are approximately linearly related, with the constant of
proportionality being termed the organic-carbon partition coefficient <Koc) of the specific
organic solute (fable 4.1). Typically practitioners assessing sorption controls now obtain
Koc values from databases ( e.g. US EPA, 1996), measure the aquifer t;,. (Heron et al.,
1997) and estimate the sorption K.i from the relationship:
(EqIL4.6)
where t;,. is in mas.5 fraction dimensionless units, e.g. expressed as 0.0002 rather than as
0.02 per cent Hence the greater the to of the aquifer deposits (e.g. higher values are often
found in shallow soils or recent sub-river, i.e. hyporheic z.one), and greater the Koc (Table
4.1), which will increase with solute hydrophobicity, the greater the K.i value, i.e.
sorptioIL Assuming ideal linear equilibrium sorption and calculation of K.i from the
above, the ret.ardation ractor R; of organic solute i may be estimated from:
(Eqn. 4.7)
where p and Tl are the bulk density and porosity of the porous media respectively.
The above hydrophobic partitioning ideal sorption approach is an approximation of
reality; it provides a reasonable first estimate. It should be recogniz.ed, however, that non-
ideal sorption proces.ses may be significant (Allen-King et al., 2002); these include slow
equilibration of sotbed and dissolved phases, dependence of the degree of sorption on
dissolved concentration magnitude and the presence of any competing, also sorbing,
solutes within multi-wntaminant plumes. Further, the nature of the~ i.e. ratio of the
carbon-hydrogen-oxygen contents, has an important control on the sorption that occurs.
It is often useful to combine some of the above parameters visually to as.5ess how
organic contaminants may comparatively behave. Figure 4.6 plots ~ a measure of
compound hydrophobicity that will indicate solubility and sorptive retardation trends
against vapour pressure, a measure of volatilization tendency. The figure indicates P AHs
are unlikely to volatiliz.e and will undergo high sorption, and this wou1d infer that
Chemicals: Health relevance, transport and attenuation 113
wit/unsaturated zone solids concentrations of PAHs could often be high (frequently the
case) and perhaps there is relatively limited development ofPAH plumes to groundwater
(often relatively small plumes are encountered). The chlorinated hydrocarbons, in
contrast, are volatile but of low sorption potential. It is likely they would vaporize (and
potentially be a vapour hazard to receptors at the soil surface) and also leach to ground-
water leaving low concentrations in soils and tmsatnrated samples (quite often the case).
Q re
G
01
a
HYDROPHOBIC
V
Vinyl -
chloride
Chlorinated
hydrocarbons
BTEX
OIrhlonobensenea
Phenols
PAHs
3
MOH •
2
VOLATILfrY
Log Vapour pressure 41 e Pa)
Figure 4.6. Polarity -volatility diagram for selected organic corsaminants
LOW
Chemical reactions
Although there are a multitude of possible chemical reactions (ablotic reactions, i.e. not
mediated by bacteria), reactions of low -concentration orgies in a water -lased
environment tend to be fairly limited. Perhaps the most common reaction is that of
slightly positively charged organic compounds (C') with Natively charged
(nucleophilic) species, such as HS-, OH- or water. The latter is a hydrolysis reaction.
Reactive organic solutes tend to be organic halides, particularly laominated compounds
and to a lesser extern chlorinated compoumds. An example of a chemical reaction is that
of the chlorinated solvent 1,1,1-TCA which was commonly used to degrease meals and
Circuit boards (as a less toxic replacement to TCI:, before concerns were raised about its
ozone -depletion potential). TCA in water will either undergo an elimination reaction to
yield 1,l-DCE_ or alternatively a sequential hydrolysis reaction replacing all the chlorine
atoms as chloride to yield ethannic acid. Interestingly it may also biodegrade to
predominantly iorm a different product, 1.1,.DCA (Klrcka et al, 1990). Further
information on chemical inactions in water may be found in Schwaraenbach ei at
(1993).

114 Protecting Groundwater for Health
Biodegradation
Bacteria degrade organic comaminants to simpler, often less toxic, products.
Biodegradation is perceived to be the primary attenuation process that may mitigate
dissolved-plume impacts to receptors by organic chemicals. monitored natural
attenuation (MNA) remedial strategies generally have their main focus upon
demonstration of OCCU1Tence ofbiodegradation (Wiedemeier et al., 1999). This not only
entails monitoring the disappearance of the organic contaminant, but also the appearance
of intermediate organic contaminants that may themselves persist or be further
biodegraded, ideally to benign inorganic products, e.g. water, carbon dioxide, chloride.
Monitoring of the inorganic hydrochemistry is also a key requirement to assessing
biodegradation occurrence. Sites may be initially aerobic/oxic (containing oxygen), and
under these conditions biodegradation of many contaminants is often the most rapid.
Dissolved oxygen concentrations in groundwater are usually low, maximally -10 mgll .
. Such levels can easily be depleted by even low to moderate levels of organic
contamination present and are not easily renewed as dispersive mixing in groundwaters
to allow oxygen re-entry is typically low. Other electron acceptors, for example sulphate,
nitrate, iron and manganese. are then used to allow biodegradation to continue under
anaerobic conditions. Finally site conditions may become so reducing that
biodegradation occurs under methanogenic conditions.
Specific examples of biodegradation are included in the sections that follow. In
general, most hydrocarbon-based compounds and most oxygenated-organics are
relatively biodegradable under a wide range of conditions, and natural attenuation of
such plumes often significant Chlorinated (halogenated) compounds are generally less
biodegradable but evidence has increasingly shown that they do biodegrade under
appropriate redox-bacterial conditions. A sequence of reactions under varying redox
conditions may be required to allow complete biodegradation to benign products. This
means that for some contaminants and sites full biodegradation 1o benign products is
difficult and there may be persistence of both the original contaminants and their
intermediate degradation products, both of which may have toxicity.
4.5.3 Organic chemicals of major concern in groundwater
Consideration of the above transport and attenuation processes, together with data on
organic chemical toxicity, use of chemicals and associated potential for release to the
subsurface, and actual chemical occurrence in groundwater data, enables identification of
groups of organic chemicals, as well as individual chemicals, thought to be of major
concern in groundwater. Two organic chemical groups of key concern include:
• aromatic hydrocarbons: benzene, toluene, ethylbenz.ene and xylenes (BTEX);
• chlorinated hydrocarbons (aliphatic and aromatic): dichloromethane (DCM),
trichloromethane (TCM, also known as chloroform), tetrachloromethane (also
known as carbon tetrachloride, crq, trichloroethene (TCE), tetrachloroethene
(PCE, also known as perchloroethylene), vinyl chloride (Vq, 1,2-
dichloroethane (1,2-DCA), 1,1-dichloroethene (1,1-DCE), l,2-dichloroethene
(cis and trans isomers, cDCE and tDCE respectively), 1,2 dichlorobenzene (1,2-
DCB) and 1,4 dichlorobenzene (1,4-DCB).
Properties of the above compounds are included in Table 4.L
Chemicals: Health relevance, transport and attenuation 115
Figure 4.7 presents data based upon 250 sit.es in Germany and 500 sit.es in the USA
(Kemdorff et al., 1992) and indi~ the prevalence of the above compounds in
groundwater. A third (predominantly) organic chemical group of key concern in
groundwater is pesticides.
T etrac:hlomelhen
T1idDoetl .. ,e
cisllrans-Dichloroelhene
1,1, 1-T,idllo!oelhai,e
Tlicl1larcmelhane
1,2--0ielllo!oetla,e
\my! chloride
Dichloromelhane
Tetradioromethane
1, 1,2-Tiichlo.oetl a,e
1.1-dciDoetlee
1.1-<lichloroethan
Chlarnbenzene
1,2-0ichkl'.llbenzene
1,4-Dichlarobenzene
1,3-0iehla'Dbenze
Benzene
i-,n-Xytene
Toluene
Naphthalene
E1hyl>enzene
o-Xylene
~
2~
2.4,&-Tnchlcnljlhenol
3,5-0inelhytphenol
Phenol -..
2-6utancne
lsophcrale
:Z.4-<limelhylphencl
0 10
~
-
--
= =
~
Frequency of de1ection {% I
20 30 40 50
Allphlllio chlorinalod hydroc:ad>ons
~ SA
--·~-
Aramatic hydrocarbons
Oxl/90,-lad hydroc:arbons
Figure 4. 7. The 25 most frequently detected organic groundwater motmninants at hlwirdous waste
sites in Germany (250 sites) and the USA (500 sites) (based on ooncentrations ~ lµg/1) (adapted
from Kemdorff et al., 1992)
Of the many organic chemicals that may potentially contaminate groID1dwater, these
three groups have perhaps received the most attention from both the groundwater
practitioner and research communities during the 1980s and 1990s. The following
Sections 4.5.4, 4.5.5 and 4.6 are hence devoted to these three groups.
1be focus upon the above three groups does not preclude the potential importance of
other organic contaminants in groundwater. Although compounds may pemaps not
occm frequently due to restricted use within specialized industries, some compoW1ds
may have low natlil'al attenuation (NA) properties and potentially develop extensive
pllil'Iles. Other chemical groups may, in contrast, have received widespread industrial
use, but were perhaps thought (sometimes mistakenly) to pose a much lower risk to
groundwater due to high NA properties. Examples of the latter may include P AHs and
PCBs that are both briefly discussed below.
116 Protecting Groundwater for Health
P AHs are a component of creosotes and coal tars frequently associated with former
gasworks and coal carbonization (coking) works (Johansen et al., 1997). They are a
diverse class of compounds of natural and anthropogenic origin. some of which show
carcinogenic properties. WHO has derived a guideline value of 0.7 µg/1 for
benzo(a)pyrene because it may be released fiom coal tar coatings of drinking-water
distnlmtion pipes. Most P AHs have extremely low solubilities in water and have a high
tendency to adsorb to the organic matrix of soils and sediments, particularly the higher
molecular mass, higher-ring PAHs, e.g. the 5-ring benz.o(a)pyrene. Thus, they are gene-
rally not found in water in notable concentrations, and hwnan exposure is mostly through
food prepared at high temperatures and air (particularly from open fires) (WHO, 2004a).
Countering the processes that may serve to attenuate gJOundwater impacts, however, are:
(i) creosote and coal tars may occur as a DNAPL ( density is composition dependent, but
may be around 1.05) and slowly migrate as a DNAPL deep into the subslD'face
potentially penetrating the water table; and fri) higher molecular ~ higher ring-
member P AHs are much more resistant to biodegradation and hence dissolved plumes,
although slow to develop, may persist and gJOW over decades (King and Barker, 1999).
PCBs are a class of stable compounds, each containing a biphenyl nucleus (two
linked benz.ene rings) with two or more substituent chlorine atoms. PCBs are produced
industrially as complex mixtures that often contain between 40 and 60 different
chlorinated biphenyls.. Similar to PAHs, most PCBs are of low solubility in water and
sorptive and hence dissolved-phase plwnes in gJOundwater tend not to be large.
However, PCB oils, historically used in electrical transformer fucilities, are DNAPLs and
may potentially penetrate deep into aquifer systems.. Dissolved PCBs are generally slow
to biodegrade and hence PCBs,. like P AHs, may serve as long-term sources of
groundwater contamination.
An emergent gJOundwater issue in the 1990s relating to hydrocarbon fuels has been
the use of oxygenates, particularly methyl tertiary-butyl ether (MfBE) and methanol or
ethanol within fuels (Squillace et al., 1996). Use ofMIBE has been most significant in
the USA with MIBE first used in gasoline at great.er than IO per cent by volwne in 1992.
MIBE has a strong taste and odolD' and is likely to impair drinking-water quality at
concentrations in the 0.01-0.1 mg/I range. However, because of its low relevance to
hwnan health, MIBE is not further discussed in this chapter; see WHO (2005c) for
further information on MIBE.
4.5.4 Aromatic hydrocarbons (BTEX)
Health aspects
Mononuclear (single-ring) aromatic hydrocarbons such as BTEX are amongst the most
common groundwater contaminal1s (Figlll'C 4. 7) and the main aromatic faction of many
hydrocarbon fuels. The key compound of health relevance \\'ithin the B1EX gJOup is
benzene, a proven carcinogen in humans. The mechanism or metabolic form by which it
exerts its action (haematological changes, including leukaemia) is not clear. WHO
(2004a) has established a guideline value for benzene in drinking-water of 10 µg/1,
corresponding to a lifespan risk of 10-5 to contract cancer by exposure to benzene via
drinking-water. Alkylated benzenes are much less toxic than benzene, and
correspondingly their WHO guideline values are 700 µg/l for toluene, 300 µg/L for
Chemicals: Health relevance, transport and attenuation 117
ethylbenzene and 500 µg/1 fur xylene (WHO, 2004a), whereas no health-based guideline
values are given for trimethylbenzenes and bulylbenzene. However, some of them may
be perceived by odour and/or 1aste at only a few micrograms per litre.
Sources and occurrence
The aromatic hydrocarbons B1EX are the primary contaminants of concern associated
with point-sources of fuels and fuel-related contamination originating from petroleum
production, refining and wholesale and retail distnbution (service stations) ofpetrolewn
products (Newell et al., 1995). They are also used as solvents and raw materials in
chemical productioIL Spills and accidental releases of gasoline (petrol), kerosene and
diesel are common sources of their occurrence in the environment The German-USA
groundwater survey data in Figure 4.7 indicate B1EX was relatively common, benzene
being the most prominent
Transport and attenuation
Most petroleum products are LNAPLs and hence the Figure 4.4 conceptual model
applies. BTEX components typically comprise just a few per cent of the LNAPL fuel.
B1EX concentrations dissolving in groundwater near fuel sources are reduced from their
pure-phase solubility values (as individual oomponent solubiliz.ed concentrations form
NAPL mixtures depend upon the mass (strictly mole) fuction of that component in the
NAPL). BTEX-aromatics, being the most soluble hydrocarbons, are st:i1l the main risk
driver for grotmdwater at hydrocarbon-contaminated sites, as solubiliz.ed concentrations
and mobility of other alkane (branched and straight-chain) and PAH hydrocarbons are
much lower. Since the early 1990s, it has been recognized that natural attenuation (NA)
of B1EX is highly significant at most sites due to the high biodegradability of BTEX
tmder a range of conditiom. M>nitored natural attenuation, i.e. monitoring of the growth,
stability and eventual decline of dissolved-phase B1EX plumes, has indeed become a
viable and cost-effective remediation option at many sites rather than active remedial
measures such as pwnp-and-treat (McAllister and Chiang, 1994).
Studies that have examined dissolved-phase hydrocarbon plwne lengths from over
600 hydrocarbon-release sites in the USA are particularly instructive on the potential for
NA applicability (Newell and Connor, 1998; Wiedemeier et al., 1999 and references
therein). Of the 604 plumes evaluated, 86 per cent were less than 300 feet (-100 m) long
with only 2 per cent of plumes greater than 900 feet One of the studies that examined
271 plumes indicated only 8 per cent of these plumes were still growing, 59 per cent of
plwnes were approximately stable as mass being dissolved from the source was balanced
by mass being depleted by attenuation (biodegradation), and 33 per cent of plumes were
shrinking as source mass inputs declined or biodegradation of contaminants perhaps
became increasingly efficient with time. These studies hence provide a strong rationale
for occurrence of NA across a variety of site conditions. Under the vast majority of
circumstances the potential for impacts of hydrocarbon plumes is limited to distances of
a few hundred meters from source zones. Thus although hydrocarbon sources can be
numerous, hydrocarbon and B1EX impacts are likely to remain local to those sources
zones. However, although dissolved-phase plume may extend to relatively short
distances from source areas, LNAPL source zones (i.e. pancake-like of hydrocarbon)
118 Protecting Groundwater for Health
themselves can on occasion be very extensive, for example Albu et al. (2002) depict
zones ofLNAPL extending over 5 km armmd oil refinay sites near Ploiesti, Romania
Much insight into the importance ofbiodegradation and associated controlling factors
has been obtained in plume studies. A controlled injection of dissolved-phase benzene,
toluene and xylene at the Borden site, Canada (Barker et al., 1987) showed complete
benz.ene, toluene and xylene biodegradation by just over 400 days with only benz.ene
persisting beyond 270 days. This study, and many real spill sites indicate BTEX are
readily degraded when dissolved oxygen is present in groundwater. Under anaerobic
conditions, rates ofbiodegradation of remaining hydrocarbon was governed by both the
rate of oxygen re-entry across the contaminant plume fringe and rates of alternative
anaerobic biodegradation pathways using less efficient electron acceptors such as nitrate,
sulphate, and iron(Ill). Many other field sites have demonstrated the importance of
anaerobic processes, primarily through changes in the groundwater geochemistry. For
example, the Plattsburgh, Hill and Patrick Air Force Bases in the USA (Wiedemeier et
al., 1999) indicate development of depleted dissolved oxygen, nitrate and sulphate
coincident with the BTEX plumes as these electron acceptors are consumed in the oxida-
tion of the BTEX pH declines as well as production ofFe(ll) and methane arising from
methanogenic activity were also evident where the most reducing conditions prevailed.
A myriad of aerobic and anaerobic BTEX biodegradation rates are available from the
literature, e.g. data and references within Wiedemeier et al. (1999) and Noble and
Morgan (2002). Rates are typically expressed as a first order rate constant or an
equivalent half-life. Table 4.2 summarizes half-life data provided in the review by Noble
and Morgan (2002) for BTEX, naphthalene and some chlorinated hydrocarbons. These
chemicals represent the most studied groundwater contaminants in relation to
biodegradation The table subdivides rate data between laboratory and field studies that
are in turn subdivided to aerobic and anaerobic conditions. Although the work of Noble
and Morgan is reasonably comprehensive and based upon many citations, it should be
noted that Table 4.2 aims to be illustrative rather than comprehensive of all literature
available on biodegradation rates for the chemicals listed; biodegradation is an active
area of study worldwide and half-life data continue to be published.
Rates selected for risk assessment modelling at other sites (where field data are
insufficient to determine rates) need to be used with care as modelling results, and hence
plume attenuation predicted and any site risk-based remediation standards computed, are
very sensitive to degradation mass-loss parameters selected. Rates may vary significantly
for individual compounds that may be a reflection of rates being lab-based or field based
and the particular aerobic-anaerobic site conditions. This is apparent from examination of
Table 42. Also, field biodegradation rates may be derived from a localiz.ed point
measurement, or more often a rate predicted from whole plume behaviour that will
average varying rates and different biodegradation processes and aerobidanaerobic
conditions occurring throughout the plume.
The laboratory half-lives in Table 4.2 are generally shorter than the equivalent field-
based values, i.e. plumes are apparently more persistent in the field presumably as a
reflection of field conditions, e.g. supply of electron acceptors being less optimal than
can be achieved in a laboratory. In general, BTEX degradation rates will be lower under
anaerobic conditions and plume may be persistent and indeed attain significant lengths
Chemicals: Health relevance, transport and attenuation 119
where groundwater is naturally anaerobic, e.g. confined aquifer conditions. This is
reasonably demonstrated by the Table 42 field data, but less so by the laboratory-based
data; the latter is in part fiom the limited studies tmdertak:en ( at least reported) for some
of the chemicals. A cautionary approach is warranted to the application to sit.es of the
half-life data provided in Table 42 (and elsewhere); clearly the range in half-life values
for a specific contaminant is large and for some contaminants and conditions insufficient
data exist to yield a reliable average and range. Some Table 42 entries are based on a
single study, in some cases no data are provided. The latter may be a reflection of a
genuine lack of data or else biodegradat:ion not being effective under the specific
conditions ( e.g. aerobic biodegradat:ion of PCE).
Table 4.2. Summary ofbiodegradation half.life data (at 10°C) for important organic groundwater contaminants (adapted from Noble and Morgan, 2002)
Chemical Aerobic -laboratory data Aerobic -field study data Anaerobic -laboratory data Anaerobic -field study data
No.of Mean Range in No.of Mean Range in No.of Mean Range in No.of Mean Range in
studies half.life half-life studies half-life half-life studies half.life half.life studies half.life half-life
(n) (daYS) (da~) (n) (days) (days) (nl (da r s) (davs) (n) (davs) (days)
Aromatic Hydrocarbons
Benzene 18 34 3-200 6 220 15-490 11 79 20-200 13 502 85-2000
Toluene 5 120 5-320 3 120 15-178 15 64 5-320 9 257 10-660
Ethyl benzene No data provided No data provided 4 239 53-548 4 519 238-693
Xylenes 1 11 11-11 3 58 1-123 5 63 j0-155 8 489 72-800
Naphthalene 11 138 10-400 No data provided No data provided No data provided
Chlorinated Hydrocarbons
PCE No data provided No data provided 6 23 4-62 4 1600 4-3600
TCE No data provided No data provided 8 43 3-99 12 1460 3-6600
OCE 5 2 O.S-3 3 39 12-S6 I 280 280-280 9 4060 42-16860
vc 7 14 84 1 10 10-10 5 81 24-124 5 948 S06-1265
DCA No data provided No data provided 5 157 16-340 2 1430 460-2400
Chemicals: Health relevance, transport and attenuation 121
4.5.5 Chlorinated hydrocarbons
Health aspects
A munber of aliphatic and aromatic chlorinated hydrocarbons are of health significance
because of their toxicity and occurrence in drinking-water, particularly as in
grouoowater, their concentrations do not decrease rapidly through volatilisation,
anaerobic degradation is slow, and in consequence contaminants may persist for some
time.
WHO (2004) gives guideline values for dichloromethane (DCM), trihalomethanes
chloromethane ( e.g. chloroform which may be generated as by-products of disinfection),
tetrachloromethane (also known as carbon telrachloride, CTC), trichloroethene (TCE),
tetrachloroethene (PCE. also known as perchloroethylene), vinyl chloride (VC), 1,2-
dichloroethane (1,2-DCA), 1,1-dichloroethene (1,1-DCE), 1,2-dichloroethene (cis and
trans isomers, cDCE and tDCE respectively), 1,2 dichlorobenzene (1,2-DCB) and 1,4
dichlorobenzene (I,4-DCB) (see Table 4.1 for selected physiochemical parameter
values). Generally, among the saturated chlorinated compounds, the 1,1-halogenated
ones (e.g. 1,1-dichloroethane) are ofless he.alth concern than the 1,2-halogenated one&
(e.g. 1,2-0CA), since they are metabolized differently. This is also true for higher
halogenated compounds ( e.g. trichloroethanes ).
Dichloromethane (DCM, also known as methylene chloride) is oflow acute toxicity,
and current evidence suggests that it is not a genotoxic carcinogen. The WHO drinking-
water guideline value for DCM is 20 µw')., based on hq,atotoxic effects observed in rats
(WHO, 2004a). Carbon tetrachloride (CTC) has a WHO guideline value of 4 µw']. based
on its liver toxicity, and fiom carcinogenic effects observed in laboratory animals it is
classified as possibly carcinogenic to humans. 1,2-dichloroethane (1,2-DCA) is
pot.entially genotoxic and a proven carcinogen in experimental animals with a WHO
guideline value of30 µ!Y'l, (WHO, 2003d; 2004a).
TrichloroetheB! (TCE) and tetrachloroethene (PCE) may degrade to the more toxic
vinyl chloride. The provisional WHO guideline value for TCE is 70 µw'). based on liver
effects in mice (WHO, 2004a; 2005d). PCE causes nervous disorders at high do~,
whereas at lower doses kidney and liver damage have been reported. It is classified as
possible hwnan carcinogen with overall evidence indicating that it is not genotoxic
(WHO, 2003e), and its WHO guideline value for drinking-water is 40 µw'). (WHO,
2004a). Vinyl chloride (VC) is genotoxic and carcinogenic in experimental animals as
well as in humans. Administered orally to experimental animals, it produced cancer at a
variety of sites (WHO, 2004d). Its WHO drinking-water guideline value is 03 µw'].
(WHO, 2004a).
1, 1-Dichloroethene (l.1-DCE) is a weak in vitro-mutagen and not classifiable as to its
carcinogenicity to humans. It is a central nervous system depressant and may cause liver
and kidney toxicity. The WHO guideline value is 30 µw']. (WHO, 2004a; 2005e). Among
the two isomers of 1,2-dichloroethene, the cis-form is detected more frequently and at
higher concentrations than the trans-form as a water contaminant, since the former is the
main anaerobic metabolite of TCE and PCE. As such it may indicate as well the
presence of vinyl chloride, the next anaerobic breakdown product, which is not only
much more toxic than all higher chlorinated ethenes but also a genotoxic human
carcinogen (see above). In contrast, both 1,2-DCEs do not seem to be genotoxic and
122 Protecting Groundwater for Health
there is no information on their carcinogenic potential. The WHO drinking-water
guideline value for each of the 2 isomers is 50 µwt (WHO, 2003:f; 2004a).
Dichlorobenzenes are the least toxic of this group of oontaminants. Their health based
guideline values in drinking-water are 300 and 1000 µwt for 1,2-and 1,4-DCB,
respectively. These exceed their odour threshold range of 0.3-30 µwt by far (WHO,
2003g; 2004a).
Sources and ocaurence
Chlorinated hydrocarbons are employed in a variety of industrial activities, including
almost any facility where degreasing, e.g. of metals, circuit boards, textiles ( dry cleaning)
and animal/leather hides. metal stripping, chemical manufacturing, pesticide production
or other activities where chlorinated solvents, cleaners, dry cleaning fluids, paint
removers are used (Chapter 11 ).
In many industrialimd oountries, chlorinated hydrocarbons are the most frequently
detected groWldwater oontaminants at hazardous waste sites (Kemdorff et al., 1°992;
Plmnb, 1992; NRC, 1994). This is highlighted by the German-USA survey data in
Figure 4.7. TCE and PCE together with their principal metabolites cOCE and VC have
been the most frequently detected chlorinated hydrocarbons at the investigated sites.
Point source release of chlorinated hydrocarbons to grolDldwater is anticipated to be the
main source of groWldwater contaminatioIL Complex mixtures of chlorinated
hydrocarbons may arise from leakages at hazmtlous waste disposal sites where many
solvent types may have been disposed. In contrast, spills at industrial
manufacturing/processing sites may well comprise liquid chlorinated hydrocarbon as a
DNAPL with a high proportion of a single chlorinated hydrocarbon component A
multitude of point sources exist in many urban areas due to the diversity and frequency
of chlorinated hydrocarbon users.
Examples of regional chlorinated hydrocarbon contamination within aquifers
underlying urban towns and cities emerged during the 1980s. Many groundwater
supplies or monitoring wells were contaminated in some irntances, particularly by TCE
and to a lesser extent PCE. Examples include Milan, Italy (Cavallero et al., 1985); the
New Jersey ooastal plain aquifer, USA (Fusillo, 1985) and Birmingham, United
Kingdom (Rivett et al., 1990); the latter example is descnbed in Box 4.2.
Transport and attenuation
Many of the chlorinated hydrocarbons will have entered the subsurface in the DNAPL
funn and may reside to significant depths within aquifers (Pankow and Cherry, 1996).
They typically have low to medium water solubility (in the range of 0.2-20 wt;
Table 4.1 ). Dissolution of DNAPL sources is expected to be slow taking years to
decades, particularly from long lengths of residual DNAPL pools that have invaded or
diffused into low-permeability strata. Dissolved-phase plumes of chlorinated
hydrocarbons can be veiy extensive, for example Mackay and Cherry (1989) depict
several plumes in the km-scale and Jackson (1998) several plumes in the alluvial aquifers
of the southwestern USA that are arolDld IO km in length. Some plumes have lead to
high profile court cases and set legal precedents on apportioning liability for historic
contamination events, e.g. the near 2-km PCE plume that caused contamination of the
Chemicals: Health relevance, transport and attenuation 123
Sawston public water supply borehole in Cambridgeshire, United Kingdom (Ashley,
19'J8).
Box 4.2. Chlorinated-hydrocarbon contamination of groundwater in
Birmingham, United Kingdom (based on Rivett et al., 1990; Rivett et al., 2005)
Birmingham is the second largest city in the United Kingdom and has a long
history of manufactming, particularly in metal-related industries. Groundwater
samples were taken during the late 1980s from 59 abstraction boreholes
typically screened over 100 min the Triassic Sandstone aquifer underlying the
city. Chlorinated solvents were found to be widespread, particularly TCE
detected in 78 per cent of abstraction boreholes with over 40 per cent of the
sampled boreholes showing concentrations over 30 µ!efl to a maximum of
5500 µ!efl. The majority of highly contaminated abstractions were located in
solvent-user sites, predominantly meta1s-related industry. The predominance of
TCE was ascn"bed to its main United Kingdom use within metal cleaning
applications since the 1930s. PCE was less evident as it has generally only been
used for dry cleaning in the United Kingdom since 1950s. Lower TCA
OCClllTeflce was ascnbed to its much later introduction in the United Kingdom
starting about 1%5 as a less toxic replacement to TCE. Greatest groundwater
contamination oa::urred in the Tame valley area that was hydrogeologically
vulnerable due to low depths to groundwater and limited aquifer protection by
low permeability drift. Moderate contamination was present in other less
vulnerable areas of the unconfined aquifer with least contamination evident in
the Mercia Mudstone confined aquifer.
The aquifer was re-visited a decade later during the late llJCJOs. Declines in
industrial use of groundwater meant only 36 abstractions were active and
available for sampling, of these 26 were from the 1980s survey. Overall
contamination detected was less and attnbuted to most of the new boreholes
being located in industry areas where solvent use appeared limited. Also, many
of the former highly contaminated abstractions had ceased operation due to
industry closure. The latter was of some concern as contamination previously
inadvertently captured by such abstractions was now able to more freely migrate
into the wider aquifer. Comparison of the 26 abstractions common to both
surveys indicated contamination at individual boreholes was at similar or greater
concentrations in the more recent survey compared to the decade-earlier survey.
· These levels are unlikely to be due to major ongoing contamination, rather, it is
reasonably assumed that incidences of new contamination will have declined
over the decade as industry has become much more environmentally aware. The
sustained level of contamination was hence ascnbed to persistent sources of
chlorinated solvents, likely DNAPL sources at depth. These will have remained
unaffected by remedial works implemented at many sites to date because under
a land redevelopment focused agenda these predominantly focused upon
shallow soil and groundwater problems.
124 Protecting Groundwater for Health
Under aerobic conditions, biodegradation of solvents such as TCE and PCE c.an be
limited to non-existent and may accomrt for the extensive plwne examples noted above.
Sorption is often limited too, particularly for the less hydrophobic compounds where
compound solubility exceeds lg,'I (fable 4.1). A controlled emplacement ofa DNAPL
chlorinated solvent source in the Borden aquifer research site, Canada, resulted in TCM
and TCE plwnes exhibiting near conservative behaviour with retardation factors in the
range of 1.0-1.2 and no evidence ofbiodegradation for these solvents and also PCE that
was more retarded at about 1.6 (Rivett et al., 2001; Rivett and Allen-King, 2003).
Dispersion of these plwnes, although moderate in this relatively homogeneous sand
aquifer, nevertheless produced leading plwne contours at concentrations in the range of
drinking-water standards that had travelled toward 100 per cent further than the mean
advection (groundwater) velocity.
In contrast, other sites have shown significant natural attenuation of chlorinated
hydrocarbons due to biodegradation activity. The most well kmwn biodegradation
pathways are those involving the sequential reductive dechlorination of chlorinated
hydrocarbons where lesser chlorinated organics, chloride and ultimately hydrocarbons
such as ethane or ethane, are formed (Vogel et al., 1987), e.g. PCE is transformed to
TCE to cDCE ( usually the predominant isomer) to VC to ethene. On average chlorinated
hydrocarbon plwnes are signi:fic.antly longer than the aforementioned B1EX plwnes. For
example, Newell et al. (1990) reported a median length of 1000 feet (about 300 m) for
chlorinated ethene (PCE, TCE, DCE, VC) plwnes (88 sites sampled).
Biodegradation of chlorinated hydrocarbons has proven to be relatively complicated
with five possible degradation )J1'ClCe&5eS (Wiedemeier et al., 1999). Most chlorinated
compounds have been observed to biodegrade by three or four of these processes, only
DCE and VC may biodegra.de via all five processes. Under anaerobic or low oxygen
conditions degradation processes include (i) dehalorespiration, in which the chlorinated
hydrocarbon is used as the electron acceptor and effectively respired, (ii) direct anaerobic
oxidation and (iii) anaerobic co-metabolism. Under aerobic conditions, further processes
are (iv) direct aerobic oxidation and (v) aerobic co-metabolism. Direct processes involve
the chlorinated hydrocarbon being used as the primary growth substrate.
Dehalorespiration and co-metabolism both require an alternative primary growth
substrate to be present That primary substrate is normally a relatively biodegradable
substrate and may include anthropogenic carbon such as B1EX contamination.
Alternatively, anaerobic conditions may be driven by high levels of na1urally occt.Dring
carbon acting as the substrate, a primary example being wetland sediments and sub
riverbed deposits, e.g. Lorah and Olsen (1999) observed TCE and 1,1,2,2-PCE
dechlorinations in the former.
Due to the complexity of biodegradation processes outlined, there is a wide
divergence in reported biodegradation rates of chlorinated hydrocarbons (Wiedemeier et
al., 1999; Noble and Morgan, 2002; Table 42). This is clearly illustrated by the Table
4.2 half-life data for the more common chlorinated hydrocarbon grolDldwater
contaminants, e.g. DCE field-based half life data vary fiom just 42 days to nearly 17 000
days. Also, Table 4.2 indicates laboratmy half-life data are generally much shorter (by 1-
2 orders of magnitude) than equivalent field data, e.g. TCE data under anaerobic
conditions indicate a laboratory half life mean of 43 days compared to a field mean of
Chemicals: Health relevance, transport and attenuation 125
1460 days. This is pemaps ascribed to the fact that optimal anaerobic reducing conditions
can be achieved in the laboratory for the whole sample, whereas in the field such
anaerobic conditions may in fact only occur in localiz.ed portions of a pllllile. Table 4.2
emphasiz.es the sensitivity of half life to aerobic and anaerobic conditions and that much
longer half life values may occur for chlorinated hydrocarbons relative to the aromatics.
The above strongly endorses the need to recogniz.e that literature half-life data have veiy
significant uncertainty when applied in a predictive ~ to sites elsewhere. The
unfortunate reality is that most sites require individual case-by-case assessment to allow
effective prediction of natural attenuation rates.
4.6 PESTICIDES
Pesticides represent a wide range of compounds used mostly as insecticides, herbicides,
and fimgicides.. Formerly a · small number of classes of chemicals included most
pesticides, ie. orgaoochlorines, organophosphates, carbamates, phenoxyacetic acids and
triazine herbicides. However, modern pesticides include other types of chemicals, and
therefore such a classification is of more limited use for descriptive purposes. Many of
the historically used pesticides, such as the organochlorines, are however
environmentally persistent and may pose a long-term groundwater problem.
Health aspects
In general, health effects associated with pesticides are specific for each chemical. This is
reflected in their different WHO guideline values for drinking-water quality (see WHO,
2004a) and in the wide range of acceptable daily intake values derived by the Food and
Agricultural Organization (F AO) for exposure through food (resulting from pesticide
uses on crops; F AO, 2004). Most health effect studies are conducted using single
compounds, little is known about effects associated with pesticide mixtures. Health
effects from acute (short-term and high level) or chronic (long-term and low-level)
exposure include liver and kidney damage, major interference with nervous, immune and
reproductive system fimctions, birth defects and cancer. In most cases the risk from food
contaminated by unduly high levels of pesticides is likely to be more significant than that
posed by pesticide levels in drinking-water.
Chronic exposure associated with pesticides has declined in Europe and North
America as many of the more persistent herbicides (such as chlordane, DDT, dieldrin,
endrin, heptachlor, y-HCH and toxaphene) have been restricted or phased ottt (Barnard et
al, 1997). They have been replaced with less persistent and more species-specific
toxicants. While acme toxicities have often increased, some important new biologically-
derived insecticides have veiy low mammalian toxicity.
Sources and occurrence
Pesticides are intentionally applied to protect crops in agriculture (Chapter 9) as well as
to control pests and unwanted vegetation in gardens, buildings, railway tracks, forests
and roadsides (Chapter 13). They may be accidentally released from production sites
(Chapter 11) or, more often, transported away from their site of application in water, air
or dust Pesticides can reach groundwater after accidental spills or excessive application
126 Protecting Groundwater for Health
in geologically sensitive settings, from contamination of poorly sealed wells by surface
nmoff after intensive rains following field application and from storage or production
sites. Though some o~ochlorine insecticides have been banned or are SUQject to
severe restrictions in many cotmtries, in several developing countries production and use
of; for example, DDT has continued because of its relatively inexpensive production and
its high efficacy against mosquitoes in malaria control. Generally the dilemma of the low
cost and high efficacy of persistent pesticides versus their long term health and
environmental effi:cts remains a contentious global issue which has been addressed by a
global convention (see Box 9.5).
As sampling has become more extensive and monitoring programmes developed,
increasing numbers of pesticide compounds are being detected in groundwater. A major
study, the National Pesticide Survey, conducted by the US EPA in the late 1980s
detected 46 pesticides in groundwater in 26 states originating from normal agricultural
practice (Williams et al, 1988) but with low frequency and usually below health-based
standards. Pesticides detected in more than five states were alachlor, aldicarb, atrazine,
cyanazine, metolachlor and simazine. More recently, extensive sampling within the
USGS National Water Quality Assessment programme has confirmed the widespread
occmrence of pesticides in both surface wat.er resources and groundwater, but generally
at concentrations below their respective allowable maximum contaminant levels (Kolpin
et al., 2000). The newer work has, however, shown that pesticides, especially
insecticides, are also reaching water resources in urban and suburban areas, including
residential sources. This work has also demonstrated widespread detection of pesticide
metabolites, often at concentrations exceeding the parent compotmd, and for which there
may not be adequate toxicity data to establish their health significance.
This picture is largely confirmed by monitoring efforts in Europe. Herbicides which
are widely used in cereal cultivation, such as MCPP and isoproturon are detected in the
cotmtries of northern Europe (Spliid and Keppen, 1998) with carbamates perhaps more
common further south. Most detected grotmdwater pesticide concentrations were in the
range 0.1 to 10 µgll. Concentrations significantly above this range can probably be
attnbuted to local point source contamination from poor disposal practices, or from non-
agricultural usage such as on railways.
Because of high analysis oosts, much less monitoring has been tmdertaken in low-
income cotmtries and data from tropical regions are scarce. However, atrazine residues
from its use in sugar cane cultivation were widely observed in groundwater in Barbados
and carbofuran was detected in shallow groundwater beneath irrigated vegetable
cultivation in Sri Lanka (Chilton et al., 1998). Elsewhere, presence of ~hlorines in
grotmdwater reflects their highly persistent nature and perhaps continuing usage even
when banned (Matin et al., 1996).
Transport and attenuation
The mobility and persistence of pesticides in the environment are well understood
because admission of a new pesticide for the market requires a series of standardiz.ed
laboratory and field experiments.
The overall likelihood of a pesticide to be a groundwater pollutant is dependent both
on its persistence and its soil sorption. Table 4.3 lists pesticides used in agriculture for
Chemicals: Health relevance, transport and attenuation 127
which WHO has derived health-based drinking-water guideline values. It provides a
classification of their leaching and nmoff potential based on their physical-chemical
characteristics, i.e. their persistence ( characteriz.ed by soil half-lives) and soil organic
carbon sorption (Koc). Other pesticides med for public health purposes (e.g. DDT,
chlorpyrifos and pyriproxyfen in malaria control) and wood conservation (e.g.
pentachloropbenol or PCP) are not listed in Table 43.
As for the organic contaminants discussed in Sections 4.1.2 and 4.5.2, soil organic
matter content., clay content and permeability all affect the potential for pesticides to
leach through soils. In general, soils with moderat.e-to-high organic matter and clay
content will absorb pesticides onto soil particles, making them less available fur leaching,
and moderate or low permeability soils allow less water infiltration.
A wide range of pesticide soil sorptionl<.i values (as defined earlier in Section 4.5.2)
exist. DDT, for example, has a Ket value roughly 20 000 times as high as that for aldicarb
and 1500 times as high as that for atrazine. This explains why aldicarb and atrazine have
been found in groundwater in agricultural areas while DDT has not
There are several processes by which pesticide may be degraded. Exposure to
sunlight may cause photolysis before they leach into soils. Hydrolysis, the degradation of
a chemical in reaction with water that may occur at surface, in the soil zone and
underlying groundwater; the longer the hydrolysis half-life the more probable it will
enter groundwater. Biodegradation, i.e. enzymatic reactions driven by microorganisms,
will occur at greatest rates in microbially active soil. Chlorinated pesticides and triazine
herbicides are the most resistant to biodegradation and may persist for years :following
application. Although the mobility of some organochlorine insecticides is limited by their
high hydrophobicity (Table 4.3), their persistence is mirrored in the accmnulation in :ratty
tissues in animals, including fish and humans, mostly from pesticides in ~water
fuod chains. Organic phosphorus pesticides tend to hydrolyse rather quickly at pH values
above neutral, thus losing their toxic properties. However, under dry conditions some
have been observed to persist for many months (Graham-Bryce, 1981). Carbamat.es are
noted for their high susceptibility to degradation (Williams et al, 1988).
Higher water . solubility does not necessarily correlate with a lower degree of
persistence, but highly sorbed pesticides tend to be more persistent The biodegradability
of pesticides depends on their molecular structure and soil half-lives can vary between a
couple of days to years (Table 4.3). Quite long halflives can occur once pesticides leave
the soil and reach the less biologically active zones of aquifers (La.vu et al., 1996; Chilton
et al., 2000). It should be noted that although many pesticide half-lives, have been
determined for soils (Table 4.3); use of such half-lives to predict aquifer behaviour may
cause misleadingly optimistic attenuation estimates.
Several other pesticide concerns remain. Little is known about the fate of pesticides in
tropical environments, most published data are from registration trials in temperate
regions. For all pesticides there is potential for incomplete transformation of the parent
compound into metabolites which may also be more or less toxic (Sawyer et al, 1994)
and may themselves be persistent enough to be detected in groundwater. When
pesticides do get into groundw'3ter, cleanup of the contamination is usually prohibitively
costly and often may not be practically feasible. The contamination can last many years
128 Protecting Groundwater for Health
and spread over a large area before dilution and degradation eventually reduce the
pesticide concentrations.
Table 4.3. Cla$mcation of leadiing potmtial for agriwltural pesticides fur which WHO bas
derived guideline values (data fiom the US Department of Agriwlture Natural Resoura:s
Conservation Service and ~wltural Research Servicel
Commonname CAS-No. WHOGVWaler Soil Koc I..eacbing Solution Adsorbal
(mg,1) solubility balf-lifl'(mL'g) potmtial runoff runoff
at20-25"C {lda ~ l f':!!mlial potential
Alachkr 1597U,0-8 .20 240 15 170 Medium Medium Low
Aldicaib ]]6-(6.3 10 (,(XX) 30 30 High Medium Low
Aldrin 3()1).00.2 0.03 0.027 365 500) Low Medium High
DieJdrin 00--57-1 0.03 o.axi JOO)" 1200) Very low Medium High
Atrazine 1912-249 2 33 60 JOO High High Medium
Cubofuran 1563-66-2 7 351 50 22 High High Medium
Oi1ortme 57-749 02 ()_(Yi() 3~ ;nm Verylow Medium High
OiJoo:toonm 15545-48-9 30 74 35 350 Medium High Low
Cyarmioe 21725-46-2 0.6 170 14 190 Medium Medium Low
2,4D 94-75--7 30 890 10 20 Medium Medium Low
2,4-JE 94-82-6 90 46 5 440 Low High Low
1,2-Dihano-3-96-12-41 100) 180 70 High High Medium
chloropupme
1,2-Dibnmodbaoe 106-934 0.4(P) 4300 100-34 High High Medium
(ethylene dtl:roome)
1,2-Diclllolqlmpan 78-87-5 40(P) Z700 700" 50 High High Medium
1,3-Dichloroprop 542-75-6 .20 2250 10 32 Medium Medium Low
I.>imabaie 00--51-5 6 ~ 7 20 Medium Medium Low
Endrin 72-~ 0.6 0.230 4300 l<XKXJ Low Medium High
2,4.5-1P 9J.72-1 OJB 140 21 300 Medium Medium Low
HCB 118-74-1 None! 0.005 l<XXJ" 5<XKXl Verylow Medium High
Jsopcmrm 34123-59-6 9 700 21 130 Medium Medium Low
~rn 58-89-9 None2 7 400 1100 Medium High High
MCPA .2039-46-5 2 ~ 25 20 High Medium Low
MCPP 708-51-00 Nooe' ~ 21 20 High Medium Low
Mdboxychloc 72-43-5 .20 0.100 120 &XXX) Very low Medium High
Metolaillor-51218-45--2 JO 530 90 ;!)() High High Medium
Molinlte 2212-67-1 6 '170 21 190 Mediwn Medium Low
Fbxmnabalin 40487-42-1 20 0275 90 500) Low Medium High
s~ 122-34-9 2 6 60 130 High High Medium
2,4.5-T 9,.76-5 9 278 30 80 High Medium Low
Temuthylazine 5915-41-3 7 9 45 ;!)() High High Medium
T rifluralin 1582-0').8 20 0.300 60 llJOO Low Medium High
• Estimated value; 1 oCWIS in drinking-water at concentrations well below those causing toxic
effects; 2 unlikely to occur in drinking-water
Chemicals: Health relevance, transport and attenuation 129
4.7 EMERGING ISSUES
4.7.1 Pharmaceuticals
There is increasing roncem about miaopollutants originating from pharmaceuticals and
active ingredients in personal care products excreted by people as complex mixtures into
wastewater systems (Kfunmerer, 2004). There are a nwnber of routes through which
phannaceuticals can impact groundwater, but primarily the sources are both untreated
and treated sewage. There is also evidence that substances of pharmaceutical origin are
not completely eliminated during wastewater treatment or biodegraded in the
environment (Daughton and Ternes, 1999; Drewes and Shore, 2001).
Health aspects
Current knowledge on the health effects of pharmaceutically active compotmds at
concentration levels fuund in groundwater samples, which are several orders of
magnitude lower than concentrations which would be therapeutically active, indicates
that there are no effects on human health reasonably to be expected from this source of
exposme (femes, 2001). However, there is an ongoing debate on how comprehensive
health effect data from short-term high dose exposure during diagnosis and treatment
should be extrapolated to long-term low dose exposure during drinking-water
consumption Moreover, the problem of correctly assessing the risk from unexpected
environmental (underground) and technical metabolites (from oxidative drinking-water
treatment) is not resolved. Investigation on the rate of pharmaceutically active substances
in drinking-water unit operations and processes is in pro~ in numerous research
studies e.g. in Europe, Australia, Japan, and North America. Additionally, proposals for
risk ~ent procedures have been suggested (Montforts, 2001 ).
Transport and attenuation
A lack of knowledge still persists regarding the rate of pharmaceuticals during travel
through the subsurface. Findings of recent studies indicate that travel through the
subsurface can substantially attenuate the majority of pharmaceutically active
compounds where surface water or domestic wastewater is used for groundwater
recharge. However, where groundwater is influenced by surface water, such as artificial
recharge, polar pharmaceutically active compotmds such as clofibric acid (blood-lipid
regulating agent), carbamaz.epine and primidone (antiepileptic drugs) and iodinated X-
ray contrast agent can migrate through the subsurface and have been detected in
groundwater samples in Germany and the USA (Heberer et al, 1998; Kuehn and
Mueller, 2000; Drewes et al, 2001).
4.7.2 Endocrine disrupting compounds
There has been increasing public concern about various environmental contaminants
which mimic estrogens and other sex-honnones and hence interfere with endogenous
endocrine systems, with potential adverse effects on human health. A global assessment
of the state of knowledge on endocrine disrupters was published by the International
Programme on Chemical Safety (Damstra et al., 2002).
130 Protecting Groundwater for Health
More than 70 000 chemicals are discussed with respect to endocrine disruptive
potential (Bradley and Zacharewski, 1998). These compounds represent both synthetic
chemicals produced industrially (such as cleaners, pesticides, food additives, birth control
pills, cosmetics) and naturally occurring compollllds (such as steroidal hormones, plant-
produced estrogens, herbal supplements and metals). Whilst endocrine disrupting
compounds (EDCs) are largely organic compounds, it should be noted that some
inorganic substances such as metals are also suspected of endocrine disrupting effects.
Although their potential occurrence in drinking-water has been the subject of public
attention and discussion in some cotmtries, it is important to note that human exposure is
chiefly through food
1be steroidal sex hormones estradioi estrone and testosterone are a class of
hormonally active agents of particular interest because they are naturally excreted into
the environment :fium human and animal sources as well as extensively used as
pharmaceuticals (e.g. birth control pills). Of the nwnerous synthetic chemicals that have
been implicated as endocrine disrupters, many are no longer used in commerce in many
colllltries, such as some organochlorine pesticides (e.g. DDT, endosulphan, dieldrin, and
toxaphene ), and PCBs. Other hormonally active compounds, such as various phenolics
and phthalates, continue to be used in a variety of industrial applications worldwide
(NRC, 1999). Alkylphenol is a biological metabolite of alkylphenol polyethoxylates
commonly used in a variety of industrial, agricultural and household applications as non-
ionic surfuctant:s. Alkylphenol and compounds are both believed to be · endocrine
disrupters (Lye el al., 1999). Another synthetic chemical that has measurable hormonal
activity is Bisphenol A used as a chemical intermediate fur nwnerous industrial products
including polymers, resins, dyes and flame retardants.
Health effects of hormonally active compounds are based on binding of these
compounds on . steroid hormone receptors which control fimdamental mechanisms of
gene regulatioIL The disruption of this process can result mainly in reproductive changes.
Developmental defects, neurobehavioral abnonnalities, immunological · deficits,
carcinogenesis and ecologic effects can also be induced (NRC, 1999).
Determining the risk ofEDCs to humans is difficult because exposure to these agents
has not been routinely monitored, and effects that might be attnbuted to background
concentrations could be complicated by endogenous hormones, pharmacological
estrogens (e.g. hormonal contraceptives), and naturally occurring hormonally active
agents (e.g. phytoestrogens) that are ubiquitous in the environment (NRC, 1999).
Although it is clear that exposures to EDCs at high concentrations can affect human
health, the extent ofhann caused by exposure to these compounds in concentrations that
are commonly found in groundwater is debated (NRC, 1999). Gene:rally, natural and
synthetic steroidal hormones are several thousand times more potent than industrial
chemicals, pesticides and metals (Khan and Ongerth, 2000).
The WHO has not yet specificalJy proposed any guidelines for the occurrence of
EDCs in drinking-water. However, some of the organochlorine compounds are regulated
as pesticides.
The relevance to human health of EDCs occurring in water is currently tmcertain.
Their occurrence in groundwater is linked to the release of sewage, manure, or spill of
specific synthetic chemicals into the enviromnent. The specific processes used in
Chemicals: Health relevance, transport and attenuation 131
wastewater treatment facilities play a key role in the introduction of EDCs into surface
water and groundwater (Drewes and Shore, 2001). The transport of EDCs to
groundwater depends on their hydrophobicity and degradability. The majority of highly
potent compounds such as steroids are hydrophobic and degradable. Degradation rates of
EDC compounds depend on temperature, soil characteristics and their molecular weights
(IUP AC, 2003).
The potential risk related to an uptake of individual EDCs present in wastewater
affect.ed groundwater by humans does not appear to be very significant The small data
set about the fate of EDCs (such as natural and synthetic hormones, surfactants and
pesticides) during percolation through the soil and aquifer and the lack of toxicity data on
long-tenn exposure oflow concentrations makes it at present impossible to finally assess
the impact of EDCs in groundwater on human health. However, contaminated
groundwater may be impacted by a mix of different compounds, which could additively
impose endocrine disrupting effects.
4.8 REFERENCES
Ahmed, F.M, Ali, MA and Adeel, Z. (2001) Technologies for Arsenic Removal From Drinking
Water, BUET, Dhaka.
Alaerts, GJ., Khouri, N. and Kooir, B. (2001) Strategies to mitigate arsenic contamination in water
supply. In United Nations Synthesis Report on Arsenic in Drinking Water,
www.who.int/water_sanitation_health/dwq/eo/arsenicun8.pdf(aca:ssedApri129. 2005).
Albu, MA., Morris, L.M., Nash. H. and Rivett, M.O. (2002) Hydrocarbon contamination of
groundwater at Ploiesti, Romania In: SILftainable Groundwater Development (eds. KM
Hiscock, M.O. Rivett and RM Davison), pp. 293-301, Geological Society Special
Publication No. 193, London.
Allen-King, RM~ Ball, W J'. and Grnthwohl, P. (2002) New modeling paradigms for the sOiption
of hydrophobic organic chemicals to heterogeneous carbonaceous matter in soils, sediments,
and rocks. Advances in Water &sour., 25, 985-1016.
Appelo, C.AJ. and Postma, D. (1993) Geochemistry, Groundwater and Pollution, Balkema,
Rotterdam.
Araya, M, Chen, B~ Klevay, L.M., Strain, JJ., Johnson, L., Robson, P ~ Shi, W ~ Nielsen, F., Zhu,
H., Olivares, M~ Pizarro, F., Haber, LT. (2003) Confirmation of an acute no-observed-
adverse-effect and low-observed-advelSe-cifect: level fur copper in bottled drinking water in a
multi-site international study. Regul. Toxicol. Pharmacol 38, 389-399.
Ashley, RP. (1998) Foreseeability of envimmnentd hazards: the implications of the Cambridge
Water Company Ca<ie. In: Groundwater Contaminants and their Migration, (ed. J. Mather),
pp. 23-26, Special Publication 128, Geological Society, London.
Bailey, K, Chilton, J., Dahi, E, Lennon, M and Jackson, P (In Press) Fluoride in Drinking Water.
WHO Drinking-water Series. 1W A Publishing, London.
Barceloux, D.G. (1999) Seleniwn J. Toxicol Clin. Toxicol, 37(2), 145-172.
Barker, J.F., Patrick, G.C. and Major, D. (1987) Natmal attenuation of aromatic hydrocarbons in a
shallow sand aquifer. Ground Water Monitor Review 7(4), 64-71.
Barnard, C~ Daberkow, S., Padgitt, M, Smith, M.E. and Uri, N.D. (1997) Alternative measures of
pesticide use. Sci. Total Environ., 203, 229-244.
Barnes, C.J. Jacobson, G. and Smith, G.D. (1992) The origin of high-nitrate groundwaters in the
Australian arid zone. J. Hydrology, 137, 181-197.
Bedient, P.B., Rifai, H.S. and Newell, C. (1999) Ground Water Contamination: Transport and
Remediation, 2nd edn, Prentice-Hall, New Jersey.
132 Protecting Groundwater for Health
Bradley, E.G. and Zacharewski, T.R (1998) Exoestrogeos: mechanisms of action and strategies for
identification and asses.5lllellt. Environ. Toxicol Contam., 17, 49-57.
Cavallero, A, Corradi, C., De Felire, G. and GillSfil, P. (1985) Underground water pollution in
Milan by industrial chlorinated organic compounds. In Effects of Landuse Upon FreshwaJers,
(ed. J.F. de L.G. Solbe), pp. 68-84, Ellis Harnood, Chichester.
CEC (2001) Commis.sion Recommendation on 1he protection of1he public against exposure to
radon in drinking water supplies. Doannent number C (2001) 4580. Official Joumal of the
European Communities, L 344/85.
Chilton, P J~ Lawrenre, AR and Stuart, ME. {1998) Pesticides in groundwater: some preliminary
results from recent research in temperate and ttopical enwonments. In Groundwater
Contaminants and Their Migration, (ed. J. Mather), pp. 333-345, Special Publication 128,
Goological Society, London.
Chilton, P J~ Stuart, M.E~ Lawrenre, AR, Gooddy, D.C., Williams, RJ. and Johnson, AC. (2000)
Asses9ng pesticide pollution of groundwater: ament knowledge and remaining gaps. In
Groundwa1er: Past Achievements and Future Challenges, (ed. O.T.N. Sililo), pp. 17-26,
Balkema, Rotterdam.
Costa, M. (2002) Potential hazards ofhexavalent dnomate in our drinking water. Toxicol Appl.
Pharmawl, 188, 1-5.
Damstra, T., Barlow, S., Bergman, A., Kavlock, R. and van der Kraak, G. (2002)
Global Assessment of the Slale-oj-the-Science of Endnaine Disruptors. Intemational
Programme on Chemical Safety.
http://www.who.int/ipcs/publicatiornv'new _ is.giesfendocrine _ disruptors/enfmdex.html
Daughton, C. and Ternes, T. (1999) Phamlaceuticals and personal care produds in the
e.nwonment agents of subtle change? EHP, 107, suppl. 6, 907-938.
Domenico, PA and Schw.irtz., R W. (1998) Physical and Chemical Hydrogeology, 2nd edn, John
Wiley & Sons, New Yotk
Drewes, J.E. and Shore, L.S. (2001) Concerns about pharmaceuticals in water reuse, groundwater
recharge, and animal waste. In Pharmaceuticals and Personal Care Products in the
Environment, (eds. Ch. Daughton and TL. Jones-Lepp), pp. 206-228, American Chemical
Society Symposium Series No. 791, Washington, DC.
Drewes, J.E., Fox, P. and Jekel, M (2001) Ocanrenre of iodimted X-ray contrast media in
domestic effluents and 1heir :rate during indirea: potable reuse. J. Environ. Sci Health Part A,
36A(9), 1633-1645.
EA (2000) Guidance on the Assessment and Monitoring of Natural Attenuation of Contaminants in
GroundwaJer. R&D Publication 95, Enwonment Agency, Bristol.
Edmunds, W.M and Gaye, C.B. (1994) F.stimating 1he spatial variability of groundwater recharge
in the Sahel using chloride. J. Hydrology, 156, 47-59.
Edmunds, W.M., Cook, JM., Darling, W.G., Kinmburgh, D.G., Miles, D.L., Bath, AH, Morgan-
Jones, M. and Andrews, J.N. (1987) Baseline geochemical conditions in the Chalk aquifer,
Berkshire UK: a basis for groundwater quality manage:mart. Appl Geochem., 2(2), 252-274.
FAO (2004) Pesticide residues in food -2004. Report of the Joint Meeting of the FAO Panel of
Experts on Pesticide Residues in Food and the Environmrot and the WHO Core Assessment
Group on Pesticide Residues. FAO Plant Production and Protection Paper 178, Food and
Agrirul.1nral Organi2.ation, Rome, Italy.
Fetter, C.W. (1999) Contaminant Hydrogeology, 2ndedn, Macmillan, New Ymk.
Aiihler, H.P., Ferlin, P., Seliin, HM., and Schulin R (1985) Transport von Fluorid, Bromid und
Chlorid in Bodensaulen und in einem natiirlich gelagertem Boden. Z Dtsch. Geol
Gesel/schqft, 136, 375-383.
Freeze, RA and Cheny, J.A (1979) GroundwaJer, Prentice-Hall, Englewood Cliffs, New Jersey.
Fusillo, T.V., Hochreiter JJ. and Lord, D.G. (1985) Distribution of volatile organic compounds in a
New Jersey coastal plain aquifer. Ground Water, 23(3), 354-360.
Gelhar, L.W. (1986) Stochastic subsurfuce hydrology ftom theory to applications. Waler Resow:
Res., 22(9), 135S-145S.
Chemicals: Health relevance, transport and attenuation 133
Graham-Bryce, IJ. (1981) The behaviour of pesticides in soil In The Chemistry of Soil Processes,
(eds. DJ. GreallandandM.H.B. Hayes),pp. 621-671, JoJm Wiley& Sons Ltd, Sussex.
Heam, W., Henderson, G., Houston, S., Wadw, A and Walker, B. (1993) Water supply and
Aboriginal and Toms Strait Ilsander health: an overview. AGSO J. Austral. Geology and
Geophysics, 14(2/3), 135-146.
Heberer, T ., Scbmich-B~ K and Stan H-J. (1998) Occum:nce and distnbution of organic
oontaminants in the aquatic system in Berlin. Part I: Drug residues and other polar
oontaminants in Berlin surrace and grolDldwater. Acta Hydrochim. Hydrobiol 26, 272-278 .
Hem, J .D. (1989) Study and Interpretation of the Chemical Charaeleristics of Natural Water, 3rd
ed, USGS, Wata-SupplyPaper2254.
Heron, G., Barcelona, MJ., Anderson, ML. and Christensen, T.H (1997) Derennination of
nonvolaille organic cadxm in aquifer solids after cmbonal:e removal by sulphurous acid
Ground Water , 3S, 6-11.
!ARC (2001) Overall Evaluations of Carcinogenicity to Humans, http://www-
cie.iarc.fr/monoeval/cr1hall.html (~ Apn129 . 2005).
IUPAC (2003) Endocrine Disrupt= in the F.nvimnment illPAC Technical Report, Pure and
Applied Chemistry, 7S(5), 631-681.
Jad{son, RE. (1998) The migration, dissolution and filte of dilorinatcd solvents in the mbanized
alluvial valleys of the southwestern. USA HydrogeologyJ., 6(1), 144-155.
Johansen, S.S., Hansen, AB., Mosbaek, H. and Arvin, E. (1997) Charactemation ofhetl:roaromatic
and other organic rompmmds in grol.Dldwater at a-eosote--contaminate sites in Demnarlc.
Groundwater Mon Remediation, 17(2), 106-115.
Kerger, BD., Finley, B.L., Cornett, G.E., Dodge, D.G. and Paustmbach, DJ. (1997) Ingestion of
chromium(VI) in drinking water by human volunteas: absoiption, distnbution, and exa-elion
of single and rq,eated doses. J. Toxicol. Environ Health, 50, 67-95.
Kerndorff; H., Scbleyer, R, Milde, G. and Plumb, R H. (1992) Geochemistry of growidwater
pollutants at German wll'lte disposal sites. In Groundwater Contamination and Analysis al
Ha::ardous Waste Sites, (eds. S. Lesage and RE. Jad{son), Marcel Dekker, Inc. New York,
Ba5d,HongKong.
Khim, S. and Ongerth, J. (2000) Hormonally Active Agents in Domestic Wastewater Treatment-A
LiJerature Review. Report prepared for CGE Australia.
King, M.W.G. and Barker, J.F . (1999) Migration and nalural. filte of a roal tar ai:osote phnne I.
Ovaview and plume development J. Contam. Hydro/., 39, 249-279.
Klecka, G.M , Gonsior, S.J. and Markham, D.A (1990) Biological transfonnations of 1,1,1-
trichloroethane in subsudllce soils and groundwater. Environ. Sci Technol., 9, 1437-1151.
Kolpin, D.W., Bmbash, J.E. and Gilliom, RJ. (2000) Pesticides in groundwater of the United
S~ 1992-%. Ground Water, 38(6), 858-863.
Kuehn, W. and Mueller, U. (2000)Riverbankfiltration-anowrview.JAWA, 92(12), 60-69 .
Kuepe:r, B.H., Redman, D., Starr, RC., Reitsma, S. and Mah, M . (1993) A field experiment to study
the behavior of tetrachloroelhylene below the waia-table: spatial distnbution of residual and
pooledDNAPL. GroundWaJer,31(5), 756-766.
Kiimmerer, K. (2004) Eootoxioological evaluation of pb.annaceulicals. In Pharmaceuticals in the
Environment: Sources, occurrence,fate and effects, 2nd ed, Springer, Heidelberg, New York
Lavy, T .L , Mattice, J.D., Massey, J.l-1., Skulman, B.W. and Smseman, SA (1996) Long-term in
situ leaching and degradation of six herbicides aged in subsoils. J. Environ. Qua/., 25, 1268-
1279.
Lawrence, C.R (1983) NitraJe-rich groundwaters in Australia. Australian Water Resources
Cmmcil Technical Paper No. 79, Australian Govemment Publishing Service, Canberra.
Lorah, MM. and Olsen, L.D. (1999) Natural attenuation of chlorinated volatile organic rompolll!ds
in a fi:eshwater tidal wetland: field evidence of anaerobic biodegradation. Water Resour. Res.,
35,3811-3827.
134 Protecting Groundwater for Health
Lye, C.M , Frid, CL.J~ Gill, ME., Cooper, D.W. and Jones, D.M (1999) Estroga:ric alkylphenols
in fish ~ sediments, and waters from the UK Tyne and Tees E5t!Jaries. Fm,iron. Sci
Technol, 33(7), J()()l)-1014.
Mackay, D.M and Cherry, JA (1989) Groundwater contamination: pump-and-treat remedialion.
Environ. Sci. Technol., 23(6), 630-636.
Mackay, DM., Freyberg, D.L., Roberts, P.V. and Chmy, J.A (1986) A natural gradient
experiment on solute transport in a sand aquifui:, 1. Approach and overview of plume
movement Water Resmu. Res., 22(13), 2017-2029.
Matin, MA, Malek, MA., Amin, MR, Ralunan, S., Khaloon, J, Ralunan, M, Aminuddio, M ..
McLadllan, MS., Horstmann, M. and Hinkel, M (1996) Polychlorinated dibaiso-p-oioxins
and di"beozofurans in sewage sludge: sources and fille following sludge applicalion to land
Sci Total Environ., 185, 109-123.
McAllister, P.M. and Chiang, C.Y. (1994) A practicaJ approach to evaluating rntural attenuation of
roo1aminants in ground water. Ground Waler Monitoring & Remediation, 14(2), 161-173.
Mendoza, CA? Johnson, RL. and Gillham, R W. (1996) Vapor migration in the vadose zone. In
Derue Chlorinated Solvents and Other DNAPLs in Groundwater, (eds. J.F. Pankow and JA
Chmy), pp. 179-201, Waterloo Press, Portland, Oregon.
Mercer, J.W. and Cohal, RM (1990) A n:viewofimmiscible fluids in the subsurfuce: Properties,
models, characa!rizatio and n::mediation. J. Contarn. Hydro/., 6, 107-163. ·
Montforts, M. (2001) Regulatory and methodologicaJ ~ conreming the risk ~t for
medicinal products; need fur research. In Pharmaceuticals in the Environmenr. Sources,
Occurrence, Fate and Effects, (ed K Kummerer), pp. 159-174, Springer, Heidelberg, New
York
Montgomeiy, J.H. (1996) Groundwater Chemicals Desk Reference, 2nd edn, CRC Press, Inc.,
Florida
NCRP (1989) ConJrol of Radon in Houses: Recommendations of the NCRP. Nalional Comcil on
Radiation Protection Report No. 103 , Bethesda, Maryland
Newell, C.J. and Coonor, JA (1998) Characteristia; of Dissolved Hydrocarbon Plumes: Results
from few-studies, API teclmicaJ transfer bulletin, American Petrolewn Institute, Washington
DC.
Newell, C.J., Hopkins, L.P. and Bedient, P .B . (1990) A hydrogeologic database fur ground-water
modelling. Ground Water, 28(5), 703-714.
Nc:m:11, C.J., Am:e, S.D., Ross, RR and Huling, S.G . (1995) LigM Non Aqueous Phase Liquids,
US EPA, Ada, Oklahoma.
Noble, P . and Morgan, P. (2002) The Effects of Contaminant Concentration on the Potential For
Natural Attenuation. EA (fur England & Wales) R&D TedmicaJ Report P2-228/IR Bristol.
NRC (1989) Irrigation-induced water quality problems, Nalional Academy Press, Nalional
Research COlDlcil, Washington, DC.
NRC (1994) Alternatives of Ground Water Cleanup , Nalional Academy Press, Naliooal R=th
Crnmcil, Washington, DC.
NRC (1999) Hormonally Active Agents in the Environment, Nalional Academy Press, Nalional
ResemdJ. Council, Washington, DC.
Pankow, J.F. and Cheny, JA (1996) Dense Chlorinated Solvents and Other DNAPLs in
Groundwater, Waterloo Pres.5, Portland, Oregon.
Plwnb, RH. (1992) The importance of volatile organic compom~ as a disposal site monitoring
parameter. In Groundwater Contamination and Anolysis at Ha:ordous Waste Sites , (eds. S.
Lesage and RE. Jackson), pp. 173-197, Marcel Dekker, Inc. New York, Basel, Hong Kong.
Rivett, M.O. (1995) Soil-gas signatures from volalile chlorinated solvents: Borden field
experiments. Ground Water, 33(1), 84-98.
Rivett, M.O. and Allen-King, RM. (2003) A controlled field experiment on gromdwater
contaminalion by a multicomponent DNAPL: dissolved-plume retardation. J. ConJam.
Hydro/., 66, 117-146
Chemicals: Health relevance, transport and attenuation 135
Rivett, M.0., Lerner, D.N., Lloyd, J.W. and Omk. L. (1990) Organic oontamination. of the
Birmingham aquirer. UKJ. Hydrology, 113, 307-323.
Rivett, M.0., Feenstra, S. and Cheny JA (2001) A controlled field experiment on groundwater
oontamination by a multicomponent DNAPL: aeation of the emplared-source and ovezview
of~lvedplumedevelopment.J. Contam. Hydrol, 49, 111-149.
Rivett, MO~ Shqnerd, KA, Keeys, L and Braman, AE~ (2005) Chlorinated Solvarts in the
Birmingham Aquiii:r, UK: 1986 -2001. Quarterly Joumal of F,ngineering Geology &
Hydrogeology, 38(4), 337-350.
Sawyer, C.N., McCarty, P.L. and Parkin, G.F. (1994) Chemistry for Environmental Engineering,
4th edo, McGraw-Hill, New York.
Sdmitzer, Mand Khan, S.U. (1972) Humic Substances in the Environment., Marcel Dekker, Inc.,
NewYork.
Schwa117, F.W. and Zhang, H. (2003) Ftmdamentals ofGrow1dwater, John Wiley & Sons, Inc.,
New York.
Sdrwam:nbach, RP., Gschffl:lld, PM. and Imboden, D.M (1993) F.nviroT1TTll!l1lal Organic
Chemistry, John Wiley & Sons, Inc, New Jersey.
Schwille, F. (1988) Dense Chlorinated So/venJs in POl'QUS and Fractured Media, (ed. J.F. Pankow),
Lewis Publishas, Oielsea, Michigan.
Senior, LA (1998) Radon-222 in the Ground Water of Chester CounJy, Per,nsylvania: USGS
Water-R.esoum:s Investigations Report98-4169 .
Smedley, PL. and Kinmomgh, D.G. (2001) Sourre and behaviour of arsenic in natural watas. In
United Nations Synthesis Report on Arsenic in Drinking Water,
www.who.int/water _sanitation_ health/dwqfenlarsenicunl.pdf (accessed April 29, 2005).
Smith, AH., Lingas, E.0. and Rahman, M (2000) Cnntamination of drinking-water by arsenic in
Bangladesh: a public health emergency. Bulletin of the WHO, 78, 1093-1103.
Spliid, N.H. and K0ppm, B. (1998) Ocrurrence of pesticides in Danish shallow groundwater.
Chemosphere,37(7), 1307-1316.
SJXN1o, G. (1989) The Chemistry of Soils, 0UP, New York.
ScJrillace, P J., ZogoISki, J.S~ Wilber, W.G. and Price, C.V. (1996) Preliminary ~ of the
ocamcnces and possible sources ofMIBE in grolllldwato-in the United States, 1993-1994.
Environ Sci. Technol.,30(,5), 1721-1730.
Stevenson, F J. ( 1994) Structural ~ of humic substances. In Humus Chemistry: Genesis,
Composition, Reactions, (ed. P J. Stevenson), 2nd edn, pp. 285-302, John Wiley & Sons, New
York.
Stumm, W. and Morgan, JJ. (1996) Aquatic Chemistry, 3rd edn, John Wiley & Sons, New Yorlc.
Ternes, T. (2001) Pharmaceuticals as New Emerging Environmental Contaminants: A Smvey. In
Proc. Second International Coeference on Pharmaceuticals and Endocrine Disrupting
Chemicals in Water, Minneapolis, Minnesota, pp. 1-9. '
Thurman, E.M. (1985) Organic Geochemistry of Natw-al Waters: Developments in
Biogeochemistry, Martinus Nijhoflmr. W. Junk Publishers, Dordn:cbt
UNSCEAR (2000) Sources, Effects and Risks of Joni::ing Radiation, UN, New York.
US EPA (1996) Soil Screening Guidance, Office of Emergency and Remedial Response,
EPN540/R95/128. httpJ/www.epa.gov/superfund/resoun:es/soil/index..htm (llCCC$Cd August
11,2005).
US EPA (1999) Screening Level Ecological lruk Assessment Protocol for Ha:.ardnus Waste
Combustion Facilities, vol. 2, Appendix A, EPA530-D-99-001B. http://www.
epa.gov/combustion/eco-risk/volume2/appx-apdf (accessed August 11, 2005).
US EPA (2000) Summary of EPA Coal Combustion Waste Damage Cases Involving Sand and
Gravel Mines/Pits/Operations, http://www.epagov/epaoswer/o1her/fussil/ sandgrav.pdf
(accessed Apnl 29, 2005).
Vogel, T.M., Criddle, C.S. and McCarty, P.L. (1987) Transfurm.alions of halogenated aliphatic
compounds. Environ. Sci Technol, 21, 722-736.
136 Protecting Groundwater for Health
Wiedemeier, T.H~ Rifai, H.S., Newell, CJ. and Wilson, J.T. (1999) Natural Attenuation of Fuels
and Chlorinated Solvents in the Subsurface, John Wiley & Sons, New Yorlc.
Williams, W.M., Holden, P. W., Parsons, D.W. and Loma, MW. (1988) Pesticides in Ground
Water Data Base, Interim Report, US EPA, Office of Pesticide Programs, Washington, OC.
WHO (1993) Guidelines/or Drinking-water Quality: Rewmmendotions, 2nd edn, vol. I , World
Health Organization, Geneva
WHO (1996a) Guidelines for Drinking-water Quality: Health criteria and other supporting
ieformation, 2nd edn, vol 2, World Health Oigaoization, Geneva
WHO (1996b) Toxicologiail Eval110tion of Certain Food Additives and Contaminants. Prepared by
the 44th Meeting of the Joiot FAO/WHO Expert Committee on Food Additives (JECFA),
Geneva
WHO (2003a) Arsenic in DrinkinJ!,water. Background Dccument for development of WHO
Guidelines for Drinkinf!,Waler Quality. World Health Organization, Geneva,
http://www.who.iot/water sanitation health/dwq/chemicals/arseoic.pdf (acres.sed September
8,2005).
WHO (2003b) Seleniwn in Drinking-water: Background Doaunent for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
h 11pJ/w\\w.111-bo.int/water sanitation bealth/dwg/chemicals/selenium.pdf (accessed
September 8, 2005).
WHO (2003c) Lead in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
hnp ://www.who.iot/water sanitation health/dwqlchemicals/leadpdf (acres.sed Sep1mlber 8,
2005).
WHO (2003d) Chromium in Drinkinf!,water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
h11p://www.who.iot/water sanitation health/dwg/chemicals/cbromium.pdf (accessed
September 8, 2005).
WHO (2003e) Vinyl Chloride in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
h11p://www.who.int/water sanitation health/cmg/chemicals/vinylchloride.pdf (accessed
September 8, 2005).
WHO (2003f) 1,2-Dichloroethene in Drinking-water: Background DocwnenJ for development of
WHO Guidelines for Drinkinf!,water Quality. World Health Organization, Geneva,
hupJ/www.who.iot/water sanitation health/dwg 2-Dichloroethene.pdf {accessed
September 8 2005}
WHO (2003g) Dichloroben::enes in Drinking-water: Background Document for developrnenl of
WHO Guidelines for Drinking-water Quality. World Health Organization, Geneva
h up://w\\w.who.int/water sanitation health/dwq/chemicals/dichlorobenz.enes.pdf (accessed
September 8, 2005}.
WHO (2004a) Guidelines/or Drinkinf!,water Quality: Rerommendations. 3rd edn, vol. I , Geneva
WHO (2004b) Fluoride in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
hnp://w\\w. who.int/water sanitation health/d\,qlchemicals/fiuoride.pdf (acres.sed September
8,2005) ..
WHO (2004c) Copper in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Geneva,
h 1tp://www.who.int/water sanitation health/dwg/chemicals/co ppcr.pdf (accessed
September 8, 2005).
WHO (2004d) Vinyl Chloride in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Health Organization, Gmeva
hup ://www.who.in1iwater sanitation health/d\\glchemicals/vinvlchloride.pdf (accessed
September 8, 2005).
Chemicals: Health relevance, transport and attenuation 137
WHO (2005a) Uranium Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Heallh Organization, Geneva,
h rtp ://www.who.int/water sanitation health/dwg/chemicals/uraniurn290605.pdf
(accessed September 8, 2005).
WHO (2005b) Nitrate in Drinking-water: Background Document for development of WHO
Guidelines for Drinking-water Quality. World Heallh Organization, Geneva,
h 1tp:f/www.who.int/water sanitation health/dwg/chemicals/en/index.html (accessed
September 8, 2005).
WHO (2005c) Methyl tertiary-butyl ether (MFBE) in Drinking-water: Background Document for
development of WHO Guidelines for Drinking-water Quality. World Heallh Organization,
Geneva, ht1p://www.who.int/water sanitation heallh/dwqlchemicals/MTBE200605.pdf
(accessed September 8, 2005).
WHO (2005d) Trich/oroethene in Drinking-water: Background Document for development of
WHO Guidelines for Drinking-water Quality. World Heallh Organiz.ation, Geneva,
htlp://www.who.int/water _ sanitation _health/dwq/chemicalsltrichloroethenemay05.doc
(accessed September 8, 2005).
WHO (2005e) Dichloroethene in Drinking-water: Background Document for development of
WHO Guidelines for Drinking-water Quality. World Heallh . Organization, Geneva,
ht1p ://www.who.int/water sanitation health/dwy/chemicalsll ldichloroethenefinal.pdf
(accessed Septembec 8, 2005).
WHO (Inl're$}Arsenic in Drinking-water, WHO Drinking-water Quality Series. !WA Publishing,
London.
Zietz, B~ Dieter, llll, Lakomek, M, Sclmeider, R, Kessler-Gaedtke, B. and Dunkelberg, R
(2003) Epidemiological Investigation on chronic copper toxicity to children exposed via lhe
public drinking water supply. Sci. Tot. &zviron., 302, 127-144.
5
Socioeconomic, institutional and
legal aspects in groundwater
assessment and protection
G. Howard, P. Chave, P. Bakir and B. Hoque
1he socioeconomic conditions in areas where groundwater is used or recharge occurs are
aitical to the development of grolilldwater protection measures.. 1he protection of
groundwater may be expensive and disruptive to the inhabitants of the land above the
aquifers. Socioeconomic conditions play an important role in determining . the likely
contaminant loads and types of contaminant that aflect the groundwater. It is also critical
in determining what types of intervention are possible, how such interventions will be
implemented and what resources (including human) will be required and are available.
Institutional and legal issues are also critical in determining the success or fuih.rre of
groundwater protection policies and strategies. Weak institutions and poor institutional
:frameworlcs are commonly identified with poor implementation of water policy (World
Bank, 1993; WELL, 1998). The development of groundwater policies and strategies
must therefore provide adequate consideration of the appropriate institutional
arrangements and consider how the needs of all stakeholders can be incorporated into the
policy. 1he latter demands that there are effective processes of public consultation and
participation in policy and strategy development Legislation is also vital to support
© 2006 World Health Organization. Protecting GroundwaJer for Health: Managing the Quality of
Drinking-waler Sources. Edited by 0. Schmoll, G. How.ird, J. Chilton and I. Chorus. ISBN:
1843390795. Published by 1W A Publishing, London, UK.
140 Protecting Groundwater for Health
effective groundwater protection. Not only should the law be supportive of groundwater
protection, effective implementation of the law is required to ensure groundwater
protection objectives are met (Caponera. 19')2; Foster et al., 19'12).
The purpose of this chapter is to discuss some of the key socioeconomic, institutional
and legal issues that are important to consider in groundwater protection. In Chapter 7,
the types of information and methods of data collection will be discussed; Chapter 20
outlines row socioeconomic issues and the institutional and legal frameworks can be
~ in developing groundwater protection.
5.1 SOCIOECONOMIC STATUS: ISSUES OF POVERTY
.AND WEALTH
Socioeconomic status is a mea&ire of the wealth of individuals, households and
oommtmities and reflects their asset<; as well as the ability of households to obtain goods
and services. Socioeconomic status is of importance when considering the level of
investment in groundwater management which individuals, oommunities and societies
are willing and able to make.
Socioeconomic oonditions influence the' capacity for different groups to protect their
environment For instance, in some oommunities short-term priorities for resource
· exploitation override the need for resource protection neces.sacy to secure a long-term
livelihood, despite the recognition in the oommunities of the need for such protection.
The poor are usually at greater risk fiom · the adverse effects of poor resource
management and it is essential that their needs be properly ~ when developing
groundwater strategies. Critical to this approach is to avoid disadvantage for the poor
caused by the implementation of groundwater protection policies and strategies. Such
disadvantage may occur, for instance, because agricultural use of land is restricted in
order to protect groundwater, which may result in reduced inoomes and decreased
security for poor farmers. Consideration must be given to compensation, financial
support, the creation of alternative employment opportunities or provision of new land
when no restrictions apply. However, the latter is often difficult to implement and should
only be considered where there is strong evidence fiom consultations that such an
approach is acceptable to the communities affected and that the proposed land for
relocation is at least the same quality as the land being left.
The implementation of groundwater protection measures will often have important
implications for the livelihoods of the households affected and this applies in all
countries. For instance, significant changes in land use regulations in developed countries
will also have a profound impact on the users of land, water and other resources.
Changes may have positive or negative impacts on some or all of the components of
livelihoods. The implications of such impacts in terms of compensation, social services
and environmental protection should be taken into account when reaching a decision
about what and how land use regulations are applied. The population affected must be
fully consulted and be willing to accept any restrictions as part of the process of
establishing protection norms. Box 5.1 outlines the problems faced in parts of Germany
in relation to controlling groundwater contamination when fumilies facing financial
hardship are able to access alternative sources of water.
Socioeconomic, institutional and legal aspects
Box 5.1. Socioeconomic fuctors and illegal use of private wells in some rural
areas ofGennany
After the political changes in Germany in 1990, the connection of rural areas in
eastern Germany to central water supplies was rapidly developed. This was
particularly urgent in some mountainous areas ofErzgebirge, Thuringia, as the
supply from individual wells was highly unsatisfuctory because aquifers in
.fractured bedrock fell dry at intervals. They were also vulnerable to short-
circuiting with surface water and sewage. Connection to central supplies ofhigh
quality and reliable quantity in the 1990s was therefore warmly welcomed by
the population Individual wells were abandoned and sometimes illegally
misused as undrained sewage pits. As local aquifers were no longer needed for
feeding household wells, their protection was no longer perceived as a priority.
However, the introduction of cost recovery for drinking-water significantly
increased prices within a few years. At the same time, unemployment rates were
very high and available work often poorly paid. As a result, many households
struggled financially. This made individual supplies attractive again, and a large
mmber were re-activated illegally. The Gennan Drinking-water Ordinance
requires annual monitoring of private wells, which typically involves partial or
total cost coverage for the analyses by the well owner, but numerous households
connected to a central supply avoided these costs by not registering their re-
activated wells for surveillance. Their use only became known because
metering showed large numbers of households that were not using any water
from the central supply.
Ensuing public health concerns include the high rate of household wells with
microbial contamination (up to 60 per cent in one survey) and a high risk of
unintentional cross-connection between self-built piping from the household
well and the public supply, potentially contaminating the public supply.
Furthermore, because of increasing stagnation in the mains due to reduced flow,
the costs of the central supply have further increased as flushing is required
more often to prevent microbial re-growth. This further reduces the
attractiveness of the central supply and encomages greater use of the re-
activated wells.
141
When actions are required by specific communities to protect grom1dwater resources,
consideration must be given to the incentives that may be required and how these can be
provided. It should be noted that in many cases such incentives do not refer to direct
monetary compensation packages but could, for example, address improved security of
tenure for poor fimners in order to promote reduced pollution loads derived from
agriculture.
The socioeconomic status of communities is likely to influence the type of
interventions that will be feasible for groundwater protection For instance, in low-
income communities in developing countries with shallow groundwater, the use of pit
latrines may not be the preferred technical solution for excreta disposal, as they lead to an
increased risk of contamination. However, alternative technologies may be too expensive
for the majority of the population to sustain. In this case, some degree of contamination
142 Protecting Groundwater for Health
of groundwater may be tolerated in order to reduce a greater health risk caused by the
lack of excreta disposal In urban areas, if contamination is deemed unacceptable, then it
is often more cost-effective t.o provide an alternative (often piped) water supply that uses
water from a more distant and protected water source (Franceys et al, 1992). In rural
areas it is often more difficult t.o implement such solutions and the use of an alternative
sanitation technology may need t.o be considered and potentially subsidiz.ed.
Similar situations may occur in developed cmmtries where balances need t.o be made
between protection of the groundwater resource and sanitation provision. In rural
communities, septic tanks may be used where shallow groundwater is tapped for
domestic supply thus representing a risk t.o the quality of the groundwater source. Off-
site methods may not be feasible because of the cost of operation and maintenance. In
this case, it is likely t.o be more cost effective to treat the water or use an alternative
source of water rather than attempt t.o change the sanitation technology.
5.1.r Livelihood concepts
The concept oflivelihoods is now used in many countries when considering the nature of
poverty. The basic concept of a livelihoods approach is that the ability ofhouseholds and
communities t.o sustain and improve their livelihood relies on income, assets, capabilities
and their vulnerability. This approach also takes int.o account gender, environmental
sustainability and cultural norms in defining sustainable livelihoods. Chambers and
Conway (1991) provided definitions of sustainable livelihoods in relation t.o both
environmental sustainability and social sustainability. DFID (2003) has combined these
t.o define a sustainable livelihood:
'A livelihood is sustainable when it can cope with and recover from stresses and
shocks and maintain or enhance its capabilities and assets both now and in the
fature, while not undermining the natural resource base. '
Stresses and shocks refer to events or changes in assets, income or vulnerability that
put pressure on the livelihood. For instance, sudden loss of employment or large
increases in price of basic goods result in a shock or stress t.o the livelihood. Equally, a
poor harvest or sudden change in allowed land use may affect the asset base of a
community. The onset of a significant health problem may ~ vulnerability
(infection with hwnan immunodeficiency virus (HIV) being a good example) of an
individual such that previous levels ofhealth protection are no longer adequate.
Water and health are both considered as assets within this frameworlc and the degree
to which households or communities have access to these assets and their resilience t.o
shocks and stresses are fimdamental components of securing a sustainable livelihood.
The livelihood approach also encompasses concepts of vulnerability and environmental
sustainability when considering poverty.
Vulnerability is composed of risks that are shared by a community (sometimes called
exposure), which includes lack of access t.o a specific water resource, and those risks
unique to an individual (often termed susceptibility) such as HIV infection. Vulnerability
may be physical, social or political (Nichol 2000). In the context of groundwater
protection, physical vulnerability may refer t.o the increased risk of contamination from
inappropriate land use. Social vulnerability arises from marginaliz.ation of parts of a
Socioeconomic, institutional and legal aspects 143
community within the larger community or society and factors such as gender-specific
restrictions to assets or decision-making. In relation to groundwater, this may result in
marginalization of women in decision-making regarding groundwater development,
management and protection. Political vulnerability typically relates to the capability of
communities to be engaged in wider decision-making proces.5eS in relation to resource
access and management
The livelihood approach ensures that the sustainability of natural resources and the
environment is given an important place in the understanding of poverty. This may be
given greater priority in rural areas where livelihood may depend on sustainable use of
natural resources (f amuno et al., 2003). However, natural resources always remain an
important component of livelihoods, as sustainability is defined in terms of a livelihood
that does not degrade the asset base. This has implications fur both rural and trban
dwellers. For instance, the protection and sustainable use of groundwater has important
implications for Uiban homeholds that rely on groundwater fur domestic sq,ply, as both
deterioration and protection of the resource may increase water costs and affect the
livelihood of the users.
5.1.2 Source of livelihoods
Understanding the source oflivelihood of communities that utiliz.e and potentially pollute
groundwater sources is important. Different means of sustaining a livelihood will result
in different types of pollution. Where commercial fanning is the principal source of
livelihood, groundwater may be vulnerable to pollution derived fiom agrochemicals such
as fertili2ers and pesticides (Chapter 9). Where irrigation is practised, contamination is
likely to increase because many irrigation systems are inefficient, resulting in significant
volumes of water infiltrating the aquifer. The water used for irrigation is :frequently
under-priced and this tends to reinforce the inefficient use of water (World Bank, 1993).
However, where irrigation is essential for growing crops, the development of
groundwater protection strategies will have to take this into account and compensation
packages and alternative irrigation practices (such as drip irrigation) promoted
(Chapter21).
Small subsistence or near subsistence fanning may make relatively little use of
agrochemicals or irrigation, but their use may be significant in countries where there are
government subsidies on agricultural production. In this case, it may be more appropriate
to remove the subsidy on agrochemical use than to tty to regulate application in
Jmlicular areas.
Where agrochemical use derives fiom private purchase, the groundwater strategy will
have to consider the capacity of regulatory bodies to develop and deliver incentives to
reduce or change applications and the cost of inspection and monitoring. Where there is
widespread small-scale private use of agrochemicals, it will be important to consider
targeting those areas where groundwater is at greatest risk fiom pollution, rather than
tiying to implement broad measures.
The situation with large commercial fanning may be simpler to regulate, as there will
be a smaller nwnber of people to deal with. Where direct actions are taken to change land
use to an economically less productive use, the land-owner would usually expect
144 Protecting Groundwater for Health
compensation and this would have to reflect their overall economic los.s. However, in
many cases, the restrictions may actually apply more directly to applications of
agrochemicals on a seasonal basis, which would not require the same level of e.conomic
recompense. It may, however, require systems of monitoring to ensure compliance.
Where the majority of the population derive an income from small-scale agriculture,
groundwater protection may be more difficult to regulate as there will be many more
farmers whose needs must be addressed. Any compensation packages that are developed
in such situations may have a lower per capita outlay than larger funns but it is likely to
result in a higher per ha cost. This will increase overall direct costs of the protection
strategy. As noted above, alternative incentives may need to be developed in some
situations. These may be related to land tenure, but also include aspects such as providing
more secure markets for produce or providing improved extension programmes as a way
of off-setting ecoromic losses.
5.2 POPULATION AND POPULATION DENSITY
Increasing population and population density can increase the risk to groundwater from
pollution and unsustainable abstraction. Balancing the needs for protection of resources
against demands from rapidly increasing populations is a key element in groundwater
protection. Population growth often provides an impetus for improving protection
strategies as the need to secure and conserve high-quality water resources for domestic
supply becomes increasingly important This can provide a strong argmnent for the need
to protect groundwater against pollution.
It should be noted, however, that the protection of particular groundwater resources is
also dependent on whether it is considered a key source of domestic water in the long-
tetm. In some cases, other resources ( either surface water or more remote growidwater)
can satisfy demands for water and the threatened groundwater will rot form a key part of
the water resources ,used for supply. This is common in wetter countries where urban
groundwater has been abandoned. In other situations, typically much drier counties,
alternatives may not exist and groundwater resources will therefore need to be protected.
5.3 COMMUNITY PARTICIPATION AND
CONSULTATION
Protection of groundwater resources is a public concern and a public responsibility and
therefore requires public participation. Participation can be defined as a process through
which all stakeholders influence and share control over development and environmental
initiatives and the decisions and resmrces which affect them. The principle of public
participation and consultation is found in developed and developing countries.
The Regional Environmental Centre for Central and Eastern Europe states that:
'The most fundamental interest that must be addressed in the process of public
participation is the basic right of individuals to have a say in matters affecting
their lives ... The basic right to participate in decisions affecting oneself .. applies
in circumstances where the rights and interests may be less recognizable [such as
Socioeconomic, institutional and legal aspects 145
right to have a clean environment] ... Taking into account the users' interests
should actively involve the users themselves.' (REC, 1995).
The American Waterworks Association Policy Statement on Public
Involvement states that: 'Involving the public in decision making ... is ... important
because ma,v, drinking water issues, including adequacy of supply, water quality,
rates and conservation, are not only technical issues, they are also social,
political, personal health, and economic issues. As such, they are best resolved
through a process of meaningful dialogue with concerned parties and the public.'
(Kusel, 1998; A WW A. 1995).
The World Bank Policy Research Working Paper states that: 'Recent evidence
from Asia, Latin America and North America suggests that neighbouring
communities can have a powerful influence on factories ' environmental
performance ... where formal regulators are present, communities use the political
process to influence the tightness of eriforcement. Where formal regulators are
absent or ineffective, 'iriformal regulation' is implemented through community
groups or NGOs.' (Afsah and Benoit-Wheeler, 1996).
Community or public participation and consultation are important aspects of resource
management as successful implementation is commonly dependent on broad agreement
with the objectives and in some cases active public participation in programmes, to
ensure these objectives are met Although the general public in most countries is aware
that pollution of surface water is caused by mismanagement of waste and inappropriate
land use, awareness is more limited when it comes to groundwater, which is often
considered 'pure' and clean. This may present particular challenges to ensuring
commitment and participation by the public in protecting groundwater resources.
The role of communities may be critical to promoting improved protection, but the
nature of the role that they will play may vary. In many situations, communities are
consulted but play limited practical roles in the implementation of groundwater
protection strategies. In other cases, communities are expected to play an active role in
the design, planning and implementation of groundwater protection
It is important to be clear about the differences in two of the principal approaches to
community involvement: consultation and participation Consultation is a process of
discussion with stakeholders about proposed actions or strategy and is geared towards
obtaining the opinion from each stakeholder about these and to review the options that
are available. However, it may not mean that the agency undertaking the consultation is
bound by the outcome of these discussions and usually does not imply a responsibility
for action by the oommunity.
Participation is a set of processes where communities and individuals play an active
role in the design, planning and implementation of programmes of water resource
development or protection. This often implies that the agency and the community have
responsibilities for ensuring agreed actions are performed. It is therefore a more long-
term and proactive process than consultation However, for successful participation there
must be efl:ective consultation and therefore the two processes are often combined.
146 Protecting Groundwater for Health
S.3.1 Consultation
It is essential that there is proper consultation with stakeholders in the development of
policy and implementation of grrnmdwater protection plans. A key activity in the initial
stages of policy development is to ensure that the views and needs of different
stakeoolders are properly reviewed and incorporated into the policy being developed as
fur as possible. The stakeoolders should also have an opportunity to oomment on the
policy and strategies developed to ensure that these reflect a position of agreement
among key stakeholding groups.
Consultation should bring in the views of Government, affected interest groups and
the views of the broader society. Therefure various oonsultation exercises may need to be
undertaken to ensure that the views of all concerned and in particular those groups whose
livelihood may be directly affected are collected and concerns addressed. Very often,
these groups are those most directly affected by water resource management through
lack of access to safe drinking-water supplies, contamination of water sources and
limited water fur irrigation. In order for policy to be effectively implemented, it is
important that there is general support fur the overall policy and strat.egy ftamework
within the country. This is an ongoing process and not something that is engaged in only
at the start of policy development It should be seen as a necessary process which
supports the development and implementation of resource management policy and
strategy.
Perceptions and cultural values attached to water are also important to understand in
the oontext of groundwater quality management and protection. Many of these concepts
provide a foundation upon which to build effective protection strategies as they attach
important religious or cultural values on the protection of the groundwater. Examples
include some aboriginal belieis about the origins and sacred nature of water in Australia
In other examples traditional beliefs may hamper the development of groundwater
protection strategies. For imtance in Uganda beliefs about the use of certain springs by
ancestral spirits prevented action being taken to improve water sources.
S.3.2 Participation ·
In wealthier industrialized countries although public participation oocurs, the emphasis
tends to be on consultation in the development of the underlying principles, policies and
plans that define the development of environmental protection. In most cases,
groundwater protection strategies are implemented by local or central government with
systems of land use restriction, compensation and appeal processes operating. Specific
activities required will often result in specific negotiated agreements with individual
land-owners.
By con~ in developing countries, the development of groundwater protection
plans and implementation of protection measures is likely to require the direct
involvement of large nwnbers of people and communities. Many of the tasks that will be
required can only be undertaken by local people taking responsibility themselves to
enforce protection measures, although this means that communities need support to
develop effective capacity. The development of community management committees or
Socioeconomic, institutional and legal aspects 147
users organmrtions is an important component in promoting effective resource use
(Subramanian et al., 1997).
By understanding these issues, appropriate strategies and plans can be developed that
identify key stakeholders, where responsibilities lie and what role is expected to be
undertaken by the community. Such decisions will be arrived at partly through
stakeholder consultation. However, during the initial stages of the development of the
policies and strategies, it is important to collect infonnati.on about communities in order
to be able to provide direction for subsequent discussions.
5.4 LAND TENURE AND PROPERTY RIGHTS
Land tenure and property rights are an important consideration when planning
interventions to protect groundwater resomces as they will directly influence the scope
and depth of consultation and negotiation regarding land use. They may also influence
what type of intervention is possible and the nature of any regulations that will need to be
developed.
One aspect ofland tenure of particular importance is the degree to which ownership
of land confers rights of ownership and use of underlying resources. In many countries,
ownership of land may confer automatic rights to exploit, although these are increasingly
subject to licensing and permitting procedmes, but ownership resides with the
Government In these cases, controls over abstraction and land use may be easier to
implement and monitor.
In other countries, resource ownership has historically resided with the land owner,
although this is being revised in many countries. In the Sultanate of Oman, for example,
private ownership of water was abolished by a Royal decree in 1988 and a centrally
regulated system of water management introduced with an associated well permit system
(Government of Oman, 1995).
Revisions to land laws may require significant transitional periods. For instance, the
Spanish Royal Decree of 1986 (No 849) considers underground waters to be in the
public domain and licences to abstract are required. Public ownership is however subject
to the right oflandowners to cany out activities on their land but these must not interfere
with groundwater quality. In order to avoid opposition to the transition from private
ownership to public resource, the Act gave extensive protection to existing rights owners,
and complete transition will not occur until 75 years have elapsed.
Land tenure is often complicated and there are many diffi:rent forms of rights
including customary rights to land, private freehold ownership and publicly owned land,
with many different variants (Payne, 1997). In addition to issues of ownership, the nature
of tenancy arrangements varies and there are further groups who lack any form of de jure
right to abode, but which may have a variety of de facto rights (Hardoy and
Satterthwaite, 1989). There are also a significant number of people who have no rights
and no security of tenure. The sections below review some key forms of tenure and
discuss their implications in relation to groundwater management
148 Protecting Groundwater for Health
5.4.1 Private land ownership
Private land-ownership is common in many pans of the world and refers to situations
where individuals own land, for instance through freehold arrangements. This may be
complicated where land is subsequently let to third parties, a common arrangement in
European agricultural areas.
This fonn of land ownership has particular consequences for the development of
· groundwater protection strategies. The large nwnber of land-owners or tenants may
make the process of consultation more cumbersome as the nwnbers of people involved
may increase the time it takes to collect and synthesiz.e local opinions and a greater range
of views may need to be accommodated Such patterns ofland ownership will also often
result in compensation packages being developed to offset loss of earnings resulting from
restrictions placed on land use. However, where such tenure is in place, it may be easier
to define a legal framework that can be transparent in its . operation and where
compulsoiy purchase or rnandatozy development controls can be enforced.
5.4.2 Customary land rights
This is common throughout much the developing world and reflects a situation where
rights to land are held by a community, although ownership is retained by an individual
or the Government An example of such an arrangement is 'common' land within a
village where all residents have the right to graze their livestock. In some parts of the
world, this may be expanded into communal owna-ship ofland.
Customaiy rights imply that decisions relating to the use of the land require
agreement with all those with rights to use the land, which may result in a more difficult
decision-making process when establishing protection strategies. However, customaiy
rights may already implicitly or explicitly restrict activities acceptable on the 'common'
land, for instance by proscnbing activities that would restrict the full enjoyment of rights
by others,
Communal rights to land can also offer benefits in terms of discussions with
communities regarding actions required to protect water resources. Firstly, the impact of
poor groundwater management is likely to be felt directly by the community as in many
cases they may be using the sources being polluted from land that is used by the
community. Secondly, it introduces the broader concept of public goods that may be
easier to accept when restrictions apply across a community rather than to specific
individuals. Thirdly, management and protection strategies can be designed to resporxl to
the demands of the community. Where communities have been active participants in
strategy development, they will be able to provide a degree of self-policing which may
ultimately prove more effective than outside inspectioIL
5.4.3 Publicly owned land
Publicly owned land is land owned by a Government for all its populatioIL Examples
of publicly owned land include national parks where the land is held for the nation,
even though some of the land may be let to individual farmers. In some European
countries, the catchment areas of major sources of water are purchased specifically for
Socioeconomic, institutional and legal aspects 149
the purpose of protecting the quality of the water source, particularly where it is used
for domestic purposes.
As most groundwater protection policies and plans are implemented by Government
bodies, publicly-owned lam is the most amenable to restriction of land use. but will still
require a process of public consultation during policy developmert. Particular issues that
are likely to need resolution will be changes in allowable use of land where part of the
land is let in long-term tenancy to farmers or where there is a public right of access. In
the former case, changes may need to be phased or supported by compensation
packages, whilst in the latter case, broad consultation should be undertaken to ensure that
there is public acceptance of the need for such restrictions. Where public access rights are
maintained within areas where there are restrictions, it is essential that appropriate
services (such as public toilets) are provided to reduce the potential for release of
contaminants into the groundwater.
5.4.4 Informal settlements
Informal settlements -situations where land tenure is 1lllclear and where rights are
limited -represent particular problems for gro1llldwater protectioIL In many cases the
residents of such settlements have little or no resources and are vulnerable both to
exploitation and to ill-heath derived fium contamination in the environment. At the same
time, such settlements may become a major source of pollution for gro1llldwater
resources as they typically lack basic sanitation, solid waste disposal or surfuce water
drainage. Where water supplies are also lacking use is likely to be made of shallow
gro1llldwater systems, potentially leading to direct impacts on the health of the
comm1lllity.
Enforcement of land use restrictions in informal areas is unlikely to be succe.55ful as
they are illegal and wilicensed settlements. However, simply trying to remove such
settlements is not only highly discriminatory agaimt the poor, but it is 1llllikely to be
effective and will result in simply shifting the problem and not resolving it. In these
situations, it is more appropriate to identify ways of working with comm1lllity groups to
make improvements in environmental health that reduces health risks.
5.5 VALUING AND COSTING GROUNDWATER
PROTECTION
An important approach to protection of groundwater is to put an economic and social
value on groundwater resources.. This value should take into acco1lllt the direct and
indirect cost of protecting the resources as a function of the direct compemation costs (if
any) and lost opportunity costs fium other, potentially more productive, uses of the land.
This should be balanced through placing a value on the aquifer in relation to its
importance in supporting economic growth. The latter should consider the current value
of groundwater to different industries and the value of each industry to the overall
economy. It should also include the incremental marginal costs caused by increased
treatment costs (either derived fium use of alternative sources or due to pollution of
150 Protecting Groundwater for Health
groundwater) and increased abstraction oosts derived from exploitation of deeper
reso~ due to contamination of shallow groundwater.
Most environmental protection activities will result in some increase in the cost of
production and distnbution of drinking-water and more generally in tams of overall
environmental protectioIL For instance, there may be a requirement to pay compensation
to existing land-users or to purchase land in drinking-water catchment areas. Within this
debate it is important to obtain the views of the public (pemaps represented by consumer
groups) on their willingness to pay for such improvements and to assess whether this will
be sufficient to off-set costs, discotmted over an appropriate period where necessruy.
Unless there is a willingness by the public (or specific water consumers) to pay the costs
of protection, it may be very difficult to sustain intervention strategies. This is discussed
:further in Chapter 20.
In addition to a direct balancing of economic costs, it is important that social aspects
such as the access to safe water supply and the burdens placed upon poor :fiunilies from
having to walk long distances to collect poor quality water should also be assigned a
value. One way of doing this is to calculate the likely public health burden derived from
poor access to water supply. This may also include a factoring in of the numbers of
people whose welfare depends on the continued exploitation of groundwater, whether for
domestic use or in agriculture or industry. An example of an approach to valuing
groundwater protection is shown in Box 52.
Box5.2. Putting a value on groundwater: Managua(basedon &:harp et al, 1997)
The city of Managua in Nicaragua is dependent on groundwater for domestic
water supplies and therefore groundwater protection is a priority. Work
tmdertaken by the StNainable Use of Water Resources project developed a
methodology to assign a groundwater protection value to groundwater sources
as an input to groundwater protection planning. The project used four criteria:
available quantity, grotmdwater quality, present or planned use and sensitivity to
changes in groundwater level. These criteria were based on the economic
valuation of water teSQurces in relation to current use, option for future use and
environmental significance.
A protection value was calculated based on scores calculated for each criteria
The scores for quantity, quality and sensitivity to changes in groundwater level
were used to define the protection value, whilst the present or planned use
a'it.eria was overlain on a final map to indicate current and planned abstractioIL
The authors note that the protection value was a relative measure based on five
classes. The data was compiled into a map on a Geographical Information
System (GIS) platform that allowed abstraction to be overlain on the protection
value. The authors concluded that the areas where there was currently greatest
use corresponded to the areas where the protection value was highest. They also
concluded that the approach provided a simple and effective tool to assist
planners to develop groundwater protection plans.
Socioeconomic, institutional and legal aspects 151
5.6 SETTING GOALS AND OBJECTIVES -HOW MUCH
WILL BE PROTECTED?
The goals and objectives of grmmdwater protection programmes must be determined
before appropriate choices can be taken. Where they existed, in the past, water resource
management and environmental protection agencies often made decisions on goals and
objectives exclusively. More recently, however, it has been increasingly recognized that
planning agencies, local government authorities, key industry groups and the general
community need to be consulted. If the inherent conflicts in land use controls are to be
resolved, then the W1derstanding of the resource should be accompanied by an
appreciation of its value to the community and of the potential impacts of specific land
uses on groundwater quality. The community as a whole should decide what needs to be
protected and how much protection it can affi>rd. The introduction of obligatory
environmental impact studies in Chile, for example, bas included the whole community
of affi:cted interests into the decision-making process for the first time (Garces, 2000). Of
course, there are still many countries where little or no protection is afforded.
Protection can either extend across an entire aquifer or be restricted to important
recharge areas, or capture z.ones, for specific water supply wells. The question of how
much protection is needed or desired depends on the characteristics of the resource. the
degree to which it is used, as well as other community social and economic goals.
Alternative macro-protection land use management policies include:
No degradation. The maintenance of the quality of grolllldwater at no worse than
existing levels. Generally, such a policy would only be applied to vital resources,
typically a resource that provides the sole source of dririking-water. For practical reasons
it can only be applied to grolllldwater resomces in undeveloped areas, or areas of very
low intensity development Further land development will normally be excluded from
the designated area
LimiJed or controlled degradation Such a policy acknowledges that existing or
proposed land uses will cause a deterioration of grolllldwater quality, but strives to
maintain the quality above certain specified limits. This policy nonnally involves
controlling the density and types of land development, and the prescription of specific
. management practices for activities that can affect groundwater quality.
Differenlial protection. Differential protection policies allow for combinations of no-
degradation and limited degradation. Land use management practices normally result in
a combination of exclusion and restriction. Such differential protection policies allow the
development of different protection objectives taking into account factors such as present
and potential uses of water resources, the tenure, z.oning and uses of land in the locality,
and the desires of the communities involved.
Cor(licts in land use management for groundwater quality protection. Restrictions on
land use for groundwater protection will always have an economic cost, and decisions
must be made about how to minimiz.e these costs while maximizing protection. For
example. any limitation on the type and amount of induslrial or urban development will
have a cost Consequently, the setting ofland use controls for grolllldwater protection can
be very controversial. Obviously, lando\\ners have an expectation that their land can be
used freely in its economic highest value use. The wider community interest, on the other
152 Protecting Groundwater for Health
hand, can require that groundwater should not be put at risk of pollutioIL Those
responsible, therefore, usually have the difficult task of trying to balance the optimum
protection of groundwater resources with the economic interests of the owners of the
overlying land surfuce.
There may be many potential complications involved in land use management, for
instance in trying to control problems such as the salinization of groundwater due to
irrigation return waters. There may be extreme examples of conflicts, as for instance in
the Doon Valley in Uttar Pradesh, fudia where limestone quanying is physically
destroying the aquifer (Shaman, 1996). Many different types ofland use may have to be
restricted to protect the quality of groundwater. It is not simply a question oflimitations
on activities involving toxic materials or the disposal of sewage. Rtm off fiom urban
areas can be a serious contaminant and it and other sotm:eS of diffuse contamination,
particularly fiom agriculture, are those that are best controlled through land use
management policies. However, it is with controls over diffilse· sources that most
conflicts will tend to arise. Toe problem is particularly acute in rapid growing cities and
in those cities in poorer countries where there is reliance on water supplies from shallow
aquifers and disposal of excreta in situ.
5. 7 INSTITUTIONAL ISSUES
The development of groundwater protection strategies and policies requires effective
institutions responsible for the planning, implementation and management of
groundwater in the country. In a great number of cases, the fuilure to protect grolilldwater
resources results not fiom a lack of appropriate legislation, but because of the poor
enforcement of existing regulations. This frequently reflects both weaknesses in the
overall institutional framework for grotmdwater protection and weaknesses within key
institutions themselves. Part of the weakness often noted is that the institutions dealing
with health, water resource or water supply fuil to collaborate to define groundwater
protection needs.
Where groundwater policies do not exist or are in need of revision, it is important that
a lead institution should be identified for policy direction, and the CO-Operation of other
relevant organizations in decision making should be sought Generally the lead
organiz.ation is placed at the central government level Even in cotmtries where local
public participation in water management is high, such as parts of the USA, local water
management plans must be consistent with national water quality management
objectives and plans.
It is essential that the different roles and responsibilities of different agencies working
in the water sector are clearly defined and that one agency is charged with the
responsibility to develop, implement and enforce a groundwater protection plaIL It is
important that the institution identified does not have any conflicts of interest that will
compromise its ability to work independently. It is usually preferred that a water
resources management body is established that is involved in the approval of
development of water sources and the control of the quality of the resource, but is not
directly involved in water source development
Socioeconomic, institutional and legal aspects 153
Institutional mandates must reflect the aims and ol!jectives of each institution with
respect 1D their roles and responsibilities within the sect.or (Alaerts, 191J7). One of the
consequences of this is 1o consider carefully the scale and soope of activities. For
instance, water and wastewater savice provision is often most effectively perfonned
when decision-making is devolved 1o decentralized bodies such as municipalities, water
companies or water users associations. National central bodies may slill retain some
responsibility for policy or. strategic development, but may have little influence on
operational matters. This implies that when considering roles and responsibilities, the
level of action (national policy or local implementation) needs to be considered as well as
the area over which the institution has a mandate for action.
The regulation and control of groundwater quality requires a somewhat different
approach, ali.hough the principles of decentralized operation are still valid However,
although implementation of regulatory activities may be decentralized, there is a need for
a strong national institution capable of providing the overall policy and strategic guidance
for groundwater protection
5.8 LEGAL FRAMEWORK
The protection of groundwater requires an adequate legal framework (Caponera, 19'J2;
Souisby et al., 191J9). As governments move 1owards the strategic management of the
colllltry's water resomces, it is often necessary to replace basic common law and
property rights with statutory provisions regulating the use, development and protection
of water (Caponera, 19'J2). Legal ism.ies related 1o water ownership, the means used 1o
control abstraction and polluting activities, and the enforcement of such legislation
become important. The framework must be supported by appropriate imtitutions that are
capable of implementing the policies and enforcing the relevant laws and regulations,
and these organizations must also have the necessary legal status and powers. The
willingnes.s to enforce compliance with pollution control measures and whether
regulatory frameworks create incentives for potential polluters to comply are critical in
ensuring effective regulation (Lane et al., 1991J). Within the general considerations of the
scope 'of environmental legislation, the legitimate demands of economic development
must be considered to ensure that a sensible balance is struck between the two (Lane et
al., 1991J).
Legal .frameworks in place or developed for water protection deal often with many
other issues apart :fium groundwater. It is possible to identify the shortcuts that relate
solely to groundwater, but it is not usually possible 1o change the laws so as to
concentrate only on groundwater quality. The use of more general laws must therefore be
accepted, and the specific legislative provisions that may be applicable to the
grolllldwater situation shouid be identified and used as appropriate, working within the
framework of all the provisions of the relevant legislation.
Legislative reform may be required in order to achieve the objectives of grolllldwater
protection This may involve the revision of existing legislation to encompass these
policy objectives or the development of new legislation geared 1owards grolllldwater.
The approach adopted depends in large part on the nature of existing legislation, the ease
with which this may be updated (bearing in mind that existing legislation may deal with
154 Protecting Groundwater for Health
broader is.5ues and updating may be time consuming) and the importance of groundwater
in the national water resources. Furthermore, as not.eel by Foster et al (1992), legislative
reform will only be effective where political will exists to ensure implementatioIL 1bese
issues are discussed further in Chapter 20.
5.9 REFERENCES
A1sah, S-L. and Benoit-Wheeler, D. (1996) Controlling Industrial Pollution: A new paradigm.
World Bank Policy Research Worlcing Paper WPS1672.
Alacts, G. (1997) Institutional arrangemaits. In Water Po/lutwn Control, (eds. R Helmer and I.
Hespaahol), pp. 219-244, E&F Spon, London.
AWWA (1995) AWWA goverrunent affairs: public involvement policy. www.awwa.org (accessed
A?il29, 2005).
Caponera, DE. (1992) frinciples of Water Law and Administration, Balkema, Rotterdam.
Chambers, Rand Conway, G. (1991) Sustainable Rural Livelihoods: Practical Concepts for the
2 I" Century, IDS Discus.5ion Paper 296, BrigbtmL
DFID (2003) Sustainable livelihoods guidance sheets. wwwJivelibood.5.mg/info/info-
guidancesbeds.html (accessed April 29, 2005).
Foster, S., Adams, B., Morales, M and Tenjo, S. (1992) F.rtrategias par la proteccion de aguas
suhterraneas, Centro Pooamericano de Ingenieria Sanitari.a y Ciencias del Ambiente, Lima
Franceys, R, Pick:furd, J. and Reed, R (1992) A Guide to the DevelopmenJ of On-site Sanitation.
World Health Organiz.ation, Genew.
Garces, JA (2000) El Sistema de Eva/uaci/Jn de lmpacto Amhenlal y el Sector Sanitario, Revista
AIDIS, No. 25, Chile.
Government of Oman (1995) Water Resowces of the Su/tanaJe of Oman, Ministry of Water
Resources, Oman.
Hardoy, JE. and Sanertb.w.rite, D. (1989) Squatter Cm=en: Life in the Urban Third World,
.Emthscan Publications, London.
Kusel, M (1998) Paspective. American Waterworks Association Mainstream, 42(3), 5.
Lane, S.N., Ricl!mds, K.S., Sinha, S. and Wy, S. (1999) Screw the lid even tighter? Warec pollution
and the enfurcement of environmental regulalion in less developed countries. In Water
Quality: Processes and Policy, (eds. S.T. Trudgill, D.E Walling and B.W. Webb), pp. 233-
255, John Wiley & Sons, Chidiesta. .
Nichol, A (2000) Adopting a Sustainable Livelihoods Approach to Water Projects: Policy and
Practical Implications, Workirig Papa-No. 133, ODI, London.
Payne, G. (1997) Urban Land Tenure and Property RighJs in Developing Countries: A review, IT
publications and ODA, London.
REC (1995) Status qf Public Participation Practices in &ivironmenlal Decision making in CenJral
and Eastern Europe Case Studies of Albania, Bulgaria, the C=ech Republic, Croatia, Estonia,
Hungary, Latvia, Lithuania, FYR Macedonia, Poland, Romania, the Slovak Republic and
Slovenia. Report of the Regional Environmental Centre fur Central and ~ Ewupe,
Budapest, http://www.rec.mg/REC/Publications/PPstatus/cover. html (accessed April 29,
2005).
Sduup, C., Alveteg, T., Johansson, P-0. and Caldera, M . (1997) ~gning a groundwater
protection value: mdhodology development. In Groundwater in the Urban Environment:
Problems, Processes and Management, (ed. J. Chilton), pp. 65~, Balkema, Rotterdam.
Shaman, D. (1996) India's Pollution RegulaJory Structure and Background, NIPR Papers, World
Bank, Washington.
Soulsby, C., Chen, M. and Malcolm, R (1999) Towards groundwater protection in the UK:
problems of integrating science and policy. In Water Quality: Processes and policy, (eds. S.T.
Trudgill, D.E. Walling andB.W. Webb), pp. 151-179,John Wiley& Sons, Chichester:.
Socioeconomic, institutional and legal aspects 155
Subramanian, A, Taggannatbaa, N.V. and Meinz.en.-Dick, R (1997) User organi=alions for
Sustainable Water Services, World Bank Technical Paper No. 354, The World Bank,
Wa-,hington OC.
TamlIDO, P.8-L., Howard, G. and Smith, MD. (2003) Ecological Risk Assessment (ERA): How
appropriate for developing countries? Environmental Irifomatics Archives, 1, 204-212.
WELL (1998) Guidance Manual on Water Supply and Sanitation Programmes, WEOC,
Loughborough.
World Bank (1993) Water Resources Management, World Bank Policy Paper, World Bank,
Washington, OC.