20080868 Ver 2_App F QA QC Protocols_20190628

Multi -Parameter Water Quality Meter Calibration
Physical parameters are measured with a YSI Pro Plus multi -parameter water quality
meter (YSI) during collection of water quality samples and during the salinity monitor
servicing and performance evaluation. Each YSI is pre -calibrated according to
manufacturer specifications in the CZR water quality laboratory the day prior to field
work for specific conductivity, dissolved oxygen, and pH. The results of this calibration
are recorded on a Field Meter Calibration/Check Sheet (Figure 1). Each YSI is also post -
checked at the end of the same week following field work for the same parameters to
verify accuracy of readings after use in the field. If there is a >15 percent difference
between the post -check readings and the standard readings, the data collected in the field
by that unit are considered unreliable and are not used for analysis. Field Meter
Calibration/Check Sheets are kept in a binder for tracking the calibration performance of
each YSI. A maintenance log that includes the date, maintenance performed, and the
initials of the person that performed the maintenance is also located in this binder (Figure
2).
The concentration of the specific conductivity standard is determined by the calibrator
based on concentrations the user expects to encounter in the field. The standard
concentration each YSI is calibrated to should be higher than the highest specific
conductivity measured in the field but still be as close as possible to that highest value.
The lowest specific conductivity concentration used for calibration is not lower than
1,000 µS due to manufacturer recommendations. In cases where the YSI will be
measuring specific conductivity concentrations in the estuary and freshwater wetlands
and/or stream sampling locations, a higher concentration standard (20,000 µS or higher)
is used for calibration. A check of at least one other lower concentration (typically 1,000
µS) is utilized to verify accuracy of readings in freshwater locations. If only freshwater
wetlands and/or streams are anticipated for measurement, the YSI is calibrated with 1,000
µS standard.
Salinity/Water Level Monitor Servicing and Performance
Creek salinity and water level are continuously monitored with In -Situ AquaTROLLs.
Each AquaTROLL is serviced and evaluated for performance every two weeks. A
Continuous Water Quality Monitor Field Form is used during each salinity monitor
service and performance evaluation (Figure 3). During this evaluation, a pre -calibrated
YSI Pro Plus unit is used to provide in -situ comparative readings to the AquaTROLL.
Because of the effects that estuarine stratification can have on the physical characteristics
of the water, the YSI probes are positioned at the same depth as the AquaTROLL probes.
Comparative readings are recorded on the Continuous Water Quality Monitor Field Form
before and after service. If the difference between the initial readings of any parameter of
the AquaTROLL and YSI vary by >15 percent, the data from the date and time of that
service and performance evaluation to the previous service and performance evaluation is
not used for analysis. Adequate time is allowed for equilibration of the YSI between
each recorded parameter.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-1
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

Calibration drift checks and recalibrations of specific conductivity and pressure (used to
calculate depth) of each AquaTROLL is also performed during the salinity monitor
service and performance evaluation. This information is also documented on the
Continuous Water Quality Monitor Field Form. The specific conductivity value used
during the calibration drift check and recalibration should be greater than the highest
specific conductivity measured by that instrument while deployed. The instrument and
calibration cup is rinsed three times prior to calibration. Following specific conductivity
calibration, the cell constant is recorded. If the cell constant is outside the range of 0.98-
1.02 and/or the instrument does not calibrate within 15 percent of standard concentration,
the unit is serviced or replaced. The specific conductivity reading in air, or zero value, is
also documented. If a reading in air reads >10 µS or <0 µS, the instrument is serviced or
replaced. Additionally, if the battery life falls below 20 percent the instrument is
replaced. If an AquaTROLL is replaced, the new instrument is calibrated and the results
of that calibration and the serial number of that unit is recorded on the Continuous Water
Quality Monitor Field Form. The Continuous Water Quality Monitor Field Forms are
kept in a binder at the CZR Wilmington office to track the performance of each deployed
AquaTROLL.
AquaTROLLs are sent in for a factory calibration following two years of deployment or
if the cell constant is outside of the factory recommended range of 0.98-1.02 resulting in
faulty calibration.
Water Quality Sample Collection
Water samples are collected every two weeks and transported in pre -labeled bottles
identified with the sample location, date, company name, and collector's initials. In
extremely shallow water conditions, a turkey baster is used to carefully siphon the water
sample. If samples are collected with a turkey baster, the baster is rinsed three times with
sample water prior to filling the sample bottle. For direct dip collection, the bottle is
submerged under water in the upstream direction with the lid attached. The lid is then
removed to allow the water to fill the bottle. The lid is replaced before removal of the
sample bottle from the water. It will be at the discretion of the biologist whether the
direct dip method or a turkey baster is utilized. If a sample cannot be collected without
disturbing the sediment via direct dip or with a turkey baster, a sample is not collected.
Notes are added on the data form about collection conditions to aid in interpretation of
results. Samples are placed on wet ice as soon as possible after collection for
preservation. If samples cannot be placed on ice within 30 minutes of sample collection
due to the length of the trail to retrieve that sample, a portable cooler is taken to the
sample site.
Water quality parameters including salinity, DO, pH, conductivity, specific conductivity,
and temperature are also measured with a pre -calibrated YSI immediately following
sample collection. These measurements are taken at 0.1 in depth. If there is insufficient
depth for the water to cover the probes and/or the probes touch the substrate in any way,
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-2
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

water is collected in a separate bottle (one not used for sample collection) for
measurement. If this method of measurement is employed, it is noted by the biologist on
the field form. Adequate time is allowed for equilibration of the YSI between each
recorded parameter. A Hanna Instruments pHep 2 pH meter is used to measure pH should
the YSI pH probe fail.
Turbidity of water samples is measured at the end of the sample collection day with a
LaMotte 2020 turbidimeter (turbidimeter). Calibration of the turbidimeter occurs
immediately before sample analyses. The turbidity standard used for calibration is higher
than the sample with the highest anticipated turbidity. At least one additional turbidity
standard is measured in the range of the anticipated sample turbidity to ensure the
accuracy of the instrument throughout the turbidity range of the samples.
Prior to measurement, samples are lightly agitated to resuspend any material fallen out of
suspension since sample collection. A disposable plastic 3 ml pipette is used to extract
sample water from the sample bottle and insert into the measuring cell. The measuring
cell is rinsed three times with sample water before being filled for measurement. A new
pipette is used for each sample to prevent cross -contamination. The exterior of the
measuring cell is wiped clean with a lint -free tissue to remove moisture from the cell
walls. The sample is then measured three times in the turbidimeter and the median value
is recorded by the biologist for that sample.
Samples are delivered by CZR in a cooler to the East Carolina University (ECU) Water
Quality Laboratory the day of collection. Enough wet ice is packed in the cooler to
ensure the samples do not exceed their preservation temperature during transport. ECU
laboratory personnel are notified by phone when CZR departs the Aurora area and of the
number of samples they will receive the day they are delivered.
East Carolina University (ECU) Chain of Custody for PCS Water Quality Samples:
Upon receipt of the samples, the samples are opened in the Central Environmental Lab
and filtered immediately. Samples are then placed in the freezer in a locked room (S 116)
until analysis.
Following is a summary of ECU's QC and calibration information from each SOP for the
different analyses:
- NH4: a standard curve and blanks are run with every set of samples processed.
- PO4: a standard curve and blanks are run with every set of samples processed.
- TDP: a standard curve and blanks are run with every set of samples processed.
- NOx: a standard curve, blanks, and QC is run with every set of samples
processed. Column efficiency is checked every 10 samples.
- DKN: a standard curve, blanks, and QC is run with every set of samples. Samples
are digested in duplicate.
- PN: a standard curve, blanks, and QC is run with every set of samples.
- PP: a standard curve, blanks, and QC is run with every set of samples.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-3
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

- Chl a: fluorometer is zeroed using a secondary solid standard before reading
samples.
In general everything run on the autoanalyzer includes QC samples, while those still done
by benchtop methods do not.
Typically, if the standard curve (run for everything but chlorophyll) yields an r> 0.99,
analysis proceeds. If not, reagents, etc. are checked and standard curves rerun. Blanks
are also used to identify possible contamination in reagent water and standards.
Water Quality TOC/DOC/POC- Procedures to ensure QA/QC at the ECU laboratory
are included in a separate section of this appendix (pages F-20 to F-73-updated
August 2018).
Water Quality- Metals
At the time that the sediment samples are taken, a 250 mL water sample is also taken for
metals analysis (backup samples are also taken at each location). The samples are
analyzed for concentrations of silver (Ag), arsenic (As), cadmium (Cd), chromium (Cr),
copper (Cu), iron (Fe), and selenium (Se) by Method 6020, and molybdenum (Mo), and
zinc (Zn) by Method 6010 (aluminum is not commonly analyzed in water samples).
Currently, the SGS laboratory in Wilmington, NC provides the bottles with HNO3
preservative, chain of custody forms, and performs the metal analyses of the water
sample.
August 2018 Update: per 2015 EPA Publication SW-846, in 2016 certified laboratories
replaced Method 6010C with Method 6010D and Method 6020A with Method 6020B.
Procedures to ensure QA/QC at SGS Laboratory are included on pages F-74
Flow
Flow in the headwater portions of the monitored streams are measured with Flowline
Products Flow Gages. No factory calibration of these gages is currently required
although as the product is refined, additional steps may need to be taken. During each
data download, any debris collected in the vicinity of the meter that inhibits flow through
the flow chamber will be removed. Pertinent conditions and observations of flow, such
as presence/absence and subjective amount of flow if present, available memory of the
data logger, percent battery life remaining for the data logger, depth of water, and time of
download will be noted. This information will be transferred to a flow gage download
sheet (Figure 4) kept in a binder at the CZR Wilmington office for reference. If the
percent battery life falls below 20 percent, the battery will be replaced. When the data
logger becomes >85 percent full, the data will be purged.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-4
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

Each gage will be installed in the stream channel paired with a wetland hydrology well.
The data from this well will be combined with the field notes to evaluate the performance
of each flow gage.
August 2018 Update: further product development of the Flowline Products did not
occur and use of the low flow gauges ceased in 2013. Since March 2012, biologists make
qualitative observations of flow (low, medium, high) and record water depths at the
locations intended for low flow gauges in the creeks. In September 2015, qualitative
observations of flow began in Huddles Cut at the three Aqua TROLLs; due to bi-
directional nature of flow in Huddles Cut, wind direction is also noted on the data form.
Videos of flow are also captured at all flow observation locations.
Wetland Hydrology
Existing wells in Tooley Creek and Huddles Cut and new wells for expanded monitoring
have been and will be installed in general accordance with the US Army Corps' ERDC
TN-WRAP-05-2 Technical Standard for Water -Table Monitoring of Potential Wetland
Sites. Existing wells at Tooley Creek and Huddles Cut are semi -continuous WM80
Ecotone monitors manufactured by Remote Data Systems. Each of the existing wells in
Tooley and Huddles Cut will be paired with an In -Situ Level TROLL 500 (Level
TROLL). Data collected with the Ecotones will be compared to Level TROLL data
collected to evaluate the accuracy of both types of equipment.
The casing for the Level TROLL unit was modified to allow for depth -to -water
measurements inside the well casing without removal of the probe (depth -to -water
measurements are not possible inside the Ecotone casing without probe removal). During
each download at each well, the depth to water is measured and this depth relative to
ground surface is compared to the real-time readings of the Level TROLLs to verify the
accuracy of the instrument. If the Level TROLL is reading >0.5 inch difference from the
true reading, the Level TROLL is calibrated according to manufacturer specifications. If
a difference of >0.5 inch exists after calibration, the Level TROLL is to be replaced.
During each download, the download time, manual water level measurement, real-time
Level TROLL measurement, file name, and any other pertinent information are recorded
in the field. This information is transferred to a well download sheet (Figure 5) kept in a
binder at the CZR Wilmington office for reference. Wells are downloaded twice a month
to minimize data loss from faulty equipment. If confidence in the Level TROLLs
increases, the download interval may be increased to monthly.
August 2018 Update: since 2011, wetland hydrology is monitored with In -Situ Level
TROLLs.
Fish Sampling
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-5
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

Fish collection is performed using paired fyke nets in narrow, shallow creeks and by
trawling near the mouths in all other creeks. A permanent anchor pole is installed in
creeks fished with fyke nets to ensure these nets are installed at the same location each
time. Fyke nets pairs (one facing upstream and one facing downstream) are set late
afternoon/early evening and retrieved the next morning. An otter trawl is pulled behind
the boat for one minute over a 75-foot trawling station. The start and end of the 75-foot
station is defined by shoreline markers (e.g. piers or spray painted trees) to ensure the
same length of creek is trawled each time. Trawling begins at the upstream end of the
interval and terminates at the downstream end of the interval or after one minute has
passed. All nets are visually inspected for holes prior to deployment. If any holes are
found, repairs are made or another net is used.
Fish information (species, number, size) is recorded in a field book dedicated to fish data.
Fish samplers carry the Peterson Field Guide to Freshwater Fishes (1991), the Peterson
Field Guide to Atlantic Coast Fishes (1986), and The Freshwater Fishes of North
Carolina (1991) to aid in identification of unknown or questionable species. For every
new fish species encountered, a representative specimen is preserved in formalin for the
reference collection kept in the laboratory at the CZR office in Wilmington.
The same YSI water quality parameters as described above are measured prior to fyke net
set and retrieval or trawling. These measurements are taken at 0.1 in depth from the
anchor pole at fyke net creeks and at the downstream end of the trawling interval in the
other creeks at a both a bottom depth (6 inches above bottom) and a top depth (within 6
inches of the water surface). Measurements are documented in the fish field book.
Adequate equilibration time for the YSI is allowed between each recorded parameter.
August 2018 Update: since 2011, percent SAV cover is estimated and shrimp and crab
are counted and tabulated.
Benthic Sampling
Prior to collection of benthic samples, the status of all netted equipment is visually
checked for net integrity and adequate sieve buckets and plastic trays are gathered. The
shelf life of the 10 percent buffered formalin is verified and an adequate supply of sample
jars and label materials are set aside. The ponar apparatus is inspected to make sure the
pin is attached and the baffle is in good condition. If the wench and davit are to be used
on the boat, they are oiled and put in the boat. All necessary maps are gathered and
cameras and GPS equipment (if used) are adequately charged.
Following a modified swamp stream sampling methodology for this study, nine timed
standing sweeps are conducted in three different creek edge habitats using a 500 µ D-net
mounted on a 60-inch wooden handle. For the sweeps, care is taken to agitate the
targeted aquatic environment to dislodge and suspend organisms and then to collect the
organisms in the net with as little sediment and detritus as possible. The contents of the
D-net are put into a plastic tray and gently sieved through the 0.5 mm sieve bucket and
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-6
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

preserved in sample jars with 10 percent formalin as preservative. The jars are labeled
inside and outside with sample number, location, and date and are put in picnic coolers
for transport to the laboratory.
Once in the laboratory, the samples are re -sieved through 0.5 mm mesh to remove
formalin and any other fine lingering debris. The organisms in the sample are then sorted
from the debris or sand under a dissecting microscope and preserved in 80 percent ethyl
alcohol (ethanol) until ready for identification. Each sample is sorted by spoonful once
and then the debris is swirled to spot check the sample a second time. All organisms are
identified to the lowest taxonomic level possible using a dissecting microscope and a
compound microscope. All organisms are saved in ethanol and debris is also saved in
ethanol if requested by client or if outside QA/QC will be performed.
An up-to-date set of identification keys, as well as recent papers and journal articles, are
consulted as necessary to insure the most accurate description and nomenclature is used
for the taxonomy. Spreadsheets are maintained that track which samples have been
sorted and which have been completed and how much time each required. A master list
is kept for each project which tracks all species identified by each year of the project and
by each creek and sample station.
Sediment Samples for Metals
Preparation for the annual sediment sampling follows the same procedure for inspection
of the ponar grab device as described above for the benthic sampling preparation. The
location of the sediment sample station is the downstream end of the fish trawling station
or at the downstream end of the fyke net location near the mouth of each sampled creek.
The ponar is carefully deployed and retrieved and the collected sediment is dumped into a
plastic tray from which —0.5 gallon of sediment is scooped from the sample into a Ziploc
bag using a plastic or stainless steel scoop avoiding sediment that may have touched the
metal of the ponar. Depending on the sediment at the sample location, more than one
ponar grab may be needed to reach 0.5 gallon. Each bag is labeled with creek name and
date and, to minimize the potential for leaks, each sample is double bagged. The ponar
itself, plastic tray, and scoop/spoon are thoroughly rinsed with deionized water between
each sample to avoid cross -contamination. A second sample is collected from each
station in case there is a problem with the shipment to the laboratory or a problem
encountered by the laboratory during analyses. The backup samples are kept at CZR
until results of the analyses are completed at which time the samples are discarded.
Metals and Bulk Density Analyses
Metals analysis and bulk density analysis of the sediment samples is run by the
laboratory according the procedures described below on Page F-75 to F-159 (August
2018 update). The sample for bulk density analysis is taken from the sediment
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-7
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

collected for metals analysis. No additional or separate procedure is required for
collection of the sample for bulk density.
Water Column Samples for Metals
Water column sample collection in each creek is done just prior to collection of the
sediment sample. Preparation of a500ml container and two backup containers is done in
the CZR laboratory prior to departure for field; all containers are washed with deionized
water and alconox and sealed. Once at the collection site, with sterile gloves, the 500 ml
container is twice rinsed with creek water collected off the bow of the boat and then the
sample is collected and poured from the 500 ml container into two 250 ml containers
(prepared in advance by the analytical laboratory and preserved with HNO3; a test bottle
for temperature fluctuation is also provided in the cooler by the lab). Each 250ml sample
is sealed and labeled and put upright in a cooler on ice; the second sample serves as a
backup in case of spill or other error. At each creek the same process is followed and new
sterile gloves are worn to prevent any cross contamination. The cooler of samples is
either shipped for overnight delivery or hand -delivered to the analytical laboratory within
24 hours.
Vegetation Surveys
To ensure a representative area of the system is monitored, ten (10) 3 meter square shrub
plots are arranged in a staggered fashion along a random compass azimuth that originates
at an electronic well. To ensure the plots are easily found from year to year and the
boundaries do not change, the shrub plot corners are marked with PVC poles and tagged
with an aluminum tree tag. The transect azimuth is also delineated with PVC poles tied
with orange flagging (or orange/black) and the alternating outside plot corners are
marked with PVC poles tied with pink flagging and aluminum tree tags. Nested 1-meter
square herb plots are located in the proximal corner of each shrub plot and delineated at
each survey by laying a portable 1-meter square PVC frame around the azimuth pole of
each shrub plot.
To ensure accurate and reproducible results, a field data collection book is created each
year for each sampling crew. Field data collection books include protocol, transect
diagram, previous year's data, list of species, and data forms for each transect (Figures 6
and 7). Explicit definitions for shrubs and herbs are given in the protocol as well as
instructions on how to measure them, particularly on problematic species such as poison
ivy and Hydrocotyle. Plant guides and photo booklets are used in the field and a
collection of pressed specimens and taxonomy guides are available onsite for
consultation. Every member of the field crew reviews photos and pressed specimens
before the sampling event and each team is led by an experienced biologist familiar with
the vegetation and sampling procedures.
If biologists are unable to identify a plant, effort is made to find the same plant outside
the plot and then to gather as much of the plant as possible, including roots, stems, fruits,
seeds, etc. Photographs are taken of any unknown that cannot be collected and as much
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-8
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

detail as possible is recorded about the species. For grasses, sedges, and rushes, dead
leaves from last year's growth are collected as well. Unknowns from each transect are
kept separate and numbered with the transect number and its own unique number. Small
specimens are immediately pressed in a temporary press. The date, specimen ID number
and any other pertinent details are written on the temporary press page with the sample.
If the plant is too big to press, flagging is wrapped around it with the identifying number
and date and it is put it in a Ziploc bag. Bagged specimens are put in a cooler upon
returning to the truck and kept cool until they can be pressed. Collected specimens are
either keyed out in the office with a plant taxonomic identification key or sent to an
expert.
Care is taken to avoid trampling any vegetation along the transect azimuth and in the
plots during the vegetation survey itself and during the monthly downloads of the
monitoring wells. If a specimen is collected, it is taken from outside of the plot. Photos
are then used to compare with plant guides and/or sent to an expert in plant identification.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-9
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

ILSI.-
TAN4
Field Meter Calibration/Check Sheet
Meter hlakeiModel: YSI Pro Plus Quatro
Date/Time:
Meter Serial #: Calibrated by:
Pre -trip calibration
S ecific Conduc ivity Calibration
Std. Value
Tern
Initial
Adjusted
Lot #
Ex iratian
H Calibration
pH
PH1
H4
Tern
Theo. PH
Initial
Adjusted
Lot #
Expiration
Post -trip check
S ecific
Conduc ivity Calibration
Std. Value
Tern
Initial
Lot #
Expiration
H Calibration
pH
PH10
PH4
Tern
Theo, PH
itial
ot #
LL
iratian
Figure 1.
Notes:
Notes:
DO Calibration
TeriBP:
Initial
Adjusted
DO %
DO m all -
Chart ❑O
Date/Time:
Checked by:
40 Calibration
TernBP:
Initial Adjusted
DO %
DO m 1L
Chart DO
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-10
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

YSI Pro Plus Quatro Maintenance Log
Serial # 1441
❑ATE MAINTENANCE PERFORME❑ I N ITI. , L
Figure 2.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-11
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

ry CZR Inc. Continuous Water Quality Monitor Field Form
1� NC PC Creeks Study (Huddles Cut and Tooley Creek)
[fix Oc]4w fE6 1745.47
Monitors Inspected By:
❑ ate:
Weather: Cold Cool Warm Hot Rain Mist Sleet Snow Humid Dry Cloudy Pt Cloudy OVC Clear Windy Breezy Calm
Comments:
STATION #: MONITOR In -Situ Aquatroll 200 100NITOR
MAKEIMODEL: SERIAL *:
Parameter
INITIAL READING
Time
Live Value Ref. Meter
FINAL READING
Time
Live 'Value Ref. I leter
Tem °C
SC nkTcm
Salinity
Depth
CALIBRATION DRIFT CHECK
SPECIFIC CONDUCTANCE
Calibration Criteria: if standard Value <1aa us,
then +1- 5 us; rf>100 us, then +1- 3%
Calibration Check[ Recall.
Time
DEPTH
Time
STANDARD
VALUE (mS)
STANDARD
LOT
EXP.
STANDARD
TEMP.
SC
READING
RECAUD.
READING
DEPTH
(INCHES)
RECALIBRATED
READING
Cell Constant: Readinq in Air. Batt. Life °fo : Time Sync?- YES NO
Comments:
STATION S: MONITOR In -Situ Aquatroll200 MONITOR
MAKEIMODEL: SERIAL #:
Parameter
INITIAL READING
Tilre
_ive `:'aloe Ref. kleter
FINAL READING
Time
Live Value Ref. Metes
Tem °C
SC slcm
Salinity
De till
CALIBRATION DRIFT CHECK
SPECIFIC CONDUCTANCE
Calibration Criteria: if standard value <1oa us,
then +I- 5 us; iF>144 us. then +1- 3%
Calibration Check[ Recal.
Time
DEPTH
Time
STANDARD
VALUE (mS)
STANDARD
LOT #
EXP.
STANDARD
TEMP.
SC
READING
RECAUD.
READING
DEPTH
(INCHES)
RECALIBRATE:'
READIN('
Cell Constant: Reading in Air. Batt. Life (%): Time Sync?: YES NO
Comments:
Figure 3.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-12
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

NCPC - 1745.47
Flow Meter Data Form
Equipment
Date: Time in: Time out: Used:
N,-)TES
My FilesJCZWF.—rJFlow Meter Form-M CPC Flow Meter Form
Revised 2JI4111
Figure 4.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-13
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

NCPC (Huddles Cut R Tooley Creek)-1745.47
Well data Form
Date: Time in: Time out: Equipment used:
Wells
serial#
well
Type
Readingffime
Manual Surface
Water Reading
Calib.
Wei
cleaned
Irrbal
NOTES:
HMW1
Manual
HMW2
11312BED
E-00
23
HMW3
Manual
HMW4
Manual
HMWS
13613188C
E-80
25.75
HMW6
136131 DCS
E-80
20
HMW7
Manual
HMW8
136BOADE
E-80
18.75
HMW9
EBDr421
E-80
30
HMW10
136A854C
E-80
12.5
HMW11
Manual
HMW12
EBDIF08
E-80
18.5
H''NW
EBD14D3
E-80
15
H'VAG
Manual
HWW4
EBD86CD
E-80
26.2E
HWW5
Manual
HWW6
Manual
HWW7
136AB38A
E-90
16
HWWB
11313644
E-90
25.75
HWW9
Manual
HS1
131524139
E-80
21
HS2-A
1314DI99
E-80
35.75
HS243
131531CC
EA
1
12.5
TW1
136AE-671
E-80
3D.5
TW2
Manual
TW3
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*Wells must be cleaned first download of each month.
My File51CZR-Admir6FormsMell Data Fam MPCRJCPC Well Data Farm
Revised 2JI1111
Figure 5.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-14
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

Huddles West Prong Well _ Herbs Name(s):
Date:
Start Time:
End Time:
Quadrat
# (%C = percent cover, #S = number of stems)
1
2
3
4
5
6
7
8
9
10
Species
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
Figure 6.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-15
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Huddles West Prong Well _ Shrubs Name(s):
Date:
Start Time:
End Time:
Quadrat
# (%C = percent cover, #S = number of stems)
1
2
3
4
5
6
7
8
9
10
Species
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
°i°c
#s
Dominant Trees (50/20 rule):
Figure 7.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-16
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

CZR Field Safety Plan
There is a level of risk to all field work. These risks cannot be eliminated; however, they
can be minimized through awareness, preparation, planning, and prudent behavior. All
CZR personnel are expected to be aware of their own limitations, as well as their
surroundings, in order to reduce the likelihood of an accident. Personnel safety is a top
priority of CZR, but ultimately, it is a personal responsibility.
Only licensed drivers are allowed to drive CZR vehicles and only pre -approved boat
operators are allowed to drive any CZR boat. All safety precautions and procedures as
identified in the 2008 CZR Office and Field Safety Manual are to be followed as directed.
No work is to be performed until all contracted obligations regarding safety are
understood and met. Prior to any job requiring fieldwork, the task or project manager in
charge is expected to:
• review the weather forecast,
• consider any specific hazards that may be encountered,
• verify that the vehicle(s) to be used is equipped with minimum first aid supplies,
• verify that field radios and/or battery mobile phones are charged and
functioning,
• ensure that all field personnel associated with the work have been provided with
adequate maps and have been informed of any known specific hazards or
impediments, and
• inform all personnel of site -specific emergency response protocols (such as
those that may exist on military property).
Each field crew is to report in to the home office at least once a day and to call that office
with their expected arrival time (or the office manager at home if it is after hours) once
they are on their way back from the field. Anyone who is in the field alone on a project
is to have a mobile phone on their person and is to have a contact person that they notify
when they come out of the field. If the contact person does not hear from the individual
by the evening, the office manager should be called.
When more than one field team is in the same general area, clear communication must
exist among the individuals regarding their whereabouts, expected tasks to be performed,
expected time of completion, and expected location to be picked up, or expected
rendezvous at the end of the field day. All field personnel must be accounted for before
leaving the job site.
Unannounced field safety checks may occur several times a year. During these checks,
field crews are monitored to see that field safety protocols are being followed.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-17
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

CZR Management of Raw Electronic Data
Hydrology data:
August 2018 Update: Maezura RDA Blue Palms to download wetland hydrology wells
were phased out in2011 when RDS Ecotone monitoring wells were replaced with In Situ
Level TROLLs at all locations except for upstream Porter Creek; these last Ecotones
were replaced with In -Situ Level TROLLS in March 2016 and use of Maezure RDA Blue
Palms become obsolete for hydrology data. Until 2018, the RuggedReader was used to
download data from Level TROLLS, but the Rugged Reader is no longer in manufacture
and replacements are difficult to find. In late 2018, Bluetooth technology and tablets for
field data retrieval were offered by In Situ. See "Water level and salinity data" in the
next paragraph below for QA/QC data management protocols of the tablet: with the
exception of exact location of saved files (hydrology vs salinity), the protocols are the
same for Level TROLLs (hydrology) as for Aqua TROLLs (water level and salinity).
For output, raw data for each well is transferred into a spreadsheet. The well data form is
reviewed simultaneously and any notes on the data form are incorporated into the
spreadsheet. For example: data gaps due to reprogramming or malfunction, well
replacements, correction factor changes, damages to equipment, battery changes. Graphs
are created from the spreadsheet and reviewed by a biologist. Raw data forms are stored
in a 3-ring binder.
Water level and salinity data:
August 2018 Update: After exporting the data from TROLL to the tablet (or backup
Rugged Reader), field crew turn in the tablet (or RuggedReader Handheld PC) and
completed Continuous Water Quality Monitor Field form upon return from field work.
The tablet files are prepared using the installed computer software and then the files are
exported from the tablet (VuSitu data files). Once confirmation that all files shown in the
zipped file on the tablet are visible in the folder of exported files, the exported files are
copied into individual tablet folders on the desktop computer and labeled with the date
the file was taken from the TROLL in the field. Once all files and data are accounted for
and properly filed, the files from each tablet are deleted.
If the RuggedReader device is used as backup, the RuggedReader is downloaded and
each data file (.wsl file) is exported from the WinSitu5 software directory to an Excel file
and stored in the Important Data Files directory within each monitoring location site
folder. The .wsl data files that reside in the WinSitu5 software directory act as an
additional backup file and are located on the C:\ drive on the computer in which the data
was downloaded. These data files in WinSitu5 are not separated into individual location
folders, they are depicted by their location name and date of download. CZR's server
backup software also creates a backup of raw data files daily and an external data storage
device is switched out weekly with one device being stored off site.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-18
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

For output, raw data from the Excel file created is transferred into a spreadsheet for each
individual monitoring station. All depths shallower than the calibration plus 3 inches are
removed from the salinity data to ensure that the conductivity sensor of the AquaTROLL
was fully submerged at the time of the reading. The data form is reviewed simultaneously
and any notes on the data form are incorporated into the spreadsheet. For example: data
gaps due to reprogramming or malfunction, equipment replacement, damages to
equipment. Certain parameters of data are extracted from the individual spreadsheet and
combined into one spreadsheet. Graphs are then created for each location from the
combined spreadsheet and reviewed by a biologist.
All raw data forms are stored in a 3-ring binder.
Rainfall data:
August 2018 update: Beginning in 2011 under the new creeks study plan, instead of
using only the Aurora NOAA Station 6N, rainfall was collected via Remote Data
Systems, Inc. (RDS) tipping bucket rain gauges installed in almost every study creek
(some creeks "share" a rain gauge with other creeks). Research on alternatives to the
RDS gauges began in 2017 when the RDS units began to fail or malfunction, the hand
held devices necessary to download the units (Maezura RDA Blue Palms with cables and
software specific to the RDS rain gauge and Ecotone monitoring wells) were no longer
available, and RDS was no longer in operation. To replace the RDS units a Texas
Electronics, Inc. tipping bucket rain gauge with HOBO data logger was deployed at all
creeks rain gauge locations by December 2018. The logger is downloaded at the office to
the computer database per step-by-step protocols to ensure that the data are properly filed
and saved prior to deletion from the logger or redeployment of the logger to the field.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-19
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks
report

East Carolina University Standard Operating Procedures for
Chemical Constituents Analyzed for the Creeks Monitoring Study for
PCS Phosphate Company, Inc.
and
East Carolina University's Standard Operating Procedure for the Analysis of
Dissolved and Total Organic Carbon CCAL 20A.0
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-20
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

July 2018
CHLOROPHYLL J PHAEOPHYTIN PROCEDURE
(Fluorometric Analysis)
References
Arar, E.J. and Collins, G.B. 1997. Method 445.0: In Vitro Determination of Chlorophyll a
and Pheophytin a in Marine and Freshwater Algae by Fluorescence. U.S. Environmental
Protection Agency. pp.1-22.
Standard Methods for the Examination of Water and Wastewater. 131" edition. 1995.
Standard Methods for the Examination of Water and Wastewater. 131" edition. American Public
Health Association. p 748.
Strickland, J.D.H. and T.R. Parsons, 1968. A Practical Handbook of Seawater Analysis.
Pigment Analysis. Bulletin 167. Fisheries Research Board of Canada. pp. 185-200.
Parsons, T., M. Yoshiaki, and C.M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Determination of Chlorophylls and Total Carotenoids:
Spectrophotometric Analysis. Pergamon Press. pp. 101-106.
Weischmeyer, N., 1994. Fluorometric analysis of chlorophyll a in the presence of
chlorophyll b and pheopigments. Limnol. Oceanogr. 39(8): 1985-1992.
Procedure: Filtering (This is usually done beforehand and filters are in freezer waiting to be
ground, if so then skip to Grinding.)
Filter through a 4.7cm diameter Whatman 934 Filter in subdued light as soon as possible
after collection or within 24 hours of collection. Filter through a Whatman 934-AH pre-
combusted glass fiber filter. Shake bottle of unfiltered sample water vigorously and pour a
measured quantity (500mL or less) into the filtering apparatus. Record volume filtered on
the outside of the foil packet and the lab sheet. The vacuum pressure should not exceed 10
in. Hg because excessive pressure can rupture cells and as a result pigment will be pulled
through the filter into the suction flask. For the same reason keep the filtration time to a
minimum (<1 min.). Do not filter to dryness. Fold filter in half so that filtrate is on the
inside of the fold. Place filter in foil packet, fold edges and store in a —200C freezer in a
Ziploc bag until processing. Can be stored up to 4 weeks without significant loss of
chlorophyll a.
Procedure: Grinding
Reagents
90% Acetone
Dilute 900mL of 100% spectrophotometric grade acetone to 1000ml- with distilled or de -
ionized water. The acetone is stored in the Flammables Cabinet.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-21
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Equipment and supplies needed to grind:
• Black rubber ice cooler w/ 2 small ice packs on bottom, covered w/ paper towel. Foils to
be placed inside after checking for volumes and order with lab sheet and Observation
sheet.
• Sample box w/ 1 large ice pack on bottom and 1 medium ice pack on side. Cover with
paper towel and place empty blue rack inside.
• Chl a Lab Sheet
• Foils with frozen filters
• Blue rack with numbered, marked and salted sample tubes
• Caps
• 90% acetone
• Red squirt bottle
• Sonics Ultrasonic probe/meter
• 2 pair forceps
• Kimwipes
• Sharpie
• Centrifuge
1. Set up data sheets (tube numbers and sample volumes) and centrifuge tubes (best to be
prepared earlier). Mark tube at 12mL and add a small amount salt (half of the scoopula
that is inside the Sodium Chloride (NaCI) bottle). The salt will help precipitate glass
fragments generated from the filters being pulverized.
2. Use either earplugs or earmuffs (located in cabinet under HPCL). In subdued light
masticate filter with ultrasonic probe until it has disintegrated (meter set for 30
seconds). Place filter in the centrifuge tube and push to bottom. Squirt enough 90%
acetone to cover. Gently move the sample tube up and down using the probe to move
the filter around and reduce it to a pulp. Rinse the probe into the tube with 1-2ml- of
90% acetone. Adjust volume in the centrifuge tube to 12mL with 90% acetone. Cap.
Gently invert the centrifuge tubes a couple of times to mix.
3. Place the centrifuge tubes in sample box (with ice packs) and close lid. This prevents the
samples from heating up and degrading the chlorophyll to phaeophytin. After all of the
samples are ground, place box of ground samples in the cold room for at least 2-24
hours.
4. Turn OFF Ultrasonic probe meter. Clean work station.
Procedure: Centrifuging - (REMAINS ON AT ALL TIMES, Temp set at 0°C, Lid securely
latched)
1. After samples have sit in cold room to steep for 2 -24 hours, they can be taken to S-116
and placed in centrifuge (in subdued light). Keep in order and make sure the numbers
have not been smeared by acetone, renumber if needed. The centrifuge buckets must
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-22
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

be loaded equally for balance, use extra blanks if needed. Make certain that the lid is
securely latched.
2. Centrifuge for 30 minutes at 2800g. When timer is set the centrifuge will automatically
begin to move. Make sure it gets up to speed.
3. Put ice packs in chest freezer to keep frozen.
4. After 30 minutes, return ice packs to box and very carefully unload samples from
centrifuge into box using care as not to disturb samples.
5. Once in Chl a room, very carefully remove tops from all tubes, do not disturb samples.
6. Complete chlorophyll a measurements on Trilogy Fluorometer.
Set up work area for reading chi a: You will need the following-
• 90% Acetone
• Acetone waste jug w/ funnel
• Box of 3mL disposable pipets
• Trilogy Fluorometer
• Turner Solid Standard
• 2 sample cuvettes
• Kimwipes
• Chla Lab Sheet
• Trash can
Place paper towels in front of Fluorometer. Pipetting and reading can be done here. On work
table next to it, the cleaning of the sample cuvettes can be done. Measure Raw Fluorescence
Unit and record on both lab sheet as well as log in back of Manual.
Trilogy Fluorometer (this can be done while samples are in centrifuge)
1. Turn ON machine (switch is on the back left side)
2. On touch screen Select chla-NA. (Non-acid chlorophyll module).
3. Confirm module. Select OK.
4. Open Lid. Remove cuvette holder. Place Solid Standard into the machine with the
lip/tip to the back. You will now make 10 measurements to warm the machine up:
• Select Tools by touching the screen. Select Settings. Select Continuous Sampling.
Change the Continuous Sampling to ON
• Frequency of measurements/second should read 1/3
• Total number of measurements should read 10
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-23
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

• Touch OK
• Touch Measure Raw Fluorescence (C-Sampling) Machine will sample 10 times over
the set period of time. Record RFU on lab sheet and in log in manual.
5. Before calibrating or continuing, you must Select Tools, Select Settings, and Select
Continuous Sampling and turn OFF Continuous Sampling.
6. Press OK
7. Place Solid Standard back into its box and return cuvette holder to position.
Reading Samples
With a kimwipe, rinse cuvettes 3x with 90% Acetone making sure that the outside is clean as
well. Shake off any excess Acetone before filling with sample. Hold cuvette with kimwipe. Using
a 3mL disposable pipet carefully transfer sample into cuvette. (Hold sample and cuvette in one
hand and transfer sample with pipet in other.) Do not disturb sample, hold up to subdued light
to make certain no filter is being transferred. Place into Fluorometer and proceed as follows:
1. Select Calibrate
2. Select Use Stored Calibration.
3. Highlight Apr16.2 and hit Select.
4. Select measure fluorescence. Read a blank (90% Acetone) as a sample first.
5. Enter volume of filtered sample water in mL followed by volume of solvent (12mL) used
in mL. It will then automatically measure and calculate fluorescence in µg/L. Record on
data sheet. Make sure readings are in correct units (µg/L).
6. Remove sample and pipet next sample while helper wastes old sample and cleans
cuvette for next sample. Continue until all samples are read.
7. Turn OFF when finished!
Calibration.
NOTE: Should calibrate when there has been an adjustment made to the instrument. At least
once per year —
1. Select/push calibrate button.
2. Select Run New Calibration.
3. Select µg/L (micrograms per liter).
4. Insert acetone blank into test tube holder (blank is 90% acetone).
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-24
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

5. Select OK.
6. You can choose to calibrate with up to 5 standards (0.2, 2, 5, 20, & 200µg chl a/L per
EPA 445 or 2, 6, 20, & 60µg chl a/L per SM 10200 H 3). Enter number of standards.
Remove acetone blank and test tube holder.
7. Insert first Standard and enter concentration.
8. Select enter more calibration standards and repeat until all desired standards have been
entered.
9. Select proceed with calibration and click YES to save calibration.
10. You can either select to name the calibration curve or not. If you select to not name the
calibration, then the machine will proceed with TEMP name.
11. Measure the solid standard to verify the standard is close to the expected reading. If
the standard readings are vastly different than the expected reading, then one may
choose to calibrate again.
12. Remove Solid Standard and place back in box holder.
Procedural Note:
The Welschmeyer method (Welschmeyer 1994) is a simplified way to measure chlorophyll a
without the need for acidification. Based on the research of Dr. Nicholas A. Welschmeyer,
the procedure is sensitive enough for oligotrophic environments; and only a single
fluorescence determination is required.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-25
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

July 2018
Total Ammonia Nitrogen (NH4/TAN) — Automated SmartChem Method
References:
SmartChem 200 Method 210-201 B, revised July 2008. Westco Scientific Instruments, Inc.
Solorzano, L., 1969. Determination of ammonia in natural waters by the phenol-hypochlorite
method. Limnology and Oceanography. 14:799-801.
Standard Methods for the Examination of Water and Wastewater. 22nd edition. 2012.
Standard Methods for the Examination of Water and Wastewater. American Public Health
Association. p 748.
Emerson, K., R.C. Russo, R.E. Lund, and R.V. Thurston. 1975. Aqueous ammonia
equilibrium calculations: effect of pH and temperature. J. Fish. Res. Board Can. 32:
2379-2383
Reagents:
1. Reagent water: Fresh deionized water from E-pure system (16 to 18 megohm/cm) in
S106.
2. REAGENT 1: Ethylenediamine Tetraacetic Acid, Disodium Salt Dihydrate (5%): In
designated 500mL volumetric flask in approx. 450mL reagent water, dissolve 25g of
EDTA (C,oH14N2Na2O8*2H2O; CAS 6381-92-6). Add 2.75g of Sodium Hydroxide (NaOH;
CAS 1310-73-2). May have to put on stir plate. Dilute to 500mL with reagent water. Mix
thoroughly. STABLE. Kept in Ammonia Tray.
3. REAGENT 2: Sodium Phenolate: In designated 100mL volumetric flask, dissolve 3.2g
of Sodium Hydroxide (NaOH; CAS 1310-73-2) in approx. 75mL of reagent water. Cool
the flask to room temperature and then add 9.3mL of liquid Phenol (C6H5OH; CAS 108-
95-2), which is kept in Flammables Cabinet. Dilute to 100mL with reagent water. Good
for 1 week. Wrap with foil and store in fridge.
4. REAGENT 3: Sodium Nitroferricyanide Dihydrate: In designated 100mL volumetric
flask, dissolve 0.075g of Sodium Nitroferricyanide Dihydrate (Na2Fe(CN)5NO*4H2O; CAS
13755-38-9) and dilute to 100mL with reagent water. Add 1 mL Concentrated Probe
Rinse Solution (Unity Scientific P/N 3AS-RN00-21). Invert, gently to mix thoroughly.
Prepare fresh weekly. Wrap with foil and store in fridge.
5. REAGENT 4: Sodium Hypochlorite (CLOROX): In designated 50mL volumetric flask,
add 25mL of Clorox (NaOCL; CAS 7681-52-9) and dilute to 50mL with reagent water.
Available chlorine should be between 2 and 3 percent. Prepare fresh daily. Kept in
Clorox cabinet.
NOTE: Purchase Concentrated Clorox and dilute per directions on designated 1 L Pyrex
bottle. Wrap in foil and keep in Clorox cabinet.
Standards:
a. 10,000W Ammonium Chloride (NH4CI; CAS 12125-02-9): Should already be
made and in designated 1 L Pyrex bottle in fridge. In 1 L volumetric flask add
0.5349g Ammonium Choride and dilute toll L with reagent water. Good for 6
months.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-26
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

b. 1,000uM Combined Standard: In designated 100mL volumetric flask pipet 10mL
of each 10,000pM component (Ammonium Chloride, Potassium Phosphate and
Potassium Nitrate) with a volumetric flask.
c. 80W Combined Standard: In designated 100mL volumetric flask dilute 8mL of
1,000pM Combined Standard to 100 mL with reagent water using a volumetric
pipette. Make fresh.
Quality Controls:
d. Q.C. #3 - 71.377uM NHaCI: In designated 100mL volumetric flask dilute 0.1 mL
(100uL) of the Certified SPEX Certi Prep Ammonia Nitrogen 1,000pg/mL
(=71,327pM) (CAT# AS-NH3N9-2Y) to 100mL with reagent water. Make fresh.
e. Q.C. #2 - 35.69uM NHaCI: In designated 100mL volumetric flask dilute 50mL of
the above Q.C. #3 to 100mL with reagent water. Make fresh.
f. Q.C. #1 - 7.14uM NHaCI: In designated 100mL volumetric flask dilute 10mL of
QC #3 to 100 mL with reagent water. Make fresh.
1. Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1 L volumetric
flask filled with -950mL of reagent water, slowly and tilted to side add 0.5mL of Probe
Rinse Solution. Fill to volume with reagent water. Mix thoroughly. Stable. Store at room
temperature behind SmartChem #1. Keep a constant supply ready.
2. Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900) In designated 2L
container add reagent water to mark. Add 30mL (one bottle) of Unity Scientific Cleaning
Solution. Rinse 30mL bottle with reagent water and pour into 2L container, repeat 2
more times. Fill to volume. Mix thoroughly. Stable. Store at room temperature next to
SmartChem #1. Keep a constant supply ready.
Procedure:
1. Samples: In duplicate, pipet approx. 3mL of sample into sample cups and discard.
Refill and place in SmartChem sample racks (racks 1-4).
2. Diluent: reagent water. Put in "Diluent #1" slot.
3. Standard/Spike: For Standard place a sample cup with -3mL of 80pM NH4-N in
SmartChem rack "RGT2 CTRL/STD", position 1. Spike goes into "RGT1" rack, slot #5
4. Place 6 empty sample cups in sample rack 5, positions 1-6. These cups will be used
to generate the standards for the standard curve.
5. Reagents: In SmartChem rack "RGT2 CTRL/STD", place Reagent #1 (EDTA) in "QC"
slot, Reagent #2 (Sodium Phenolate) in slot "11 ", Reagent #3 (Sodium Nitroprusside) in
slot "12" and Reagent #4 (Sodium Hypochlorite) in slot "l3".
6. Q.C.'s: These are poured into designated SmartChem bottles and placed in rack
"RGT1" in spaces 6-8 in descending order (Q.C. #3 to #1).
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-27
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

7. Check fluid levels in cuvette Wash Solution, DIW, and Probe Rinse Solution bottles, refill
if necessary.
8. Place waste tubes into designated waste jar to collect waste from SmartChem.
SmartChem:
1. Turn on computer; turn on SmartChem.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select "NH3-1-1-LauraLow" from Profile list.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure
to include the empty cups for standard dilutions before starting.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "print" and "print" again.
2. Real time sample data can be displayed by selecting the "Results" button. Once all
samples have been run, the print option will appear in the upper left menu bar, clicking
the button will automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a) Select "options" from left menu bar, then "data retrieval".
b) Find and select the plan of interest. A split screen will appear — click on the
method in the box in the lower half of the page. This will highlight the symbols
just to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select
"print".
ii. Click on the graph and this will bring the standard curve forward. Select
"print'.
Cleaning:
1. All samples and Diluent can be safely disposed of down the drain
2. All Reagents, Q.C.s, and Standards must be collected for pick-up by EH&S.
3. Empty SmartChem sample cups can be disposed of in the trash can.
4. SmartChem reagent bottles should be washed out 5x with deionized water and inverted
on rack/paper towel to dry.
5. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack
above sink next to SmartChem to dry.
Principle:
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-28
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

An intensely blue compound, indophenol, is formed by the reaction of ammonia with alkaline
phenol and then hypochlorite (Berthelot reaction), catalyzed by sodium nitroprusside. The
color (absorbance) measured at 630nm is proportional to the ammonia + ammonium
concentration.
This method measures both ammonia (NH3; unionized ammonia) and ammonium (NH4+;
ionized ammonia) in the sample and therefore results can be reported as total ammonia
nitrogen (TAN). Water temperature and pH will affect the ratio of these two forms of
ammonia in aquatic systems (see Emerson, et al.,1975, for a table
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-29
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
Nitrate + Nitrite (NOx) — Automated SmartChem Method
References:
SmartChem 200 Method 375-100E-1, revised July 2008. Westco Scientific
Instruments, Inc.
NOTE: Run Efficiency Test first, to check coil status.
Reagents:
1. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in S106.
2. REAGENT #1: Ammonium Chloride-EDTA Buffer Solution: Dissolve 85g of Ammonium
Chloride (NHaCI; CAS 12125-02-9) and 0.1g of Disodium Ethylenediamine Tetracetate
(C,oH,4N2Na2O8*2H2O; CAS 6381-92-6) in approximately 900mL of reagent water. Adjust
the pH to 8.5 with Concentrated Ammonium Hydroxide (NHaOH; CAS 1336-21-6), which is
kept under sink in Bases cabinet. Dilute to 1 Liter with reagent water. Store in 1,OOOmL
Pyrex bottle with screw cap, in Nitrate/Nitrite tray. STABLE, but check pH every few
months and adjust as necessary.
3. REAGENT #2: Color Reagent: In a 250mL volumetric flask with approximately 150mL of
reagent water, slowly add 25mL Concentrated Phosphoric Acid (H3PO4; CAS 7664-38-2).
Cool to room temperature. Add and dissolve 10.Og of sulfanilamide (4-NH2C6H4SO2NH2;
CAS 63-74-1). Swirl to mix. Add 0.5g of N-(1-napthyl) Ethylenediamine Dihydrochloride
(NNED) (C,oH7NHCH2CH2HN2*2HCL; CAS 1465-25-4) and dissolve. Slowly and at an
angle to reduce foaming add 2mL of concentrated Probe Rinse Solution (Westco part #
3AS-RN00-21) and slowly dilute to 250mL with reagent water. Invert and mix well. Store in
designated brown glass bottle and keep refrigerated. Good for one week.
4. Nitrate Module Reservoir buffer solution, 30%: In designated 1 L volumetric flask kept in
Nitrate/Nitrite tray dilute 300mL of the Ammonium Chloride-EDTA Buffer Solution (Reagent
#1) to 1 L with reagent water. (This solution is used to flush the reduction coil between
sample analyses and is not used in the reaction chemistry.) Reservoir container is kept
next to the SmartChem, stored in white plastic 2L bottle at room temperature w/ tube
connected to SmartChem. STABLE, but check pH every few months and adjust back to
8.5 as necessary.
Standards:
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Central Environmental Laboratory
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East Carolina University
100W Combined Standard: (KNO3 + KH2PO4 + NH4CI) Using a volumetric pipete, dilute
10mL of the 1,000pM Combined Standard to 100mL in designated volumetric flask. Good
for 1 week, blue taped flask kept in fridge.
2. 50W Combined Standard: Using a volumetric pipete, dilute 5mL of the 1,000pM Combined
Standard to 100mL in designated volumetric flask. Prepare fresh.
3. 1,000wM Sodium Nitrite: (NaNO2) Using a volumetric pipete, dilute 10mL of the 10,000pM
NaNO2 Standard to 100mL with reagent water in designated volumetric flask. Good for 1
month. Store in fridge.
4. 50wM Sodium Nitrite: (NaNO2) Using a volumetric pipete, dilute 5mL of the 1,000pM
NaNO2 Standard to 100mL with reagent water in designated flask. Prepare fresh.
Quality Control:
1. 35.7uM N: In designated volumetric flask dilute 0.10 mL (100uL) of the Astoria Pacific
((1,000 mg/L NH3-N (=71,393 uM N) kept in Nitrite cabinet) to 200 mL with reagent water.
Prepare fresh daily.
2. 3.57W N: In designated volumetric flask dilute 10mL of the 35.7pM N to 100mL. Prepare
fresh daily.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) Concentrate stored in S.C.
cabinet. In designated 1 L volumetric flask filled with -950mL of reagent water, slowly and tilted
to side add 0.5mL of Probe Rinse Solution (Westco). Fill to volume with reagent water. Mix
thoroughly. Stable. Store at room temperature behind SmartChem #1. Keep a constant supply
ready.
Cuvette Wash Solution: Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
Concentrate stored in S.C. cabinet. In designated 2L container add reagent water to mark. Add
30mL (one bottle) of Unity Scientific Cleaning Solution. Rinse 30mL bottle with reagent water
and pour into 2L container, repeat 2 more times. Fill to volume. Mix thoroughly. Store at room
temperature next to SmartChem #1. Stable.
Procedure:
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Central Environmental Laboratory
Department of Biology
East Carolina University
1. Pour or pipet approximately 3mL of sample into sample cup and discard. Pour or pipet
3mL of sample into the cup and place in SmartChem sample rack (racks 1-5)
Diluent: reagent water. Place in "Diluent 1" spot
Standard and Q.C.'s:
2. Place a sample cup with -3mL of the 100pM Combined Standard in SmartChem "RGT
2 Ctrl/Std" rack, position 1 (NO3-N).
3. "RGT 2 Ctrl/Std" rack, place QC #2 in position 2. (35.7pM)
4. "RGT 2 Ctrl/Std" rack, place Q.C. #1 in position 3. (3.57pM)
5. "RGT 2 Ctrl/Std" rack, place 50pM Nitrite in position 4. (EFF2)
6. "RGT 2 Ctrl/Std" rack, place 50pM Combined Std. in position 5. (EFF)
7. "RGT 2 Ctrl/Std" rack, place reagent water in position 6. (CCB)
8. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5.
9. These cups will be used to generate the standard dilutions for the standard curve.
Reagents:
10. In "RGT1" rack, place Reagent #1= Ammonium Chloride-EDTA buffer in the Q.C.
position.
11. "RGT1" rack, place Reagent #2= Color reagent, in position 11.
12. "RGT1" rack, place extra Reagent #1 bottles in positions 12 and 13.
13. Do Not Overfill Reagent bottles, fill just below shoulder and make sure there are no
bubbles.
14. Check fluid levels in cuvette wash solution, DIW, and probe rinse solution bottles, refill if
necessary.
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Central Environmental Laboratory
Department of Biology
East Carolina University
15. Collect waste in NOx jug, place in sink/tub.
Software Procedure:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select Profile List on middle of screen. Select "NO3-Nitrate Long Run" from the list of
methods at the bottom of the page.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the empty cups for standard dilutions.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "print" and selecting "print"
again.
Real time sample data can be displayed by selecting the Results button under the cuvette
wheel. Once all samples have been run, the print option will appear in the upper left menu
bar, clicking the button will automatically print out the results.
Cleaning:
1. All reagents, standards, QC's, and blanks must be collected for pick-up by EH&S. Collect
these into Nitrite Hazardous Waste bottles.
2. SmartChem samples can be dumped down drain and cups disposed of in the trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and
inverted on rack/paper towel to dry.
4. Wash SmartChem racks with tap water and scrub with brush, hang on drying rack above
sink next to SmartChem to dry.
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Central Environmental Laboratory
Department of Biology
East Carolina University
Coil activation/cleaning: (see SmartChem 200 method WCd-COIL, revised July 2008):
• Hydrochloric Acid, 2N: In a 500mL volumetric flask slowly add 83mL of
Concentrated Hydrochloric Acid (HCL; CAS 7647-01-0) to approximately 300mL of
reagent water. Cool to room temperature and dilute to 500mL with reagent water.
Stable. Transfer to glass container with stopper and store in Acid cabinet.
• Hydrochloric Acid, 6N: In a 100mL volumetric flask slowly add 50mL of
concentrated hydrochloric acid (HCL; CAS 7647-01-0) to approximately 30 mL of
reagent water. Cool to room temperature and dilute to100mL with reagent water.
Stable. Transfer to glass container with stopper and store in Acid cabinet.
Copper Sulfate solution, 1.5%: In a 500mL volumetric flask dissolve 6.5g of
Copper Sulfate Pentahydrate (CUSO4*5H2O; CAS 7758-99-8) in approximately
400mL of reagent water. Dilute to 500mL. Stable. Kept in glass container with
stopper in Nitrate Tray.
*Check that the NOx Reservoir Bottle is filled, the siphon tube in place, and that no air
bubbles are present in the delivery line.
*Collect waste in NOx coil waste jar.
1. On the main menu on the bottom right of screen press "Diagnostics".
2. Press the "NO3" tab.
STEM
• Check Box 2 (5N HCI) and enter 3 cycles.
• Check Box 3 (2% CuSO4) and enter 3 cycles.
• Press "Start".
STEP 2:
• Check Box 3 only and enter 6 cycles.
• Press "Start"
TEP 3:
• All boxes unchecked, Run "Prime" 5 times.
1. Press "Diagnostics" to exit.
2. Can run a second time if efficiency is still bad.
Principle:
This method determines the combined Nitrate (NO3) and Nitrite (NO2) present in the sample.
This sum is also known as Total Oxidized Nitrogen (TON) and is also referred to as NO,
Nitrate is reduced to Nitrite by passage of a filtered sample through an open tubular copperized
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Central Environmental Laboratory
Department of Biology
East Carolina University
cadmium redactor column. The resulting Nitrite plus any Nitrite originally present in the sample
is then determined as NO2 by diazotizing with Sulfanilamide followed by coupling with N-
(Napthyl)-Ethylenediamine Dihydrochloride to form a highly colored Azo Dye, which is
measured colorimetrically at 550 nm.
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Central Environmental Laboratory
Department of Biology
East Carolina University
Particulate Kjeldahl Phosphorus (PP, PKP) — Automated SmartChem Method
(Usually run as "PN,PP Method" (TKNM+OP4M))
References:
SmartChem 200 Method 420-200E, revised July 2008. Westco Scientific Instruments, Inc.
Digestion Reagent: (*Should already be made and in Kjeldahl cabinet)
,. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in S106.
2. Digestion Reagent (Mercury free): (AKA "BLUE JUICE") Can make one 2L or more.
Easier to weigh out both the Cupric Sulfate and the Potassium Sulfate first (x 3). Use
three 2L volumetric flasks found in Kjeldahl cabinet.
a. In the fume hood dissolve completely 9.5g Cupric Sulfate (anhydrous; CUSOa;
CAS 7758-98-7) in each of the 2L volumetric flasks. Using a full squirt bottle,
rinse weigh boat and funnel, then add the remainder of the bottle to the flask
(approx. 500 mL). Swirl to completely dissolve.
b. Add 268g of Potassium Sulfate, (K2SO4; CAS 7778-80-5) which will only partially
dissolve. Repeat the squirt bottle step. Swirl to mix.
c. Set up an ice bath in the fume hood using a white tray half full of ice.
d. Measure out 268mL concentrated Sulfuric Acid using both a 250mL, and a
25mL graduated cylinder marked at 18mL.
e. Place volumetric flask on ice, and slowly add the concentrated Sulfuric Acid
(H2SO4; CAS7664-93-9) to the volumetric flask 5-10mL at a time while
continuously swirling — Will get very, very hot! There is a fine line between too
hot and too cold, you want to keep it warm enough to get the chemicals to
dissolve and not so cool that the chemicals will come out of solution. Continue
adding the acid, swirling and mixing. Can use some reagent water if gets too hot.
Once everything is dissolved take it out of the ice bath and allow to cool to room
temperature. Using dei water that is at room temperature slowly bring up to
volume while mixing. The volume can fluctuate so be careful not to add too
quickly and make sure that the temp of reagent water is the same as what you
are adding to. If making more than 1 batch at a time, mix batches together and
aliquot among the four designated 4 Liter bottles in Kjeldahl cabinet. Mix
thoroughly. STABLE.
Standards:
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Central Environmental Laboratory
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East Carolina University
• 500wM N/200W P (Combined medium range standard): In designated 200mL
volumetric flask, using volumetric pipettes, dilute 10mL of the 10,000pM Ammonium
Chloride (NHaCL) Standard, kept in fridge, and 4mL of the 10,000pM Potassium
Phosphate (KH2PO4)to 200mL with reagent water. Good for 1 week.
Quality Control (QC's):
1. 89.22uM N/26.3uM P: In designated volumetric flask, using the 1000pL pipet, dilute
0.250mL of the Certified SPEX Certi Prep Ammonia Nitrogen 1,000pg/mL
(=71,327pM N) (CAT# AS-NH3N9-2Y) and 0.5mL (500pL) of the Certified SPEX Certi
Prep 1,000pg/mL Phosphate (=10,525pM P)to 200mL with reagent water. Mix
thoroughly. Prepare fresh daily.
2. 178W N/52.6uM P: In designated volumetric flask, using the 1000NL pipete, dilute
0.5mL of the Certified SPEX Certi Prep Ammonia Nitrogen 1,000pg/mL (=71,327pM)
and 1 mL of the Certified SPEX Certi Prep 1,000pg/mL Phosphate (=10,525pM P) to
200mL with reagent water. Mix thoroughly. Prepare fresh daily.
3. 356W N/105uM P: In designated volumetric flask, using the 1000NL pipete, dilute
1mL of the Certified SPEX Certi Prep Ammonia Nitrogen 1,000pg/mL (=71,327pM)
and 2mL of the Certified SPEX Certi Prep 1,000pg/mL Phosphate (=10,525pM P) with
reagent water to 200mL. Mix thoroughly. Prepare fresh daily.
Procedure: (For Digestion Block)
NOTE: Tubes are kept on wooden shelf, should already be in order with boiling chips
added and covered w/ saran wrap. Racks and Digestion Block hold 40 tubes.
Use Kjeldahl tubes that were baked in drying oven (1051C) for at least 4 hours, usually
overnight. Add -r11 boiling chips (Henger granules part # 136-CC sieved through #16
Mesh) to each Kjeldahl tube.
1. Prepare lab sheet with tube numbers and sample ID's.
2. Unwrap filters from foil one at a time, tearing each into 3 or 4 pieces on the foil with
forceps and placing as far into the kjeldahl tube as possible, before moving on to the next
sample filter. When all sample filters are in the kjeldahl tubes use a plastic rod to push the
filter pieces to the bottom of the tube such that the 10ml of digestion reagent will cover
the filter.
3. With a freshly rinsed (x15) graduated cylinder measure 25mL of reagent water for blanks,
add to tubes (x2).
4. Measure 25mL of each Q.C. (89.22uM N/26.3uM P, 178W N/52.6uM P, 356W
N/105uM P), in order from low to high, and add to tubes.
5. Measure 25mL of the 500W N/200W P Standard and add to tube.
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East Carolina University
6. Add 10mL of Kjeldahl Digestion Reagent to all sample/blank/standard/QC tubes using
designated repeat pipettor. Be careful not to spill/splash digestion reagent, use goggles.
7. Cap all tubes with green stoppers.
8. Wipe each tube with damp towel and dry completely. Place tubes in digestion rack as
each row is wiped. Make certain that a green tray is under the rack and a "Work in
Progress" (W.I.P.) card is attached. Also record what samples they are. Ex: CZR4Mar17
(#1-40) on bottom of card. NOTE: Can let sit overnight to digest next morning.
9. Take green stoppers out of tubes and place in tan tray in order, to replace in order.
10. Put on two side panels.
11. Lift up over head to check bottom of tubes for liquid, cracks, and boiling chips.
12. Place the rack into the digestion block very carefully making sure that all tubes go into the
block simultaneously. Close sash on hood.
Set Digestion Control Box:
1. Turn ON, switch on right side of box. Make sure display reads "J1" when first turned on
and then the current temperature is displayed.
2. To Program: (On upper part of the display with ramp information)
• Press TEMP key, Enter 210 and press ENTER;
• Press TIME key, Enter 1.8 and press ENTER;
• Press TEMP key, Enter 385 and press ENTER;
• Press TIME key, Enter 1.5 and press ENTER.
• Press START/STOP kev.
3. Make sure all 6 lights turn on. If not, turn controller off and start over.
4. NOTE: Set timer for 28 minutes. After timer goes off put on orange heat resistant gloves
(temp. should be-160°C) and watch for liquid bubbling to the top of the tubes. If
bubbling does occur CAREFULLY lift digestion rack -1 inch off the bottom of the
digestion block (enough for bubbling to cease reaching the top of the tube). May have to
keep lifting rack until boiling calms down. DO NOT ALLOW THE TUBES TO BOIL OVER
AS THIS WILL LIKELY CAUSE THE TUBES TO EXPLODE!
5. The controller will beep twice when the digestion has completed (about 3.5 hours total).
Wearing orange heat resistant gloves, CAREFULLY remove the two side panels and lift
the digestion rack with tubes and place on the metal tray inside the fume hood. Be
careful the tubes do not hit anything in the hood.
6. Set a timer for 9 minutes. During this time, fill the designated brown square repeat
pipeter (kept in Kjeldahl cabinet) with fresh reagent water and prime. Check to see if it is
set to 25mL.
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Department of Biology
East Carolina University
7. When timer goes off, pipet 25mL of reagent water into each of the
sample/standard/blank/QC tubes. NOTE: pipeter will only reach 3 tubes deep, so the
rack needs to be rotated to reach the other '/2 of the tubes. There will be tubes in two
middle rows that you cannot access.
8. Remove rack from fume hood and place on bench top. Remove the tubes water was not
added to and put in blue rack. Add water and return to digestion rack. Cap each tube with
a rubber stopper.
9. Using the vortex genies, mix each tube about 30 seconds each. Return tubes, in order,
to the blue rack inside the green tray.
10. Samples need to be mixed 3x with an hour between mixes before they can sit overnight
and be read the next morning.
11. If a second digestion is planned for the same day, turn OFF controller. Turn back ON.
Allow the digestion block to cool to 160°C., about 2-3 hours with fan blowing into hood. If
tubes have been wiped down check them and place on block, timer to be set to 10
minutes because block is already hot. After timer goes off check temp and watch for
bubbling until sure they are calm and continue through steps.
Reagents For SmartChem:
*FOR PN, should only have to make Reagent #3 and #4 fresh)
STOCK Buffer Solution: (Should already be made and stored in a 1L Pyrex Bottle with
screw cap in DKN/TKN tray.)
OR if not already made:
In 1 L volumetric flask dissolve 134g of Sodium phosphate, Dibasic Heptahydrate
(Na2HPO4.7H2O; CAS 7782-85-6) in approximately 250 mL of reagent water. Add and
dissolve 20g of Sodium Hydroxide, cool and then dilute to 1 L. Mix thoroughly. STABLE.
Store in designated IL Pyrex bottle with screw cap in DKN/TKN tray.
2. REAGENT #1: WORKING Buffer Solution: (Should already be made and stored in a
1,000mL Pyrex Bottle with screw cap in DKN/TKN tray.)
OR if not already made:
In a 1 Liter volumetric flask, combine the reagents in the following stated order:
• 200mL of Stock Buffer Solution
• 250mL of the 20% Sodium Potassium Tartrate solution
• Mix by swirling.
• Add 120mL of the 20% Sodium Hydroxide solution
• Mix by swirling.
• Dilute to 1 Liter with reagent water
• Mix thoroughly.
NOTE: THIS IS A MEDIUM RANGE BUFFER (0-5mg/L; 0-357pM
3. Sodium Hydroxide, 20%: (Only needed to make Working Buffer Solution) (Should
already be made and stored in 1,000mL Pyrex bottle with screw cap in Bases cabinet.)
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East Carolina University
OR if not already made:
Under hood in a 1 Liter volumetric flask filled to approximately 800mL of reagent water,
slowly add 200g of Sodium Hydroxide (CAS 1310-73-2). Excessive heat will be
generated. Mix well to dissolve. Cool to room temperature and dilute to 1 Liter with
reagent water. Mix thoroughly. STABLE. Stored in 1,000mL Pyrex bottle with screw cap
in Bases cabinet.
4. Sodium Potassium Tartrate, 20%: (Only needed to make Working Buffer Solution) In
designated 250mL volumetric flask (kept in TKN/DKN tray) filled to approximately 200mL
of reagent water, slowly add and dissolve 50g Sodium Potassium Tartrate Tetrahydrate
(KOCO(CHOH)2COONa.4H2O; CAS 6381-59-5). Solution will become cold. Bring to room
temperature and dilute to 250mL with reagent water. Mix thoroughly. *Do not want any
NH4 in chemical.
5. REAGENT #2: (Sodium Salicylate): (Should already be made and stored in designated 250mL
volumetric flask in TKN/DKN tray.)
OR if not already made:
In designated 250mL volumetric flask, kept in TKN/DKN tray, dissolve 5g of Sodium
Salicylate (2-HOC6H4CONa; CAS 54-21-7) in approximately 225mL reagent water. Add
(very slowly and at an angle to keep from foaming) 1.5mL of concentrated Probe Rinse
Solution (Westco part # 3AS-RN00-21) and dilute to 250mL with reagent water. Mix
thoroughly. Store solution in same volumetric flask covered with foil, inside TKN tray.
Prepare fresh every 2 weeks.
*REAGENT #3: (Sodium Hypochlorite Solution - Clorox): Pour about 10mL of the diluted
5.25% Clorox into a 50mL beaker. In designated 50mL volumetric flask, add 3mL of the
diluted 5.25% Clorox, NaOCI) using a glass volumetric pipet. Add approximately 45mL of
reagent water. Add (slowly and at an angle to keep from foaming) 0.5mL concentrated
probe rinse solution. Slowly bring to volume with reagent water. Mix thoroughly. Prepare
fresh daily.
6. *REAGENT #4: (Sodium Nitroferricyanide Solution): In designated 50mL volumetric flask,
dissolve 0.4g Sodium Nitroferricyanide(Nitroprusside) Dihydrate (Na2Fe(CN)5NO.2H2O;
CAS 13755-38-9) in approximately 40mL of reagent water and dilute to 50mL with
reagent water. Cover flask with aluminum foil and let dissolve. Mix well. Add 0.5mL of
concentrated probe rinse solution. Mix thoroughly. Prepare fresh every 2 days, refrigerate
overnight.
*FOR PP
Sodium Dodecyl Sulfate (SDS), 17.7%: Use only purest grade, with Phosphate
concentrations <_0.0001% Phosphate. In designated bottle, using a 100mL graduated
cylinder and a disposable pipette dissolve 15g SDS (CH3(CH2)11OS03Na; CAS #151-21-
3) in 85mL of reagent water very carefully (Use antistatic brush and wear MASK).
Requires mixing on stir plate with stir bar to dissolve completely. Good for two weeks.
Kept in PP/TKP tray.
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Central Environmental Laboratory
Department of Biology
East Carolina University
2. REAGENT #1: (Salt solution): (Should already be made and stored in designated glass
bottle in PP/TKP tray.)
a. In designated 500ml- volumetric flask, dissolve 2.65g of Sodium Chloride (NaCI;
CAS 7647-14-5) in 450ml- of reagent water. Very slowly and at an angle add
15mL of 17.7% SIDS and slowly dilute to 500ml- with reagent water. Mix
thoroughly. Stable. Store in same flask at room temperature in PP/TKP Tray.
3. REAGENT #2: (Molybdate/Antimony Solution): (Should already be made and stored in
designated glass bottle in PP/TKP tray.)
a. In designated 250ml- volumetric flask dissolve 2g of Ammonium Molybdate
Tetrahydrate ((NH4)6Mo7O24*2H2O; CAS 12054-85-2) in about 200ml- of reagent
water. Add 0.05g of Antimony Potassium Tartrate (K(SbO)C4H4O6*1/2H2O; CAS
28300-74-5). Swirl to mix. Slowly and at an angle add 5mL of 17.7% SIDS and
dilute to 250mL with reagent water. Mix thoroughly. Cover with foil. Store in
same 250mL flask at room temperature in PP/TKP Tray. Make every two weeks.
4. REAGENT #3: (Ascorbic Acid Solution): In designated 50mL volumetric flask dissolve 3g
of Ascorbic Acid (C6H8O6; CAS 50-81-7) with reagent water. Prepare fresh daily.
NOTE: Wrap all small plastic SmartChem reagent bottles containing light sensitive
reagents with foil before putting in SmartChem.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1 L volumetric
flask filled with —950ml- of reagent water, slowly and tilted to side add 0.5mL of Probe
Rinse Solution (Westco). Fill to volume with reagent water. Mix thoroughly. Stable. Store
at room temperature behind SmartChem #1. Keep a constant supply ready.
Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
In designated 2L container add reagent water to mark. Add 30mL (one bottle) of Unity
Scientific Cleaning Solution. Rinse 30mL bottle with reagent water and pour into 2L
container, repeat 2 more times. Fill to volume. Mix thoroughly. Stable. Store at room
temperature next to SmartChem #1.
Procedure- SmartChem Set Up:
(Set up the Reagents, empty cups, Std, QC's and Diluent, etc. before samples, when the
first rack of samples are poured the SmartChem can be started and the rest of the
samples can be added.)
1. Samples: Using a 5mL automatic pipet, carefully pipette sample from digestion tube
being careful not to disturb bottom of tube. Use a clean pipette tip for each sample.
Rinse a SmartChem sample cup with a small amount of sample and discard into waste
container. Fill cups in duplicate and place in SmartChem sample rack (racks 1-5).
NOTE: All Kjeldahl waste has to be collected and disposed of via EH&S
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Central Environmental Laboratory
Department of Biology
East Carolina University
2. Diluent: Using a 5mL automatic pipet fill the designated small plastic SmartChem bottle
with both blanks, alternating back and forth between the two tubes of Blank. Usually
about 3 times from each tube. Mix. Place "Diluent 1" slot.
3. Q.C's: Using a 5mL automatic pipet fill one cup with each of the Q.C.'s. These will be
placed in descending order in cups 2-4 on "Reag 2 Ctrl/Std" rack.
4. Stds/Spike: Using a 5mL automatic pipet fill two cups with the 500pM Standard (one is for
the Spike). This will be placed in cup 1 of the" Reag 2 Ctrl/Std" rack. The Spike will be
placed in 51" slot of "Rgt 1 rack".
5. Place 6 empty sample cups in positions 1-6 in SmartChem Sample rack 5. These cups
will be used to generate the standards for the standard curve.
6. Reagents: (All reagents are placed in "Reag 2 Ctrl/Std" rack) Do not overfill bottles, pop
any bubbles.
• Position 1 - Reagent #1 - DKN Working Buffer (TKBU)
• Position 2 - Reagent #2 - Sodium Salicylate (TKSA)
• Position 3 - Reagent #3 - Clorox (TKHY)
• Position 4 - Reagent #4 - Sodium Nitroprusside (TKNI)
NOTE: Fill each small plastic SmartChem reagent bottle only just before shoulder of
bottle. If they are too full or there is a bubble present the SmartChem will register it as
"empty".
7. Check fluid levels in Cuvette Wash Solution, DEI, and Probe Rinse Solution resevoir
bottles, refill if necessary.
Software Procedure:
1. Turn ON computer turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select Profile List on middle of screen. Select "PN/PP-TKNM+OP4M" from the list of
methods at the bottom of the page. This screen will show where the Std/Spike, Q. C.'s,
Reagents, Diluent and empty cups are to be placed.
Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the empty cups for standard dilutions.
Printing Data:
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-42
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "Print" and selecting
"Print" again.
2. Real time sample data can be displayed by selecting the "Results" button under the
cuvette wheel. Once all samples have been run, the "Print option" will appear in the
upper left menu bar, clicking the button will automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a. Select "options" from left menu bar, then "data retrieval".
b. Find and select the plan of interest. A split screen will appear — click on the
method in the box in the lower half of the page. This will highlight the symbols just
to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select
"print".
ii. Click on the graph and this will bring the standard curve forward. Select
"print".
Cleaning:
1. All samples, reagents, standards, QC's, and blanks must be collected into designated
hazardous waste bottles.
2. SmartChem sample cups can be disposed of in the trash can
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and
inverted on rack/paper towel to dry.
4. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack
above sink next to SmartChem to dry.
Principle:
The method is based on the conversion of all forms of phosphorus in the digested sample to
orthophosphorus. Ammonium molybdate and antimony potassium tartrate react in an acid
medium with dilute solutions of phosphorus to form an antimony-phospho-molybdate
complex. This complex is reduced to an intensely blue colored complex by ascorbic acid.
The color (absorbance) measured at 660nm is proportional to the phosphorus concentration
in the original sample.
Total Kjeldahl nitrogen is the sum of free ammonia nitrogen and organic nitrogen compounds
which, through digestion, are converted to ammonium sulfate. The method is based on the
conversion of the digested ammonium sulfate to ammonia. In the presence of sulfuric acid,
potassium sulfate, and a cupric sulfate catalyst, amino nitrogen of organic materials is
converted to ammonium. The ammonium cation is then converted to ammonia by
neutralization with a concentrated buffer. Once converted to ammonia, an intensely blue
compound, indophenol, is formed by the reaction of ammonia with alkaline phenol and then
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-43
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
hypochlorite (Berthelot reaction), catalyzed by sodium nitroprusside. The color (absorbance)
measured at 660nm is proportional to the Kjeldahl nitrogen concentration in the original
sample.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-44
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
Particulate Kjeldahl Nitrogen (PKN) — Automated SmartChem Method
References:
SmartChem 200 Method 390-200E, revised July 2008. Westco Scientific Instruments, Inc.
Standard Methods 4500-Norg. Nitrogen (Organic) (20th Edition). American Public Heath
Association, American Water Works Association, Water Environment Federation. pp. 4-
123/128.
Reagents and Standards:
1. Reagent water: Fresh deionized water from E-pure system in S106
2. Digestion reagent (mercury free): in a 2L volumetric flask, dissolve completely 9.5 g
cupric sulfate (anhydrous; CUSO4; CAS 7758-98-7) in 1600 mL of reagent water. Add
268 g of potassium sulfate (K2SO4; CAS 7778-80-5), only partially dissolves. In the fume
hood, set up an ice bath, place volumetric flask on ice, and slowly add 268 mL (5-10 mL
at a time) of concentrated sulfuric acid (H2SO4; CAS7664-93-9) while mixing — will get
very, very hot!. Allow to cool to room temperature, mix. Allow to sit overnight. In the
morning, adjust volume to 2 liters as needed. If making more than 1 batch at a time, mix
batches together and aliquot among the designated 4 liter bottles in Kjeldahl cabinet.
2. Sodium Hydroxide, 20%: In a 1 Liter volumetric flask, slowly add 200 grams of sodium
hydroxide (NaOH; CAS 1310-73-2) to approximately 800 mL of reagent water. Excessive
heat will be generated. Mix well to dissolve. Cool to room temperature and dilute to 1
Liter with reagent water. Stable.
3. Sodium Potassium Tartrate, 20%: In a 250 mL volumetric flask, dissolve 50 g sodium
potassium tartrate tetrahydrate (KOCO(CHOH)2COONa.4H2O; CAS 6381-59-5) in
approximately 200 mL of reagent water. Dilute to 250 mL with reagent water. Solution
will become cold. Do not want any NH4 in chemical.
4. Stock Buffer Solution: In a 1 Liter volumetric flask, dissolve 134 g of Sodium phosphate,
dibasic heptahydrate (Na2HPO4.7H2O; CAS 7782-85-6) in approximately 800 mL of
reagent water. Add and dissolve 20 g of Sodium Hydroxide, cool and then dilute to 1
Liter.
NOTE: if anhydrous sodium phosphate, dibasic (CAS 7558-79-4) is used to
prepare the stock buffer solution, dissolve 71 g of anhydrous Na2HPO4 in
approximately 800 mL of reagent water. Add 20 g sodium hydroxide, cool, and
then dilute to 1 Liter.
5. REAGENT 1: Working Stock buffer solution: In a 1 Liter volumetric flask, combine the
reagents in the following stated order:
a. 200 mL of Stock Buffer Solution
b. 250 mL of the 20% Sodium Potassium Tartrate solution
c. Mix
d. Add 120 mL of the 20% Sodium Hydroxide solution
e. Dilute to 1 Liter with reagent water
THIS IS A MEDIUM RANGE (0-5 mg/L; 0-357 uM N) BUFFER
6. REAGENT 2: Sodium Salicylate: In a 250 mL volumetric flask, dissolve 5 g of Sodium
Salicylate (2-HOC6H4CONa; CAS 54-21-7) in 200 mL reagent water. Add 1.5 mL of
concentrated probe rinse solution (Westco part # 3AS-RN00-21) and dilute to 250 mL
with reagent water. Invert to mix 5 times. Transfer and store solution in a dark glass
bottle. Store in TKN tray. Prepare fresh every 2 weeks. Prepare 1 day ahead of use.
a. NOTE: wrap the SmartChem reagent bottle containing this reagent with foil when
used in the analysis
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
7. REAGENT 3: Sodium Hypochlorite Solution (Clorox): In a 100 mL volumetric flask, add 6
mL of Clorox (6% NaOCI) add 1 mL of concentrated probe rinse solution. Bring to
volume with reagent water. Invert to mix 5 times. Prepare fresh daily.
8. REAGENT 4: Sodium Nitroprusside Solution: In a 50 mL volumetric flask, dissolve 0.4 g
sodium nitroprusside dehydrate (Na2Fe(CN)5NO.2H2O; CAS 13755-38-9) in
approximately 40 mL of reagent water and dilute to 50 mL with reagent water. Add 0.5
mL of concentrated probe rinse solution. Invert to mix 5 times. Cover flask with
aluminum foil. Prepare fresh every 2 days, refrigerate overnight.
a. NOTE: wrap the SmartChem reagent bottle containing this reagent with foil when
used in the analysis
9. STANDARDS:
a. 10,000 uM Ammonium Chloride (NH4CI; CAS 12125-02-9): weigh out 0.26745g of
NH4CI and dilute to 500 mL with reagent water. Good for 6 months.
b. 10,000 uM Potassium Phosphate (KH2PO4; CAS 7778-77-0): weigh out 0.68045g
of KH2PO4 and dilute to 500 mL with reagent water. Good for 6 months.
c. Combined medium range standard (500 uM N/200 uM P): using volumetric
pipettes, dilute 10.0 mL of the 10,000 uM NH4CL standard and 4.0 mL of the
10,000 uM KH2PO4 to 200 mL with reagent water. Good for 1 week.
10. QUALITY CONTROL:
a. 89.22 uM N/16.13 uM P: dilute 0.250 mL (250uL) of the Certified Certi Prep
10,000 mg/L NH4CI (=71,377 uM N) and 0.1 mL (100uL) of the Certified Certi
Prep 10,000 mg/L KH2PO4 (=32,258 uM P) to 200 mL with reagent water.
Prepare fresh daily.
11. Probe Rinse Solution: Add 0.5 mL of probe rinse solution (Westco) to -950 mL of
reagent water in a 1 L volumetric flask. Add reagent water to mark. Invert, gently, five
times to mix. Store at room temperature. Stable.
12. Cuvette Wash Solution: Add 50 mL of cuvette cleaning solution (Westco) to
approximately 800 mL of reagent water in a 1 L volumetric flask. Add reagent water to
mark. Invert, gently, five times to mix. Store at room temperature. Stable.
Procedure:
9. Use Kjeldahl tubes that were baked in drying oven (1050C) for at least 4 hours.
10. Add 8-10 boiling chips to each Kjeldahl tube (Henger granules part # 136-CC)
11. Tear sample filter into quarters, being careful not to touch sample, and place in bottom of
digestion tube. Include at least 2 ashed but unused filters as filter blanks.
12. Measure 25 mL of the 500 uM N/200 uM P combined standard (x2), 25 mL of the 89.22
uM N/16.13 uM P QC (x2), and 25 mL of reagent water for reagent blanks (x2).
13. Add 10 mL of digestion reagent to all sample/blank/standard/QC tubes using designated
repeat pipettor.
14. Place tubes in digestion rack and lift up to check bottom of tubes for liquid, cracks, and
boiling chips.
15. Place, carefully, the rack into the digestion block, put on two side panels.
16. Set digestion control box. Turn on switch on right side of box. Make sure display reads
"J1" when first turned on. Press temp key, enter 210, press enter;
Press time key, enter 1.5, press enter;
Press temp key, enter 385, press enter;
Press time key, enter 1.5, press enter.
Press start/stop key.
Make sure all 6 lights turn on. If not, turn controller off and start over.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-46
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
NOTE: keep careful watch 30-40 minutes (160°C+) into digestion for bubbling to the top of
the tubes. If bubbling does occur, put on autoclave gloves, and CAREFULLY lift digestion
rack -1 inch off the bottom of the digestion block (enough for bubbling to cease reaching the
top of the tube). Replace rack and add a few more boiling chips to the sample. DO NOT
ALLOW THE TUBES TO BOIL OVER AS THIS WILL LIKELY CAUSE THE TUBES TO
EXPLODE!
17. The controller will beep twice when the digestion has completed (about 3 hours total).
Wearing autoclave gloves, CAREFULLY lift the digestion rack with tubes out and place
on the metal tray inside the fume hood. Remove the two side panels.
18. Set a timer for 9 minutes. During this time, fill the designated repeat pipettor with fresh
reagent water.
19. When timer goes off, pipet 25 mL of reagent water into each of the
sample/standard/blank/QC tubes. NOTE: pipettor will only reach 4 tubes deep, so the
rack needs to be rotated to reach the other '/z of the tubes.
20. Remove rack from fume hood and place on bench top. Cap each tube with a rubber
stopper.
21. Using the vortex genies, mix each tube about 30 seconds each. Return tubes, in order,
to a regular tube rack. Make sure each rack has a note with sample date, digestion date,
and number of mixes (needs to be done 3 times). Allow samples to sit at least 12 hours
before running samples.
22. If a second digestion is planned for the same day, allow the digestion block to cool to at
least 1800C before placing second set of samples on the block. The box fan can be used
to speed up cooling.
23. Using a 5 mL pipet, carefully pipette sample from digestion tube, being careful not to
disturb bottom of tube.
24. Rinse a SmartChem sample cup with a small amount of sample and discard into waste
container. Repeat this process 3 times, if possible. Fill cups in duplicate and place in
SmartChem sample rack (racks 1-5)
NOTE: All Kjeldahl waste has to be collected and disposed of vis EH&S. Place waste
into designated waste containers.
25. Cups need to be pipetted for standards, blanks, and QC's as well. The first and last five
sample cups in the run should consist of: 1) water 2) reagent blank 3) filter blank 1, 4)
filter blank 2, 5) QC. Mix contents of multiple standard/QC/Blank tubes before pouring
into into sample cups in the same manner as done with samples.
26. Diluent: digested blank (reagent)
27. Standard: place a sample cup with -3mL of the combined digested 500 uM N in
SmartChem rack RGT2, position 1. Place 7 empty sample cups in positions 1-7 in
SmartChem sample rack 5. These cups will be used to generate the standards for the
standard curve.
28. Reagents: in SmartChem rack RGT1, place #1= Working Stock Buffer (TKBU), #2=
Sodium Salicylate (TKSA), #3= Clorox (TKHY), #4= Sodium Nitroprusside (TKNI), #5=
500 uM N Std, used as a spike. The volume required can be found on the printout from
SmartChem.
29. Check fluid levels in cuvette wash solution, DIW, and probe rinse solution bottles, refill if
necessary.
Software Procedure:
1. Turn on computer; turn on SmartChem. Beeping noise indicates that the two are
communicating with one another.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-47
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
2. Open SmartChem software. Allow bulb to warm up for at least 20 minutes if system was
off.
3. Go to SAMPLE ENTRY, located on left menu bar.
4. Select "TKNM med range" from the list of methods at the bottom of the page
5. Enter the total number of samples in the open box at top of page (include the DIW's,
Blanks and QC's). Click on the green check button at top of page. This will create a
spreadsheet.
6. Enter the sample ID's into the spreadsheet on the right side of the screen. *SmartChem
will not allow two samples to have the same ID*
7. Click on the save button (red floppy disk, upper left)
8. Follow through the plan description process: ie., title your sample run, print out the report
(this will tell you where everything goes as well as the volumes required for each of your
reagents).
9. Go to SYSTEM MONITOR, located on left menu bar.
10. Select your saved plan. The SmartChem configuration of samples/reagents/etc. will be
displayed on the screen.
11. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the empty cup for standard dilutions.
12. Click on the green arrow (run) key. Always run a water blank (WBL) between runs. If
switching between two methods/chemistries, wash cuvettes in addition to the WBL.
13. Keep an eye out during the run. If a reagent runs low, a box will appear on the screen
giving the option of pausing the system to refill. If either the probe rinse, DIW, or cleaning
solution run low, an audible beep will accompany the box on the screen (this can be
turned off by pressing the blinking red button on the front of the SmartChem).
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "print" on upper left side
of screen.
2. Real time sample data can be displayed by selecting the "???" button. Once all samples
have been run, the print option will appear in the upper left menu bar, clicking the button
will automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a) Select "options" from left menu bar, then "data retrieval".
b) Find and select the plan of interest. A split screen will appear — click on the
TKNW in the box in the lower half of the page. This will highlight the symbols
just to the right.
iii. Click on the erlynmeyer flask and this will bring the data forward. Select
"print".
iv. Click on the graph and this will bring the standard curve forward. Select
"print".
Cleaning:
1. All samples, standards, QC's, and blanks must be can be collected for pick-up by EH&S.
Collect these into designated hazardous waste bottle.
2. SmartChem sample cups can be disposed of in the trash can
3. All reagents can be safely disposed of down the drain. Remember to record the number
of samples and type of analysis on drain log.
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
4. SmartChem reagent bottles should be washed out 5x with deionized water and inverted
on rack/paper towel to dry.
5. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack
above sink next to SmartChem to dry.
Principle:
Kjeldahl nitrogen is the sum of free ammonia nitrogen and organic nitrogen compounds
which, through digestion, are converted to ammonium sulfate. The method is based on the
conversion of the digested ammonium sulfate to ammonia. In the presence of sulfuric acid,
potassium sulfate, and a cupric sulfate catalyst, amino nitrogen of organic materials is
converted to ammonium. The ammonium cation is then converted to ammonia by
neutralization with a concentrated buffer. Once converted to ammonia, an intensely blue
compound, indophenol, is formed by the reaction of ammonia with alkaline phenol and then
hypochlorite (Berthelot reaction), catalyzed by sodium nitroprusside. The color (absorbance)
measured at 660nm is proportional to the Kjeldahl nitrogen concentration in the original
sample.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-49
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
Dissolved Orthophosphate (PO4) — Automated SmartChem Method
References:
SmartChem 200 Method 410-20013, revised July 2008. Westco Scientific Instruments, Inc.
Standard Methods for the Examination of Water and Wastewater. 4500-P-E (18th and 19th
Editions). American Public Heath Association, American Water Works Association,
Water Environment Federation.
EPA. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020.
Method 365.3. Environmental Monitoring and Support Laboratory, Cincinnati, OH.
Reagents:
1. Reagent water: Fresh deionized water from E-pure system in S106 (16-18 megohm/cm).
2. REAGENT #1: Reagent Water.
3. REAGENT #2: (Color Reagent): See Below........
In designated brown bottle mix 50mL of 5N Sulfuric Acid (H2SO4) and 15mL of the
Ammonium Molybdate Solution. Swirl to mix. Add 5mL of the APT Solution (Antimony
Potassium Tartrate). Swirl to mix. Add 10mL of 17.7% SDS. Swirl to mix. Add 20mL
reagent water. Invert gently five times to mix. Store in fridge. Prepare fresh weekly.
To Make Reagent #2 you will need:
TWO IN FRIDGE - REAGENTS MUST BE BROUGHT TO ROOM TEMPERATURE:
a. Antimony Potassium Tartrate Solution: (Should be made and in fridge) Weigh out
1.3715g Antimony Potassium Tartrate (K(SbO)C4H406*1/2H2O; CAS 28300-74-5)
and dilute to 500mL with reagent water. Stable for 6 months. Store in designated
brown bottle in fridge.
b. Ammonium Molybdate Solution: (Should be made and in fridge) Dissolve 20g of
Ammonium Molybdate ((NH4)6Mo7024*4H2O; CAS 12054-85-2) to 500mL with
reagent water. Stable for 6 months. Store in designated red bottle in fridge. If
precipitate forms, filter solution and scrub out bottle with reagent water and brush.
c. Sodium Dodecyl Sulfate (SDS) 17.7%: (Should be made and in Phosphate tray)
Use only purest grade, with Phosphate concentrations :50.0001 % Phosphate. In
designated bottle, using a 100mL graduated cylinder and a disposable pipette
dissolve 15g SDS which is kept in Flammables cabinet (CH3(CH2)11OS03Na; CAS
#151-21-3) in 85mL of reagent water. Very carefully (Use antistatic brush and
wear MASK. Requires mixing on stir plate with stir bar to dissolve completely.
Good for two weeks. Kept in Phosphate tray.
d. Sulfuric Acid, 5N: (Should be made and in Acid cabinet) In the fume hood, in a
500mL volumetric flask slowly add 70mL of concentrated sulfuric acid (H2SO4;
CAS 7664-93-9) to approximately 400mL of reagent water. Cool to room
temperature and dilute to 500mL with reagent water. Stable. Kept in Acid cabinet
in designated glass bottle.
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
4. REAGENT #3: (Ascorbic Acid, 0.1 M): In designated volumetric flask dissolve 0.88g of
Ascorbic Acid (C61-18O6; CAS 50-81-7) in 50mL of reagent water. Add 0.5mL of 17.7%
SDS. Invert, gently, five times to mix. Prepare fresh daily.
Standard/Spike:
a. 40PM KH2PO4: In designated volumetric flask, dilute 4mL of 1,000pM Combined
Standard to 100mL with reagent water. Make fresh.
Quality Controls (QC's):
b. Q.C. #1: (5.26uM KH2PO4): In designated volumetric flask dilute 0.1mL (100uL) of
the SPEX Certi Prep 1,000pg/mL Phosphate = 10,525NM) to 200mL with reagent
water. Prepare fresh daily.
c. Q.C. #2: (15.79uM KH2PO4): In designated volumetric flask dilute 0.3mL (300uL)
of the SPEX Certi Prep 1,000pg/mL Phosphate = 10,525pM) to 200mL with
reagent water. Prepare fresh daily.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1 L volumetric
flask add 0.5mL of Unity Scientific Probe Rinse solution to -r950mL of reagent water. Add
reagent water to volume. Mix thoroughly. Stable. Store at room temperature next to
SmartChem #1. Keep constant supply.
Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900) In designated 2L container
add reagent water to mark. Add 30mL (one bottle) of Unity Scientific Cleaning Solution.
Rinse 30mL bottle with reagent water and pour into 2L container, repeat 2 more times.
Fill to volume. Mix thoroughly. Stable. Store at room temperature next to SmartChem #1.
Procedure: (While samples are being poured, wash and WBL can be done.)
1. Pour or pipet approximately 3mL of sample into sample cup and discard. Pour or pipet
3mL of sample into the cups and place in SmartChem sample rack (racks 1-5). Usually
run duplicates.
2. In "Reag2 Ctr/Std" Rack, place the 40pM Std, in slot 1, QC 2 in slot 2, QC 1 in slot 3.
3. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5. These cups
will be used to generate the standards for the standard curve.
4. Diluent: reagent water. Place in "Diluent 1" spot.
5. Reagents: in SmartChem rack "RGT1", place #1= Reagent water, #2= Color reagent, #3=
Ascorbic Acid and Spike. Do Not Overfill bottles.
6. Check fluid levels in Cuvette Wash Solution, DIW, and Probe Rinse Solution bottles, refill
if necessary.
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Central Environmental Laboratory
Department of Biology
East Carolina University
SmartChem:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. In Methods select "CPO4" from the list of methods at the bottom of the page.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the 6 empty cups for standard dilutions.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "print" and selecting
"print" again.
2. Real time sample data can be displayed by selecting the Results button under the cuvette
wheel. Once all samples have been run, the print option will appear in the upper left
menu bar, clicking the button will automatically print out the results.
Cleaning:
1. All reagents, standards, QC's, and spikes must be collected for pick-up by EH&S. Collect
these into designated hazardous waste bottles.
2. Samples can be disposed of down drain and sample cups can be put in trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and
inverted on rack/paper towel to dry.
4. Wash SmartChem racks with tap water and scrub with brillo pad/brush, hang on drying
rack above sink next to SmartChem to dry.
Principle:
Ammonium Molybdate and Potassium Antimonyl Tartrate react in acid medium with
Orthophosphate to form a Heterpoly Acid — Phosphomolybdic Acid that is reduced to intensely
colored Molybdenum Blue by Ascorbic Acid. The color (absorbance) measured at 880nm is
proportional to the Orthophosphorus concentration.
Final Plan of Study for Potential Effects of Headwater Wetland Reduction on Downstream Aquatic Functions F-52
PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Central Environmental Laboratory
Department of Biology
East Carolina University
Total and Dissolved Kjeldahl Nitrogen (DKN/TKN) — Automated
SmartChem Method
References:
SmartChem 200 Method 390-200E, revsed July 2008. Westco Scientific Instruments, Inc.
Standard Methods 4500-Norg. Nitrogen (Organic) (20th Edition). American Public Heath
Association, American Water Works Association, Water Environment Federation. pp. 4-
123/128.
Digestion Reagent: (*Should already be made and in Kjeldahl cabinet)
1. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in
S106.
2. Digestion reagent (Mercury free): (AKA "BLUE JUICE") Can make one 2L or more.
Easier to weigh out both the Cupric Sulfate and the Potassium Sulfate first (x 3). Use
three 2L volumetric flasks found under digestion block.
a. In the fume hood dissolve completely 9.5g Cupric Sulfate (anhydrous; CUSO4;
CAS 7758-98-7) in each of the 2L volumetric flasks. Using a full squirt bottle,
rinse weigh boat and funnel, then add the remainder of the bottle to the flask
(approx. 500 mQ. Swirl to completely dissolve.
b. Add 268g Potassium Sulfate, (K2SO4; CAS 7778-80-5) which will only partially
dissolve. Repeat the squirt bottle step above. Swirl to mix.
c. Set up an ice bath in the fume hood using a white tray half full of ice.
d. Measure out 268mL concentrated Sulfuric Acid using both a 250mL, and a
25mL graduated cylinder marked at 18mL.
e. Place volumetric flask on ice, and slowly add the concentrated Sulfuric Acid
Standard:
(H2SO4; CAS7664-93-9) to the volumetric flask 5-10mL at a time while
continuously swirling — Will get very, very hot! There is a fine line between too
hot and too cold, you want to keep it warm enough to get the chemicals to
dissolve and not so cool that the chemicals will come out of solution. Continue
adding the acid, swirling and mixing. Can use some reagent water if gets too
hot. Once everything is dissolved take it out of the ice bath and allow to cool to
room temperature. Using dei water that is at room temperature slowly bring
up to volume while mixing. The volume can fluctuate so be careful not to add too
quickly and make sure that the temp of reagent water is the same as what you
are adding to. If making more than 1 batch at a time, mix batches together and
aliquot among the four designated 4 liter bottles in Kjeldahl cabinet. Mix
thoroughly. STABLE.
500W Ammonium Chloride: From the 10,000pM Ammonium Chloride that is kept
in fridge pour out approximately 20mL into a beaker and leave covered with
parafilm to warm to room temperature. Using a 10mL glass volumetric pipet, rinse
and then pipete 10mL of the Ammonium Choride to designated 200mL volumetric
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East Carolina University
flask and bring to volume with reagent water. Mix thoroughly. Good for 1 week,
store in fridge.
**To make the 10,000uM Ammonium Chloride (NH4CI; CAS 12125-02-9): Weigh
out 0.5349g of NH4CI and dilute to 1,000mL in volumetric flask with reagent water.
Mix thoroughly. Transfer to 1 L Pyrex bottle with screw cap and store in fridge.
Good for 6 months.
Quality Control (QC's):
1. 89.22W N: (Using the 1000pL pipet, dilute 0.250mL of the Certified SPEX Certi Prep
Ammonia Nitrogen 1,000pg/mL (=71,327pM) (CAT# AS-NH3N9-2Y) to 200mL with
reagent water in designated volumetric flask. Mix thoroughly. Prepare fresh daily.
2. 178uM N: (Using the 1000pL pipete, dilute 0.5mL of the Certified SPEX Certi Prep
1,000pg/mL (=71,327pM) to 200mL with reagent water in designated volumetric flask.
Mix thoroughly. Prepare fresh daily.
3. 356uM N: (Using the 1000pL pipete, dilute 1mL of the Certified SPEX Certi Prep
1,000pg/mL (=71,327pM) to 200mL with reagent water in designated volumetric flask.
Mix thoroughly. Prepare fresh daily.
Digestion Block Procedure: ***NEVER LEAVE RACKS OF KJELDAHL TUBES ON
CARTS!
Use Kjeldahl tubes that were baked in drying oven (1050C) for at least 4 hours (usually
overnight). Tubes are kept on wooden shelf, should be in order with boiling chips added and
covered w/ saran wrap. Racks and Digestion Block hold 40 tubes.
1. Add -11 boiling chips (Henger granules part # 136-CC sieved through #16 Mesh) to each
Kjeldahl tube.
2. Prepare lab sheet with tube numbers and sample ID's. (Duplicate whatever fits in rack of
40- ie; 9 dups for 25 samples.)
3. Measure 25mL of sample (x2), with graduated cylinder that has been rinsed 15 times with
fresh reagent water, and add to tubes. (EX.A full set of 25 CZR samples - will only be
able to duplicate 9 samples, the rest will be singles plus 2 Blanks (dei water), 3 QC's and
Standard = 40 tubes, in that order.)
4. With another freshly rinsed (xl5) graduated cylinder measure 25mL of reagent water for
blanks, add to tubes (x2).
5. Measure 25mL of each Q.C. (89.22pM, 178pM and 356pM), in order from low to high,
and add to tubes.
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East Carolina University
6. Measure 25mL of the 500pM Standard and add to tube.
7. Add 10mL of Kjeldahl Digestion Reagent to all samples/blanks/QC's/Std tubes using
designated repeat pipettor, kept in Kjeldahl cabinet. Be careful not to spill/splash
digestion reagent, use goggles.
8. Cap all tubes with green stoppers, kept in wall of drawers.
9. Wipe each tube with damp towel and dry completely. Place tubes in digestion rack as
each row is wiped and dried. Make certain that a green tray is under the rack and a "Work
in Progress" (W.I.P.) card is attached. Also record what samples they are. Ex:
"CZR4Marl 7 (#1-40)" on bottom of card. Can let sit overnight to digest next morning.
10. Take green stoppers out of tubes and place in tan tray in order, to replace in order.
11. Put on two side panels, kept inside digestion fume hood.
12. Lift up over head to check bottom of tubes for liquid, cracks, and boiling chips.
13. Place the rack into the digestion block very carefully making sure that all tubes go into the
block simultaneously. Close sash on hood.
Set Digestion Control Box: Turn ON, switch on right side of box. Make sure display reads
"J1" when first turned on and then the current temperature is displayed.
To Program: (On upper part of the display with ramp information)
1. Press TEMP key, Enter 210 and press ENTER;
2. Press TIME key, Enter 1.8 and press ENTER;
3. Press TEMP key, Enter 385 and press ENTER;
4. Press TIME key, Enter 1.5 and press ENTER.
5. Press START/STOP key.
Make sure all 6 lights turn on and stay on. If not, turn controller off and start over.
NOTE: Set timer for 28 minutes. After timer goes off put on orange heat resistant gloves
(temp. should be-160°C) and watch for liquid bubbling to the top of the tubes. If bubbling
does occur CAREFULLY lift digestion rack -1 inch off the bottom of the digestion block
(enough for bubbling to cease reaching the top of the tube). May have to keep lifting rack
until boiling calms down. DO NOT ALLOW THE TUBES TO BOIL OVER AS THIS WILL
LIKELY CAUSE THE TUBES TO EXPLODE!
1. The controller will beep twice when the digestion has completed (about 3.5 hours total).
Wearing orange heat resistant gloves, CAREFULLY remove the two side panels and lift
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East Carolina University
the digestion rack with tubes and place on the metal tray inside the fume hood. Be
careful the tubes do not hit anything in the hood.
2. Set a timer for 9 minutes. During this time, fill the designated brown square repeat
pipeter (kept in Kjeldahl cabinet) with fresh reagent water and prime. Check to see if it is
set to 25mL.
3. When timer goes off, pipet 25mL of reagent water into each of the
sample/standard/blank/QC tubes. NOTE: pipeter will only reach 3 tubes deep, so the
rack needs to be rotated to reach the other '/z of the tubes. There will be tubes in two
middle rows that you cannot access.
4. Remove rack from fume hood and place on bench top. Remove the tubes water was not
added to and put in blue rack. Add water and return to digestion rack. Cap each tube with
a rubber stopper.
5. Using the vortex genies, mix each tube about 30 seconds each. Return tubes, in order,
to the blue rack inside the green tray.
6. Samples need to be mixed 3x with an hour between mixes before they can sit overnight
and be read the next morning.
7. If a second digestion is planned for the same day, turn OFF controller. Turn back ON.
Allow the digestion block to cool to 1600C., about 2-3 hours with fan blowing into hood. If
tubes have been wiped down Repeat Steps #11 through #14, EXCEPT timer to be
initially set to 10 minutes because block is already hot. After timer goes off check temp
and watch for bubbling until sure they are calm and continue with Steps #15 through #21.
SmartChem Reagents:
1. Stock Buffer Solution: (Should already be made and stored in a 1L Pyrex Bottle with
screw cap in
TKN/DKN/PKN tray.)
OR to make:
In 1 L volumetric flask dissolve 134g of Sodium Phosphate, Dibasic Heptahydrate
(Na2HPO4.7H2O; CAS 7782-85-6) in approximately 350mL of reagent water. Add and
dissolve 20g of Sodium Hydroxide, add more reagent water, let cool and then dilute to 1 L.
Mix thoroughly. STABLE. Store in designated 1L Pyrex bottle with screw cap in
TKN/DKN/PKN tray.
2. REAGENT #1: Working Buffer Solution: (Should already be made and stored in a
1,000mL Pyrex Bottle with screw cap in TKN/DKN/PKN tray.)
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East Carolina University
OR to make:
In a 1 Liter volumetric flask, combine the reagents in the following stated order:
• 200mL of Stock Buffer Solution
• 250mL of the 20% Sodium Potassium Tartrate solution
• Mix by swirling.
• Add 120mL of the 20% Sodium Hydroxide solution
• Mix by swirling.
• Dilute to 1 Liter with reagent water
• Mix thoroughly.
• Sodium Hydroxide, 20%: (For Reagent #1) (Should already be made and stored
in 1,000mL Pyrex bottle with screw cap in Bases cabinet.
OR to make:
Under hood in a 1 Liter volumetric flask filled to approximately 800mL of reagent
water, slowly
add 200g of Sodium Hydroxide (CAS 1310-73-2). Excessive heat will be
generated. Mix well to
dissolve. Cool to room temperature and dilute to 1 Liter with reagent water. Mix
thoroughly.
STABLE. Stored in 1,000mL Pyrex bottle with screw cap in Bases cabinet.
Sodium Potassium Tartrate, 20%: (For Reagent #1) In designated 250mL
volumetric flask (kept in TKN/DKN/PKN tray) filled to approximately 200mL of
reagent water, slowly add and dissolve 50g Sodium Potassium Tartrate
Tetrahydrate (KOCO(CHOH)2COONa.4H2O; CAS 6381-59-5). Solution will
become cold. Bring to room temperature and dilute to 250mL with reagent water.
Mix thoroughly. *Do not want any NH4 in chemical.
3. REAGENT #2: Sodium Salicylate: (Stored in designated 250mL volumetric flask in
TKN/DKN/PKN tray.)
OR to make:
In designated 250mL volumetric flask, kept in TKN/DKN/PKN tray, dissolve 5g of Sodium
Salicylate (2-HOC6H4CONa; CAS 54-21-7) in approximately 225mL reagent water. Add
(very slowly and at an angle to keep from foaming) 1.5mL of concentrated Probe Rinse
Solution (Westco part # 3AS-RN00-21) and dilute to 250mL with reagent water. Mix
thoroughly. Store solution in same volumetric flask covered with foil, inside TKN tray.
Prepare fresh weekly. NOTE: wrap the small plastic SmartChem reagent bottle
containing this reagent with foil when used in the analysis.
4. *REAGENT #3: Sodium Hypochlorite Solution (Clorox): Pour about 10mL of the
diluted 5.25% Clorox into a 50mL beaker. In designated 50mL volumetric flask, add 3mL
of the diluted 5.25% Clorox, NaOCI) using a glass volumetric pipet. Add approximately
45mL of reagent water. Add (slowly and at an angle to keep from foaming) 0.5mL
concentrated probe rinse solution. Slowly bring to volume with reagent water. Mix
thoroughly. Prepare fresh daily.
5. *REAGENT #4: Sodium Nitroferricyanide Dihydrate Solution: In designated 50mL
volumetric flask, dissolve 0.4g Sodium Nitroferricyanide (Nitroprusside) Dihydrate
(Na2Fe(CN)5NO.2H2O; CAS 13755-38-9) in approximately 40mL of reagent water and
dilute to 50mL with reagent water. Cover flask with aluminum foil and let dissolve. Mix
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East Carolina University
well. Add 0.5mL of concentrated probe rinse solution. Mix thoroughly. Prepare fresh
every 2 days, refrigerate overnight. NOTE: wrap the small plastic SmartChem reagent
bottle containing this reagent with foil when used in the analysis.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1 L volumetric
flask filled with
-r950mL of reagent water, slowly and tilted to side add 0.5mL of Probe Rinse Solution
(Westco). Fill to
volume with reagent water. Mix thoroughly. Stable. Store at room temperature behind
SmartChem #1.
Keep a constant supply ready.
Cuvette Wash Solution: Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
In designated 2L container add reagent water to mark. Add 30mL (one bottle) of Unity
Scientific Cleaning Solution. Rinse 30mL bottle with reagent water and pour into 2L
container, repeat 2 more times. Fill to volume. Mix thoroughly. Stable.Store at room
temperature next to SmartChem #1.
Procedure: *Remember to use Kjeldahl waste jug and perform a Wash and WBL first.
(NOTE: Set up the Reagents, empty cups, Std, QC's and Diluent, etc. before pipetting samples.
When the first rack of samples are poured the SmartChem can be started and the rest of the
samples can be added.)
1. Samples: Using a 5mL automatic pipet, carefully pipette sample from digestion tube
being careful not to disturb bottom of tube. Use a clean pipette tip for each sample.
Rinse a SmartChem sample cup with a small amount of sample and discard into waste
container. Fill cups in duplicate and place in SmartChem sample rack (racks 1-5).
NOTE: All Kjeldahl waste has to be collected and disposed of via EH&S, use the
waste beaker while pipetting.
2. Diluent: Using a 5mL automatic pipet fill the designated small plastic SmartChem bottle
with the Blanks,
alternating back and forth between the two tubes of Blanks. Usually about 3 times from
each tube. Mix.
3. Q.C.'s: Using a 5mL automatic pipet fill one cup with each of the Q.C.'s. Fill to top line on
cups. These will be placed in descending order in slots 2-4 on "Reag 2 Ctrl/Std" rack.
4. Standard/Spike: Using a 5mL automatic pipet fill two cups with the 500pM Standard (one
is for the Spike). Fill to top line on cup. This will be placed in cup 1 of the "Reag 2
Ctrl/Std" rack. The Spike will be placed in 51" slot of "Rgt 1" rack in a supporting half
bottle.
5. Place 6 empty sample cups in positions 1-6 in SmartChem Sample rack 5. These cups
will be used to
generate the standards for the standard curve.
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Central Environmental Laboratory
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East Carolina University
6. Reagents: (All reagents are placed in "Reag 2 Ctrl/Std" rack) Do not overfill bottles, pop
any bubbles.
a. Position #1 - DKN Working Buffer (TKBU)
b. Position #2 - Sodium Salicylate (TKSA)
c. Position #3 - Clorox (TKHY)
d. Position #4 - Sodium Nitroprusside (TKNI)
NOTE: Fill each small plastic SmartChem reagent bottle only to shoulder of bottle. If they
are
too full or there is a bubble present the SmartChem will register it as "empty".
7. Check fluid levels in Cuvette Wash Solution, DEI, and Probe Rinse Solution bottles, refill
if
necessary. Keep an eye on Probe Rinse as this needs to be filled regularly.
SmartChem:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select "TKNH-RgtsMoved+ExtraBuffer" from the "Profile list".
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the empty cups for standard dilutions.
******NEVER LEAVE RACKS OF KJELDAHL TUBES ON CARTS!
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "print" and selecting
"print" again. Then press Exit.
2. Real time sample data can be displayed by selecting the Results button under the cuvette
wheel. Once all samples have been run, the print option will appear in the upper left
menu bar, clicking the button will automatically print out the results.
Cleaning:
1. All samples, reagents, standards, QC's, and blanks must be collected for pick-up by
EH&S. Collect these into designated hazardous waste bottles.
2. SmartChem sample cups can be disposed of in the trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and
inverted on rack/paper towel to dry.
4. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack
above sink next to SmartChem to dry.
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East Carolina University
Principle:
Total Kjeldahl nitrogen is the sum of free ammonia nitrogen and organic nitrogen compounds
which, through digestion, are converted to ammonium sulfate. The method is based on the
conversion of the digested ammonium sulfate to ammonia. In the presence of sulfuric acid,
potassium sulfate, and a cupric sulfate catalyst, amino nitrogen of organic materials is converted
to ammonium. The ammonium cation is then converted to ammonia by neutralization with a
concentrated buffer. Once converted to ammonia, an intensely blue compound, indophenol, is
formed by the reaction of ammonia with alkaline phenol and then hypochlorite (Berthelot
reaction), catalyzed by sodium nitroprusside. The color (absorbance) measured at 660nm is
proportional to the Kjeldahl nitrogen concentration in the original sample.
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Total and Total Dissolved Phosphate (TP/TDP) — Automated SmartChem Method
(NEVER DO TOTAL PHOSPHATE IN SMARTCHEM IF SAMPLES ARE NOT FILTERED)
References:
SmartChem 200 Method 410-200B, revised July 2008. Westco Scientific Instruments, Inc.
Standard Methods for the Examination of Water and Wastewater. 4500-P-B (191" Edition).
Sample Preparation. American Public Health Association, American Water Works
Association, Water Environment Federation. pp. 4-109/111.
Standard Methods for the Examination of Water and Wastewater. 4500-P-E (181" and 191n
Editions). American Public Health Association, American Water Works Association, Water
Environment Federation.
EPA. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020. Method
365.3. Environmental Monitoring and Support Laboratory, Cincinnati, OH.
Reagents:
1. Reagent Water: Fresh deionized water from E-pure system in S106 (16-8megohm/cm).
2. Digestion Reagent: In designated 500mL volumetric flask dissolve 10g Low Nitrogen
Potassium Persulfate (K2S2O8; CAS# 7727-21-1) in about 300mL of reagent water. Dissolve
1.5g Sodium Hydroxide (NaOH; CAS# 1310-73-2) and dilute to 500mL with reagent water.
May have to put on stir plate to mix well. Prepare fresh daily. Use designated volumetric
flasks for smaller amounts, which are kept in gray tray above SC#1. Use repipeter to add to
tubes.
3. Borate Buffer: (Should already be made and stored in white tray above SC #1)
a. In designated 500mL flask dissolve 30.9g Boric Acid (CAS #10043-35-3) in approx.
300mL reagent water. Add 4g Sodium Hydroxide and bring to volume with reagent
water. May have to put on stir plate to dissolve. Stable. Use repiteter to add to tubes.
4. Phenolphthalein, 1 %: Phenolphthalein Solution (CAS 77-09-8) Stored in flammable cabinet.
Fill dropper bottle, kept in TDP drawer, as needed. Stable.
5. Sodium Hydroxide, 50%: (Should already be made)
a. Ina 500mL volumetric flask containing 350mL of reagent water dissolve 250g of
Sodium Hydroxide. Gets HOT! Allow to cool to room temperature and bring to 500mL
with reagent water. May have to use stir plate to dissolve. Stable for several months.
Store in Bases cabinet in IL Pyrex bottle with screw cap. Fill dropper bottle, stored in
TDP drawer, as needed.
6. Sulfuric Acid, 11 N: (Should already be made) ALWAYS ADD ACID TO WATER!
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a. To approximately 600mL of reagent water in a 1 L volumetric flask, very slowly add
306mL of concentrated Sulfuric Acid (H2SO4; CAS# 7664-93-9). Allow to cool to room
temperature and dilute to 1 L with reagent water. Stable for several months. Stored in
Pyrex bottle w/ screw cap in acid cabinet. Mix before using. Fill dropper bottle, stored
in TDP drawer, as needed.
7. REAGENT #1: Fresh deionized water from E-pure system in S106.
8. REAGENT #2: (Color Reagent): See below ..... All reagents must be at room temperature
and must be mixed in the order given.
a. In designated brown bottle mix 50mL of 5N Sulfuric Acid - H2SO4, (should already be
made and stored in acid cabinet), add 5mL of the Antimony Persulfate Tartrate - APT
Solution (should already be made and stored in brown bottle in fridge). Swirl to mix.
Add 15mL of the 40% Ammonium Molybdate Solution (should already be made and
stored in red bottle in fridge). Swirl to mix. Add 3 mL of 17.7% SDS, may already be
made and stored in white TKP/DKP/PKP tray. Mix gently. Store in fridge. Prepare
fresh weekly.
*Reagents needed to make Color Reagent (Reagent #2), IF NOT ALREADY MADE, are
as follows:
a. Sulfuric Acid, 5N: Perform in the fume hood. In designated 4L jug with 1720mL
deionized water SLOWLY add 280mL of Sulfuric Acid (1-12SO4, CAS 7664-93-9) and
swirl as it is added. Mix thoroughly. Stable. Kept in 4L bottle in acid cabinet.
b. Antimony Potassium Tartrate Solution: In a 500mL volumetric flask add 1.3715g
Antimony Potassium Tartrate (K(SbO)C4H4O6*1/2H2O; CAS 28300-74-5) and dilute to
500mL with reagent water. Store in dark bottle in fridge. Stable for 6 months.
c. Ammonium Molybdate Solution, 40%: Dissolve 20g of Ammonium Molybdate
((NH4)6M07O24*4H2O; CAS 12054-85-2) to 500mL with reagent water. Store in red
bottle in fridge. Stable for 6 months. If precipitate forms, dump in waste, scrub out
bottle with reagent water and brush and remake.
d. Sodium Dodecyl Sulfate (SDS), 17.7%: (Use only purest grade, with Phosphate
concentrations :50.0001% Phosphate.) In designated bottle (stored in TKP/DKP/DKP
tray) add 15g SDS (stored in flammables cabinet) (CH3(CH2)1 1 OSO3Na; CAS 151-
21-3). In graduated cylinder measure out 85mL of reagent water. Using a disposable
3mL pipete, rinse weigh boat and funnel into bottle. Add remaining reagent water.
Will require stirring on stir plate to dissolve completely. Make every two weeks.
13. REAGENT #3: (Ascorbic Acid, 0.1 M): In designated 50mL volumetric flask dissolve 0.88g
Ascorbic Acid (C61-18O6; CAS 50-81-7) with reagent water. Bring to volume. Add 1mL of
17.7% SIDS. Mix well. Prepare fresh daily.
Standards:
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10,000uM Potassium Phosphate (KH2PO4; CAS 7778-77-0): Should already be made and
in fridge.
a. In a 1 L volumetric flask add 1.3609g of Potassium Phosphate Monobasic and dilute to
1 L with reagent water. Mix well. Good for 6 months. Store in Pyrex bottle w/orange
cap in fridge.
2. 1,000uM Potassium Phosphate:
a. Can use 1,000pM Combined Standard (NH4C1, KH2PO4, KNO3). Good for 1 month.
Store in fridge.
3. 401aM Potassium Phosphate: In designated 100mL volumetric flask add 4mL of 1,000pM
Combined Standard OR 4mL of 1,000pM KH2PO4 Standard and bring to volume with
reagent water. Prepare fresh daily.
Quality Controls:
1. QC1: 5.26uM P: In designated 200mL volumetric flask add 0.1mL (100pL) SPEX Certi Prep
1,000pg/mL Phosphate (=10,525pMP, CAT# AS-PO49-2Y) and bring to volume with reagent
water. Prepare fresh daily.
2. QC2: 15.79uM P: In designated 200mL volumetric flask add 0.3mL (300pL) SPEX Certi Prep
1,000pg/mL Phosphate (=10,525pMP, CAT# AS-PO49-2Y) and bring to volume with reagent
water. Prepare fresh daily.
Probe Rinse Solution: (Should already be made and stored next to SC#1- If not already on sink
counter, it is stored in SmartChem supplies cabinet)
In designated 1 L volumetric flask, with -950mL of reagent water, slowly add 0.5mL Probe Rinse
Solution (Unity Scientific, P/N# 3AS-RN00-21) and bring to volume with reagent water. Mix
thoroughly. Stable. Store at room temperature in designated flasks. Keep steady supply.
Cuvette Wash Solution: (Should already be made and stored next to SC#1- otherwise, stored in
SmartChem supplies cabinet)
In designated 2L container add 1 bottle (30mL) cuvette cleaning solution (Unity Scientific P/N#
365-0366-900) to approximately 1500mL of reagent water. Rinse bottle 3x with reagent water, add
to container and fill to mark. Invert, gently, five times to mix. Store at room temperature. Stable.
Keep steady supply.
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Set Up:
Culture tubes; one per sample and one for each: QC #1, QC #2 and Standard.
Six tubes for Blanks (= diluent).
10mL pipeter and pipet tip.
Procedure (Digestion):
Pipet 10mL of shaken sample and discard down the drain. Pipet a second 10mL into a 30mL
culture tube. Repeat this process with a rinse of next sample between each sample. Be sure
the culture tube has a screw cap with a rubber or Teflon liner so a tight seal can be made.
2. Repeat the above process with six Blanks (deionized water), one QC#1, one QC#2 and one
40pM P Standard.
3. Add 5mL of Phosphate Digestion Reagent to each sample/standard/QC/blank using
designated repeat pipetter. Stored in white tray above SC#1.
4. Cap culture tube tightly. Mix by gently inverting culture tube three times. Do not shake;
shaking the culture tube vigorously does not allow for complete mixing of sample and
digestion reagent.
5. Heat tubes in autoclave for 30 minutes at 250°C. Note: you can stop here and finish the
following day. Let samples and autoclave cool to room temperature (overnight). There may
be a white precipitate in the bottom of the tube at this stage, okay.
6. Add 1 mL of Borate Buffer to each tube using designated repeat pipeter, kept in white tray
above SC #1. Keep track of caps so that the same cap can be returned to the same tube.
7. Cap tubes and mix by gently inverting tube three times. If precipitate does not dissolve, filter
the sample.
8. Remove caps, keeping them in order. Add 1 drop of 1 % Phenolphthalein to each tube
(including samples/standard/QCs/blanks). Steps #8 and #9 can be added on top of each
other.
9. Add 1 drop of 50% Sodium Hydroxide to each tube (including samples/standard/QCs/blanks).
Cap tubes and mix by gently inverting tube three times. If sample does not turn pink, add
another drop of Sodium Hydroxide and mix sample again.
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10. Add 1 drop of 11 N Sulfuric Acid to each tube (including samples/standard/QCs/blanks). Cap
tubes and mix by gently inverting tube three times. If sample does not return to clear, add
another drop of Sulfuric Acid and mix sample again.
Procedure (SmartChem):
1. Samples: Pipet approximately 3mL of sample into SmartChem sample cup and discard into
waste beaker. Pipet 3mL of sample into (two cups per sample) cups and place in SmartChem
sample rack (racks 1-5). Also, add 1 sample cup containing only DEI water at the beginning
and at the end.
2. Diluent/Blank: Combine the six Blanks in an erlynmeyer flask and swirl to mix. Pour mixed
Blanks into Diluent bottle and insert into SmartChem "Diluent #1" slot.
3. Standard/Spike: Pipete 3mL of the digested 40pM PO4-P into two sample cups rinsing as
with samples. Place Standard in SmartChem rack "RGT2 Ctrl/Std", position 1.
4. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5. These cups will
be used to generate the standards for the standard curve.
Reagents:
• In SmartChem rack "RGT1", place Reagent #1 (Dei. water) in position 1.
• "RGT1" rack, place Reagent #2 (Color Reagent) in position 2.
• "RGT1" rack, place Reagent #3 (Ascorbic Acid) in position 3.
5. Check fluid levels in cuvette wash solution, DIM and probe rinse solution bottles, refill if
necessary.
Software Procedure:
1. Turn on computer; turn on SmartChem.
2. Select "WP2W" from the list of methods at the bottom of the page.
3. Follow procedures for SmartChem Set -Up posted next to equipment.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to
include the empty cup for standard dilutions.
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Printing Data:
1. After all standards have been made and run, a standard curve will automatically be
generated and appear on the screen. Print this out by selecting "Print" and "Print" again.
2. Real time sample data can be displayed by selecting the "Results" button. Once all samples
have been run, the print option will appear in the upper left menu bar, clicking the button will
automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a) Select "Options" from left menu bar, then "Data Retrieval".
b) Find and select the plan of interest. A split screen will appear — click on the method in
the box in the lower half of the page. This will highlight the symbols just to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select
"Print".
ii. Click on the graph and this will bring the standard curve forward. Select
"Print".
Cleaning:
1. All samples and reagents need to be collected in Waste jug.
2. Wash culture tubes as discussed on CEL washing protocol.
3. Rinse out designated Phosphate Digestion repeat pipettor with deionized water and replace
on shelf in white tray.
4. Rinse the pump portion only of the designated Borate Buffer repeat pipettor with deionized
water and replace with remaining buffer on shelf in white tray.
5. Other glassware needs to be rinsed with tap water and placed in vat to be washed according
to CEL washing protocol.
6. SmartChem sample cups can be disposed of in the trash can.
7. SmartChem reagent bottles should be rinsed until clean with deionized water and inverted on
rack/paper towel to dry.
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8. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack above
sink next to SmartChem to dry.
Principle:
Total Phosphorus (TP) is run on unfiltered water. Total Dissolved Phosphorus (TDP) is run on
filtered water. The Potassium Persulfate Digestion coverts all form of Phosphorus to
Orthophosphate (at concentrations less than 40 uM). Ammonium Molybdate and Potassium
Antimonly Tartrate react in acid medium with Orthophosphate to form a Heterpoly Acid —
Phosphomolybdic Acid that is reduced to intensely colored Molybdenum Blue by Ascorbic Acid. The
color (absorbance) measured at 880nm is proportional to the Orthophosphorus concentration.
Using this method on samples with salinities above 18-20ppt creates a precipitate that clouds the
solution. It is very difficult to remove this cloudiness from the samples. If the cloudiness is not
removed, 1) inflated absorbance values are generated which in turn results in over -estimation of
concentration, 2) pump lines in SmartChem could become clogged.
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Standard Operating Procedure for the
Analysis of Dissolved and Total
Organic Carbon
CCAL 20A.0
Cooperative Chemical Analytical Laboratory
Forestry Sciences Laboratory
Oregon State University
3200 SW Jefferson Way
Corvallis, Oregon
Prepared by Kathryn Motter and Cameron Jones
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

Standard Operating Procedure for the
Analysis of Dissolved and Total
Organic Carbon
CCAL 20A.0
Table of Contents
1.0 Scope and Application...............................................................................3
2.0 Summary of Method..................................................................................3
3.0 Definitions...................................................................................................3
4.0 Interferences...............................................................................................4
5.0 Safety...........................................................................................................4
6.0 Equipment and Supplies...........................................................................5
7.0 Reagents and Standards............................................................................5
7.1 Preparation of Reagents...........................................................................5
7.2 Preparation of Standards..........................................................................6
8.0 Sample Handling and Storage..................................................................6
9.0 Quality Control..........................................................................................6
10.0 Calibration and Standardization............................................................7
11.0 Procedure..................................................................................................7
11.1 Shimadzu TOC-VCSH Instrument Operating Parameters ..............7
11.2 Procedure.................................................................................................8
11.3 System Notes............................................................................................8
12.0 Data Analysis and Calculations..............................................................8
13.0 Method Performance...............................................................................8
14.0 Pollution Prevention................................................................................8
15.0 Waste Management..................................................................................9
16.0 References.................................................................................................9
17.0 Tables, Diagrams, Flowcharts, and Validation Data ............................10
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Standard Operating Procedure for the
Analysis of Dissolved and Total
Organic Carbon
CCAL 20A.0
1.0 Scope and Application
1.1 This method details the determination of Dissolved Organic Carbon and Total Organic
Carbon in fresh waters by oxidative combustion and infrared analysis. This practical range of
determination for this method as reported by the manufacturer is 0 — 25000 ug/L. The reported
Method Detection Limit is 4 ug/L.
2.0 Summary of Method
2.1 Samples are acidified with hydrochloric acid and sparged with zero air to remove
inorganic carbon. The sample is then injected into a heated reaction chamber packed with platinum
catalyst. The water is vaporized and the organic carbon oxidized to carbon dioxide and water by
catalytic combustion. The CO2 formed is transported to the detector in a carrier gas stream and
measured directly by an infrared detector. The amount of CO2 is directly proportional to the
concentration of carbonaceous material in the sample.
3.0 Definitions
3.1 DI water: Water that has been through a deionization system to produce water similar to
ASTM Type I reagent with 16.7 Mohms resistivity (ASTM) (Reference 16.3).
3.2 Method Detection Limit (MDL): The minimum concentration of an analyte that can be
measured and reported with 99% confidence, based on a one-sided 99% confidence interval (t-
value at a significance level of 0.01 and n-I degrees of freedom) from at least seven repeated
measurements of a low concentration standard measured within an analysis run. Where,
t = Student's t value at a significance level of 0.01 and n-1 degrees of freedom
s = standard deviation of at least seven repeated measurements of a low level standard
4.0 Interferences
4.1 This method is actually for analysis of NPOC (non-purgable organic carbon) which refers
to organic carbon present in a non-volatile form. In most literature for water analysis, the terms are
used interchangeably because the amount of purgeable organic substances in natural waters is
small. Because purgeable organic substances may be lost during sparging, true TOC may be
determined by calculating the difference between TC and IC.
4.2 Any contact with organic material may contaminate a sample. Care must be taken in
sample handling and storage to minimize exposure.
4.3 This procedure is applicable only to homogeneous samples which can be reproducibly
injected by microliter syringe into the instrument; the inner diameter of the syringe and injection
tubing limit the maximum particlute size that may be included with the sample aliquot.
4.4 Inorganic carbon is considered an interference in the analysis and must be removed or
accounted for in the final calculation.
4.5 Removal of inorganic carbon by acidification and sparging may result in the loss of
volatile organic substances.
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4.6 Combustion temperatures above 950°C are required for decomposition of same
carbonates. Acidification may aid decomposition. Elemental carbon is not oxidized at the lower
temperature used in this method, but it is generally not present in fresh water samples.
5.0 Safety
5.1 The toxicity or carcinogenicity of each reagent has not been precisely determined;
however, each chemical should be regarded as a potential health hazard. Exposure to these
chemicals should be reduced to the lowest possible level. Cautions are included for known
extremely hazardous materials.
5.2 The following chemicals have the potential to be highly toxic or hazardous. For detailed
explanations, consult the MSDS.
5.2.1 Sulfuric acid (used in regeneration of the suppressor)
5.2.2 Oxalic acid (used in regeneration of the suppressor)
6.0 Equipment and Supplies
Note: Brand names, suppliers and part numbers are for illustrative purposes only. No endorsement is
implied. Equivalent performance may be achieved using apparatus and materials other than those
specified here, but demonstration of equivalent performance that meets the requirements of this method
is the responsibility of the laboratory.
6.1 Shimadzu TOC-VCSH Analyzer
6.1.1 Shimadzu ASI-V Autosampler
6.1.2 Instrument Controller
6.1.3 Data Collection Software
6.1.4 High Sensitivity Catalyst
6.1.5 CO2 Absorber (soda lime)
6.2 "Zero Air" compressed air and regulator
6.3 40 mL borosilicate vials and septum caps
6.4 Laboratory glassware and pipettes
6.5 Balance
6.6 Safety glasses
6.7 Nitrile gloves
6.8 Lab coat or apron
6.9 Laboratory exhaust fume hood
6.10 High Density Polyethylene (HDPE) bottles
7.0 Reagents and Standards
7.1 Preparation of Reagents
7.1.1 2NHC1
Dilute one part concentrated hydrochloric acid with five parts DI water. A final
concentration accuracy of t 2% is acceptable.
7.2 Preparation of Standards
7.2.1 Calibration Standards:
Standards are prepared by of single element standards purchased from vendors that
provide traceability to NIST standards. Working standards for calibration are prepared at
concentrations stated below.
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Standard #
mg C/L
1
0.20
2
0.50
3
1.00
4
2.00
5
5.00
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ECU Laboratory Guide
How To Use The TOC-V analyzer
Turning the Machine on:
First turn the compressed air tank on. Then the button in the lower right hand corner of the machine.
If you are just doing Carbon analysis that is all you need to turn on. If you are also doing Nitrogen
analysis, turn on the nitrogen measuring unit on the right of the TOC.
Before you Begin:
Make sure there is 2N Hydrochloric acid and ultra pure water in the two containers to the left of the
analyzer. The larger container holds the — while the smaller container holds the --.
Next, open the TOC, and check that the humidifier and the Cooler Drain Container are filled with
water. The Humidifer should be filled to the line labeled Hi. To fill: take off small plastic cap and
fill with pure lab water. The Cooler Drain Container should be filled to right below the arm and tube
on the right side of container. To fill: use little blue tube connected to container or pull the container
out of its holder gently, remove the black cap and fill. ***If you see bubbles in this, DO NOT RUN
SAMPLES!!!!
Creating a Sample Table:
1. Double Click TOC-Control Von the desktop.
2. Choose Sample Table Editor.
3. Click New in the Sample Tab Viewer.
4. Select the correct instrument. Click OK.
If you are creating a new Calibration Curve:
5. Click Calibration curve in Sample Tab viewer.
6. Click New.
7. Select the system then click next. (use NPOC for Carbon analysis)
8. Select "Edit Calibration points manually" and "Div. Standard Solutions (auto correction of dilution
factor" then next.
9. Select the analysis Type (NPOC or TN for Nitrogen)
10. Deselect zero shift.
11. Enter a name for the curve then click next.
12. Select units (mg/L), select 3/5 injections, make acid addition 0 and make the sparge time 1:30 then
click next.
13. Make sure the injection is at 100ul- then click add and enter the calibration point concentration in the
cal. Point conc. Field then ok. Repeat. Then click Finish.
If you are creating a method file:
5. Click Method File in Sample Tab Viewer.
6. Click New.
7. Choose the Correct Instrument then click next.
8. Choose the analysis type then create a file name. Click Next.
9. Then enter the calibration curves you would like to use. Click next.
10. Select 3/5 injections and set acid addition at 0. Click Finish.
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Editing a Sample Table:
1. Insert the Calibration Curve (if running a calibration curve) or the Method File (if running an
unknown or blank) into the sample table by dragging. (when running multiple unknowns
simply copy and paste)
2. Click the Birthday Cake Button in the top right hand corner of the sample table.
3. Enter the vial #'s being used. Click on the lower right part of the cell and drag downward to
enter a series of sample vial #'s all at once.
4. Click ok to close window and automatically set all the #'s in the sample table. Make sure to
save.
Connecting to the Instrument and Conducting Analysis:
1. Open sample table to be used and click connect. (-30 min are required for the furnace to rise
and stabilize.
2. To check the furnace temperature click monitor. The furnace should be around 720 degrees to
run. Also check the flowmeter and the bubblemeter on the outside of the machine. The
flowmeter should be at 150 and the bubblemeter at 0.5. When the sample table says ready in
the upper right hand corner, click start.
3. Select what you want it to do after: shutdown, sleep (restarts at a specific date and time), or
keep running. Click Start.
4. To view peaks, click the peaks button next to the birthday cake.
5. When finished: If you told the machine to shut down, it will automatically shut down after
—30 min. After —30 minutes you must turn off the air tank and turn off the nitrogen analyzer.
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-SGS1
SGS North America Inc. — Orlando
Laboratory Methods for Analysis
of
Metals in Sediment (pages F-77 — F-117) and Metals in Water (pages F-118 — F-138),
Total Organic Carbon in Sediment (pages F-139 — F-154), and
Bulk Density of Sediment (pages F-155—F-160)
SGS - Orlando Orlando 4405 Vineland Road Orlando, FL 32811, USA t [phone] www.sgs.com
Member of the SGS Group (SGS SA)
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SGS - ORLANDO STANDARD OPERATING PROCEDURE
FN: MET 104.13
Rev. Date: 02/2018
Pages F64-F74
TITLE: DIGESTION OF SOILS FOR ICP ANALYSIS
REFERENCES: 3050B
REVISED SECTIONS: removed all references to Accutest
1.0 SCOPEAND APPLICATION, SUMMARY
1.1 This method is applicable for the digestion of sediments, soils, sludges and solid
wastes. After digestion, the samples can be analyzed by ICP. This digestion
method is based upon SW846 method 3050B.
1.2 An aliquot of a homogenized soil is digested with repeated additions of nitric
acid and hydrogen peroxide. Hydrochloric acid is added and the sample is refluxed
for an additional 15 minutes. The sample is cooled to room temperature and diluted
to 50 ml. If particulate matter is present, the sample is filtered.
2.0 PRESERVATION
All soils must be refrigerated at < 6 °C. All bottleware used by SGS - Orlando is
tested for cleanliness prior to shipping to clients. Analysis results must be < '/2 RL to be
acceptable. Please refer to SOP SAM104, current revision for further instruction.
3.0 HOLDING TIME
All samples should be digested and analyzed within 6 months of the time of collection.
4.0 INTEREFRENCES
Sludge and soil samples can contain diverse matrix types, which may contain a
variety of interference. Spiked samples can be used to determine if this interference is
adequately treated in the digestion process. For discussion of other interference, refer to
specific analytical methods.
5.0 APPARATUS
The apparatus needed for this digestion procedure are listed below.
5.1 Automatic repipettor (s)
5.2 Fisher Brand 0.45 micron (um) filter or equivalent. Filter lots are checked for
cleanliness through the Method Blank process. All Method Blank analytical results
must be <'/2 RL to be acceptable, if not, the contaminated lot must be identified and
removed from laboratory use. Samples filtered through the contaminated filters
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must be re -filtered through acceptable filters.
5.3 Top loader balance- capable of accurately weighing 0.01g. Refer to SOP QA005,
current revision for balance calibration information.
5.4 Thermometer- capable of measuring to at least 1250C and checked against NIST
traceable thermometers. Refer to SOP QA002, current revision for further
information.
5.5 Environmental Express Hot Block or equivalent capable of maintaining a
temperature of 90-95°C.
5.6 Environmental Express digestion vessels or equivalent, 65ml capacity. Each Lot of
digestion tubes comes with a Certificate of Analysis which demonstrates
cleanliness as well as volume accuracy at the 50ml mark. Please refer to
Digestion Tube Certificate Logbook for further information. Tube Lots are also
checked through the Method Blank process. All Method Blank analytical results
must be < '/2 RL to be acceptable, if not, the contaminated lot must be identified
and removed from laboratory use. Re -digestion is required for all samples
prepared with the contaminated tube lot.
5.7 Fisher Brand disposable 10 ml syringes or equivalent. Syringe lots are
checked for cleanliness through the Method Blank process. All Method Blank
results must be < '/2 RL to be acceptable, if not, the contaminated lot must be
identified and removed from laboratory use. Samples filtered through the
contaminated syringes must be re -filtered through acceptable syringes.
5.8 Fisher Brand wooden spatulas or equivalent.
5.9 Eppendorf Pipette (s) - Pipette (s) are checked daily for accuracy and to ensure
they are in good working condition prior to use. Volumes are checked at 100% of
maximum volume (nominal volume). Pipettes are checked within the metals
department and results are stored electronically in the "Pipette Calibration Log".
Refer to SOP QA006, current revision for further information regarding pipette
calibration. BIAS: mean must be within 2% of nominal volume. Precision: RSD
must be < 1 % of nominal volume based on three replicates.
5.10 Class A volumetric flask (s)
5.11 Class A volumetric pipette (s)
5.13 Teflon Chips
5.14 Solid Standard Reference Material (SRM) as required per project/client specific
requirements.
5.15 Environmental Express ribbed watch glasses or equivalent.
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6.0 REAGENTS
All chemicals listed below are trace metal grade unless otherwise specified. Refer
to Acid Certificate of Analysis logbook for Certificate of Analysis and compliance with
specifications of the grade listed. De -ionized (DI) water should be used whenever water
is required. SGS - Orlando produces DI water to the specifications for the ASTM Type II
standard designation based on the system manufacturer's performance specifications.
The DI water is used exclusively for laboratory purposes. Refer to SOP QA037, current
revision for more information regarding testing and monitoring.
6.1 Hydrochloric acid, Fisher Trace metal grade or equivalent
6.2 Nitric acid, Fisher Trace metal grade or equivalent
6.3 Hydrogen peroxide, reagent grade ,30%
6.4 Metals spiking solutions commercially purchased:
Environmental Express Multielement spiking solution or equivalent made with 5%
HNO3 and a trace of HF.
Inorganic Ventures 5000 mg/I Mineral solution.
Prepared Metals Standards:
100ppm Molybdenum, 100ppm Tin, 100ppm Strontium, and 100ppm Titanium
spiking solution prepared as follows: Using a 10ml class A volumetric pipette,
add 10mis of 1000ppm stock Molybdenum, 10mis of 1000ppm stock Tin, 10mis
of 1000ppm Strontium, and 10mis of 1000ppm Titanium to a 100ml class A
volumetric flask containing approximately 50mis of DI water and 3mis of
concentrated Nitric acid and 5mis of concentrated HCL. Dilute to volume with DI
water and mix well. This standard must be prepared every 6 months or before
stock standard expiration date, whichever comes first. Refer to Metals Standard
Prep Logbook for further information. Some of the information included in the
logbook is as follows: standard name, elements in mix, manufacturer, lot number,
parent expiration date, acid matrix, stock concentration, volume of standard
added, total volume, final prepared concentration, prep date, initials, MET
number, and prepared standard expiration date.
7.0 PROCEDURE
7.1 Decant any free liquid from the solid sample. Remove any foreign objects such as
twigs or rocks. The sample container must have enough room to move the matrix
around with the wooden spatula. Mix the sample thoroughly using the wooden
spatula. Make certain the entire sample is mixed well. The wooden spatula must
reach the bottom of the original container and be able to be moved through the
entire sample to ensure proper mixing. If the sample is packed tightly or matrix is
dense and cannot be efficiently moved around in the original jar, a secondary
container such as a porcelain dish must be used. Remove the sample from the
original container and place in the clean secondary container. While in the
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secondary thoroughly mix sample around until appearing uniform in consistency.
Upon completion the sample is re -packed into the original container. Refer to SOP
QA034, current revision for more information on sample homogenization. Using
a wooden spatula weigh out approximately 1.0 gram of a homogeneous sample
on a top loading balance and place in the digestion vessel.
7.2 The sample identification must be accurately recorded on the digestion vessel and
sample digestion log. In addition to the samples, a serial dilution (performed at
the analytical bench), a post digestion spike (performed at the analytical bench),
a matrix spike (MS), matrix spike duplicate (MSD), blank spike, duplicate (DUP)
and a method blank should be set up with each batch of 20 samples. Refer to
Table 1 for the spiking solution levels to use for each matrix spike, matrix spike
duplicate, and blank spike. For the method blank and blank spike, 1.0 g of Teflon
chips should be used. Refer to scheduling sheets and/or project specific QAPP for
further information regarding client specific QC requirements.
7.3 Add 2.5 ml of concentrated nitric acid to all quality control and samples.
Cover all samples and matrix QC with ribbed watch glasses. Keep all samples
covered throughout entire digestion procedure except during reagent additions.
7.4 Pre heat the Hot Block to 90 to 95°C. Place the labeled digestion vessels into the
heating apparatus. Heat the samples at a gentle reflux for 10-15 minutes at 90 to
95°C. Allow the samples to cool.
7.5 Add an additional 2.5 ml of concentrated nitric acid to all quality control and
samples. Heat the samples at a gentle reflux for an additional 30 minutes. Allow
samples to cool.
NOTE: If brown fumes are generated, which indicates oxidation of sample by HNO3,
then repeat step 7.5 until no brown fumes are present.
7.6 Heat at 90 to 950C without boiling until sample volume is reduced to
approximately 2.5mis. Do not allow sample to go to dryness.
7.7 Allow samples to cool. Add 2 ml of DI water and 3 ml of 30% hydrogen peroxide
to each sample and reflux until effervescence subsides.
7.6 Continue to add 30% hydrogen peroxide in 1 ml aliquots with warming until the
effervescence is minimal or until the general sample appearance is unchanged.
Do not add more than a total of 10 mis of 30% hydrogen peroxide.
7.8 Heat at 90 to 950C for 2 hours. Do not allow sample to go to dryness.
7.9 Allow samples to cool. Add 5 ml of concentrated HCI and reflux for an
additional 15 minutes.
7.10 Allow the sample to cool. Dilute to final volume of 50 mis using DI water, cap and
shake vessel. The sample is now ready for analysis by ICAP. If particulate
matter is present, uncap the vessel and filter using Fisher Brand disposable
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syringe and 0.45 micron (um) filter or equivalent. The method blank and blank
spike for the filtered sample's prep group must be filtered as well. All samples are
filtered at the analytical bench.
8.0 QC REQUIREMENTS
For each digestion batch of 20 samples, a serial dilution (performed at the analytical
bench), a post digestion spike (performed at the analytical bench), a matrix spike
(MS), a matrix spike duplicate (MSD), a duplicate (DUP), a blank spike (LCS), and a
method blank should be prepared. Re -digestion is suggested for QC that does not
meet the SGS - Orlando QC limits. The appropriate lab supervisor or lab manager will
notify the analyst of samples that need re- digestion. Please refer to TABLE 1 in this
SOP for spiking volumes and concentrations. Refer to scheduling sheets and/or project
specific QAPP for further information regarding client specific QC requirements.
9.0 GLASSWARE CLEANING
All glassware should be washed with soap and tap water and then soaked in a 5%
nitric acid bath. It should then be rinsed at least 3 times with de -ionized water. Refer
to SOP GN196, current revision forfurther information regarding glassware cleaning.
10.0 DOCUMENTATION REQUIREMENTS
All digestion information should be completed in the Metals Digestion Log. The
information required includes: the sample identification (including bottle number), the
initial sample weight, the final sample volume, the acids (including the lot number
and manufacturer), the spiking solutions used, the observed temperature, the
corrected temperature, the thermometer ID, the digestion vessel lot number, the filter lot
number, the Teflon chips lot number, analysts signature, and the digestion date. The
analyst should write additional information such as unusual sample characteristics in the
comment section.
11.0 SAFETY
The analyst should follow normal safety procedures as outlined in the SGS - Orlando
Laboratory Safety Manual which includes the uses of safety glasses and lab coats. In
addition, all acids are corrosive and should be handled with care. Flush spills with plenty
of water. If acids contact any part of the body, flush with water and contact supervisor.
12.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
12.1 Pollution Prevention
Users of this method must perform all procedural steps in a manner that controls the
creation and/or escape of wastes or hazardous materials to the environment.
The amounts of standards, reagents and solvents must be limited to the amounts
specified in this SOP. All safety practices designed to limit the escape of vapors,
liquids or solids must be followed. All method users must be familiar with the waste
management practices described in Section 12.2.
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12.2 Waste Management
Individuals performing this method must follow established waste management
procedures as described in the Sample and Laboratory Waste Disposal SOP
SAM108, current revision. This document describes the proper disposal of all
waste materials generated during the testing of samples.
13.0 GENERIC DEFINITIONS
13.1 Batch: A group of samples which are similar with respect to matrix and the
testing procedures being employed and which are processed as a unit. A sample
batch is limited to a maximum of 20 samples or 24 hours whichever comes first.
13.2 Blank Spike (BS): An analyte-free matrix spiked with a known amount of
analyte(s), processed simultaneously with the samples through all the steps of the
analytical procedure. Blank Spike Recoveries are used to document laboratory
performance for a given method. This may also be called a Laboratory Control
Sample (LCS).
13.3 Continuing Calibration Verification (CCV): A check standard used to verify
instrument calibration throughout an analytical run. A CCV must be analyzed at the
beginning of the analytical run, after every 10 samples, and at the end of the run.
13.4 Holding Time: The maximum times that samples may be held prior to preparation
and/or analysis and still be considered valid.
13.5 Initial Calibration (ICAL): A series of standards used to establish the working
range of a particular instrument and detector. The low point should be at a level
equal to or below the reporting level.
13.6 Initial Calibration Verification (ICV): A standard from a source different than that
used for the initial calibration. A different vendor should be used whenever
possible. The ICV is used to verify the validity of an Initial Calibration. This may
also be called a QC check standard.
13.7 Matrix Spike (MS): A sample aliquot spiked with a known amount of analyte(s),
processed simultaneously with the samples through all the steps of the analytical
procedure. The matrix spike recoveries are used to document the bias of a
method in a given sample matrix.
13.8 Matrix Spike Duplicate (MSD): A replicate sample aliquot spiked with a known
amount of analyte(s), processed simultaneously with the samples through all the
steps of the analytical procedure. The matrix spike recoveries are used to
document the precision and bias of a method in a given sample matrix.
13.9 Method Blank (MB): An analyte-free matrix to which all reagents are added in the
same volumes or proportions as used in sample processing. The method blank is
processed simultaneously with the samples through all the steps of the analytical
procedure. The method blank is used to document contamination resulting from the
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analytical process.
13.10 Sample Duplicate (DUP): A replicate sample which is used to document the
precision of a method in a given sample matrix.
13.11 Preservation: Refrigeration and/or reagents added at the time of sample
collection (or later) to maintain the chemical integrity of the sample.
14.0 METHOD PERFORMANCE
Method performance is monitored through the routine analysis of negative and positive
control samples. These control samples include method blanks (MB), blank spikes (BS),
matrix spikes (MS), and matrix spike duplicates (MSD). The MB and BS are used to
monitor overall method performance, while the MS and MSD are used to evaluate the
method performance in a specific sample matrix.
Blank spike, matrix spike, and matrix spike duplicate samples are compared to method
defined control limits. Statistical control limits are stored in the LIMS for QA purposes only.
Additionally, blank spike accuracy is regularly evaluated for statistical trends that may be
indicative of systematic analytical errors.
15.0 HOT BLOCK MAINTENANCE
Clean surface area of hot block periodically to prevent sample and reagent build up
on the surface of the block. If the hot block cannot maintain a temperature between 90-
95 degree C or the user experiences any other type of mechanical or electronic error a
service representative will need to be contacted. Any hot block that is not functioning
properly must be tagged as "Out of Service".
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TABLE 1: ICP METALS SPIKING LEVELS
(Suggested levels, may vary depending on instrumentation used)
ELEMENT INITIAL CONC VOLUME USED FINAL CONC FINAL VOL.
(ppm) (ml) (mg/1) (ml)
Ba
200
0.50
2.0
50
Be
5
0.50
.05
50
Cd
5
0.50
.05
50
Cr
20
0.50
.20
50
Cu
25
0.50
.25
50
Co
50
0.50
0.50
50
Mn
50
0.50
0.50
50
V
50
0.50
0.50
50
Zn
50
0.50
0.50
50
As
200
0.50
2.0
50
Se
200
0.50
2.0
50
Pb
50
0.50
0.50
50
TI
200
0.50
2.0
50
Sb
50
0.50
0.50
50
Mo
100
0.25
0.50
50
Sn
100
0.25
0.50
50
Al
200/5000
0.5/0.25
27
50
Fe
200/5000
0.5/0.25
26
50
Mg
5000
0.25
25
50
Ca
5000
0.25
25
50
K
5000
0.25
25
50
Na
5000
0.25
25
50
Ag
5
0.50
0.05
50
Ni
50
0.50
0.50
50
Sr
100
0.25
0.50
50
Ti
100
0.25
0.50
50
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/_1» =1►1I] P:I_1
1.0 Application
Appendix A designed to supplement SOPs MET104.xx and MET105.xx for the preparation
of soil samples for compliance with DoD and certain state -specific projects
2.0 Background
A theory of particulate sampling was developed by geologist Pierre Gy to improve the
quality of data gathered in support of mineral exploration and mining. The MIS approach
described herein is based upon Gy's theories and is applicable to environmental sampling
at contaminated sites.
A large portion of sampling error is a result of compositional and distributional heterogeneity.
Compositional heterogeneity describes the variability of contaminant concentrations
between the particles that make up the population in the sample. This type of
heterogeneity results in fundamental error (FE).
Distributional heterogeneity occurs when particles are not randomly distributed
across the population due to slight spatial variations. Spatial variability will be missed if
all samples are collected from one place. This type of heterogeneity results in grouping
and segregation error (GSE).
Gy found that fundamental error is directly proportionate to maximum particle size and
inversely proportionate to sample size, therefore it is beneficial to collect and analyze a
sample of sufficient size that consists of particulate matter where majority of
contamination is present. In order to manage FE under 15%, particulate matter size must
be under 2 mm and minimum sample mass above 30g.
To minimize GSE, it is imperative to collect sample increments randomly and in enough
locations to capture the spatial variability, even within sample that already has been
collected from the field.
3.0 Subsampling for Metals
Some projects require that metals analysis be performed on the multi -incremental sample
that was collected for 833013. The technique used should be listed in the project QAPP or
SOW. Consult the client if this information is not available.
See flow chart below for various subsampling techniques:
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Air-dry at room temperature approxiimately
1 kg of field sample
Pass through #10 meshsieve
it /
Ring -and -Puck MiII Grinding Required Ring -and -Puck Mill Grinding Prohibited
Grind entire field sample in ring -
and -puck mill
Subsample using Gy's method
Collect about 50g and label
sample for metals arelysis
Digest for ICP or CVAA
Analyze by ICP or CVAA
Gubsomple about 101:Ig using
Gy's method
Grind entire portion by n-orbDr
and pestle to c 250 bum
Pass thrOLigh #60 rnes h sieve
Colectand label sample for
metals analysis
Digest for ICP or CVAA
Arnlyne by IC P or CVAA
If Ring and Puck Mill grinding is required, then proceed with the grinding procedure listed in SOP
OP046 for explosives. The metallic components from the Ring and Puck Mill may introduce
chromium and iron into the sample.
After grinding, place a baking tray on the downdraft table. Transfer the entire sample to the tray.
Shape the sample into an elongated pile with flattened top surface that it is approximately 1 cm
thick. Using a rectangular scoop, collect multiple top -to -bottom cuts across the sample (see
figure below). A minimum of 4 cuts should be made through each sample. Combine the cuts in
an appropriately labeled container. Minimum sample size should be 50 grams. Close the jar and
repeat this procedure for each sample including the MB.
Transfer the samples to the metals department for analysis.
If Ring and Puck Mill grinding is not required then follow the procedure listed below.
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Transfer the sample to a large ziplock bag after it has been air dried and sieved. Sample
should be transferred over the downdraft tables to minimize dust contamination. Seal the
bag and thoroughly mix the sample.
Place a baking tray on the downdraft table. Transfer the entire sample to the tray. Shape the
sample into an elongated pile with flattened top surface that it is approximately 1 cm thick.
Using a rectangular scoop, collect multiple top -to -bottom cuts across the sample (see figure
below). A minimum of 4 cuts should be made through each sample. Combine the cuts in an
appropriately labeled container. Minimum sample size should be 50 grams. Close the jar
and repeat this procedure for each sample including the MB.
Return the remaining sample to the ziplock bag or mixing bowl.
Grind each sample and MB to a particle size less than 250 um with a non-metallic mortar
and pestle.
Place a baking tray on the downdraft table. Sieve each sample through a #60 sieve onto a
tray. Collect and label the samples. Transfer the samples to the metals department for
analysis.
For digestion withdraw approximately 5 g of sieved material. If mortar -and -pestle grinding
was specified per QAPjP, 1 g is sufficient. Follow digestion procedure outlined in the body of this
SOP.
x
�c�. � Tra�r,ro,^aal SuhsarnplE�
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SGS - ORLANDO STANDARD OPERATING PROCEDURE
FN: MET 108.03
Rev. Date: 02/2018
Pages F-76 — F-105
TITLE: METALS BY INDUCTIVELY COUPLED PLASMA ATOMIC
EMISSION SPECTROMETRY (ICP)
REFERENCES: SW846 6010D7 2014
INSTRUMENT. THERMO 6500, SERIAL # 20100903 SSTRACE 1
INSTRUMENT: THERMO 6500, SERIAL # 20103825 SSTRACE 2
AUTOSAMPLER: CETAC 240 POSITION, SERIAL # 031038A520 SSTRACE 1
AUTOSAMPLER: CETAC 240 POSITION, SERIAL # 041048A520 SSTRACE 2
SUGGESTED WAVELENGTH (S): TABLE 2
REVISED SECTIONS: removed all references to Accutest
1.0 SCOPE AND APPLICATION SUMMARY
SW-846 methods, with the exception of required method use for the analysis of method -
defined parameters, are intended to be guidance methods which contain general
information on how to perform an analytical procedure or technique which a laboratory can
use as a basic starting point for generating its own detailed Standard Operating Procedure
(SOP), either for its own general use or for a specific project application. The performance
data included in this method are for guidance purposes only, and are not intended to be
and must not be used as absolute QC acceptance criteria for purposes of laboratory
accreditation.
1.1 This method is applicable for the determination of metals in water, sludges, sediments,
and soils. Elements that can be reported by this method include: Aluminum, Antimony,
Arsenic, Barium, Beryllium, Cadmium, Calcium, Chromium, Cobalt, Copper, Iron, Lead,
Magnesium, Manganese, Molybdenum, Nickel, Potassium, Selenium, Silver,
Sodium, Strontium, Titanium, Thallium, Tin, Vanadium, and Zinc.
1.2 Sample matrices are pretreated following SW846 and EPA methods for digestion of
soil, sediment, sludge or water samples. Refer to specific metals department digestion
SOP's for more information on digestion techniques.
1.3 This inductively coupled argon plasma optical emission spectrometer (s) (ICP-OES) uses
an Echelle optical design and a Charge Injection Device (CID) solid-state detector to
provide elemental analysis. Control of the spectrometer is provided by PC based FEVA
software. In the instrument, digested samples are introduced into the Thermo 6500 ICP,
passed through a nebulizer and transported to a plasma torch. The element -specific
emission spectra are produced by a radio frequency inductively coupled plasma. The
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spectra are dispersed by a spectrometer, and the intensities of the emission lines are
monitored with the solid state detector.
1.4 Reporting limits (RL)(LLOQ) are based on the extraction procedure. Reporting limits
may vary depending on matrix complications, volumes and by client needs, but the
reporting limits must always be verified with a low check which meets the criteria
outlined in this SOP. Solid matrices are reported on a dry weight basis. Refer to
table 1 of this SOP for SGS - Orlando typical reporting limits. Refer to scheduling
sheets and/or project specific QAPP for further information regarding client specific
reporting limits.
1.5 MDLs must be established for all analytes, using a solution spiked at approximately 3
to 5 times the estimated detection limit. To determine the MDL values, take seven
replicate aliquots of the spiked sample and process through the entire analytical
method. The MDL is calculated by multiplying the standard deviation of the replicate
analyses by 3.14, which is the student's t value for a 99% confidence level. MDLs
must be determined approximately once per year for each matrix and
instrument. Please refer to SGS - Orlando QA SOP QA020, current version for
further information regarding method performance criteria and experimental method
detection limits.
MDLs are generated for each matrix on both ICP instruments. The higher of the
two statistically calculated MDL's is entered into LIMS as the MDL. The verified
MDLs are stored in the LIMS and must be at least 2 to 3 times lower than the RL.
Exceptions may be made on a case by case basis; however, at no point shall the
MDL be higher than the reported RL.
1.6 LLOQ verification. LLOQ is the lowest point of quantitation. The LLOQ is initially
verified by the analysis of 7 replicate samples, spiked at the LLOQ and processed
through all preparation and analysis steps of the method. The mean recovery should
be within +/- 35 percent of the true value with an RSD < 20 percent.
1.7 Ongoing Lower limit of quantitation (LLOQ) check sample. The lower limit of
quantitation check sample should be analyzed on a quarterly basis to demonstrate
the desired detection capability. The LLOQ sample is carried through the entire
preparation and analytical procedure. The mean recovery should be within +/- 35
percent of the true value with an RSD < 20 percent.
1.8 Compounds detected at concentrations between the RL and MDL are quantitated
and qualified as estimated values and reported with either a "J" or "I" qualifier. Some
program or project specifications may require that no values below the RL be reported.
1.9 Instrument Detection Limits (IDL). It is suggested that IDL's be completed upon
initial instrument installation, whenever instrument conditions have significantly
changed, or at a minimum annually. Instrument detection limits can be estimated as
the mean of the blank results plus 3 times the standard deviation of 10 replicate
analyses of the reagent blank solution. (use zero for the mean if the mean is
negative) Each IDL measurement shall be performed as though it were a separate
analytical sample. IDLs shall be determined and reported for each wavelength used
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in the analysis of the samples.
2.0 PRESERVATION AND BOTTLEWARE
All samples should be preserved with nitric acid to a pH of <2 at the time of collection. All
sample pH are checked in sample receiving and within the metals department. Samples that
are received with a pH >2 must be preserved to pH <2 and held for 24 hours prior to
metals digestion to dissolve any metals that absorb to the container walls. Refer to SOP
SAM101, current revision for further instruction. Final pH of TCLP extracts are checked
and recorded in SGS - Orlando Extractions Department. Please refer to TCLP (1311)
fluid determination logbook and SPLP (1312) fluid determination logbook for further
information. TCLP extracts received from SGS - Orlando Extractions Department are
prepared as soon as possible, no longer than 24 hours from time of receipt. If precipitation is
observed during the sample preparation process the sample(s) are immediately re -prepped
on dilution until no precipitation is observed. Samples received for dissolved metals
analysis should be filtered and preserved to pH<2 as soon as possible and held for 24 hours
prior to digestion. Refer to SGS - Orlando Sample Filtration Logbook for further
information.
All soil samples must be stored in a refrigerator at < 60C upon receipt. Refer to SOP
SAM101, current revision for further instruction.
All bottleware used by SGS - Orlando is tested for cleanliness prior to shipping to clients.
Analysis results must be less than one half the reporting limit (LLOQ) to be acceptable.
Refer to SOP SAM104, current revision for further instruction.
3.0 HOLDING TIME AND BATCH SIZE
All samples must be prepared and analyzed within 6 months of the date of collection. Refer
to appropriate SGS - Orlando digestion SOP, current revision for batch size criteria.
4.0 INTERFERENCES
Several types of interferences can cause inaccuracies in trace metals determinations by ICP.
These interferences are discussed below.
4.1 Spectral interferences are caused by overlap of a spectral line from another
element, unresolved overlap of molecular band spectra, background contribution from
continuous or recombination phenomena, and background contribution from stray light
from the line emission of high concentration elements. Corrections for these
interferences can be made by using interfering element corrections, by choosing an
alternate analytical line, and/or by applying background correction points. The
locations selected for the measurement of background intensity will be determined
by the complexity of the spectrum adjacent to the wavelength peak. The locations
used for routine measurement must be free of off-line spectral interference or
adequately corrected to reflect the same change in background intensity as occurs at
the wavelength peak.
Note: Refer to section 17.0 of this SOP for further instruction regarding interfering
element correction factor generation.
4.2 Physical interferences can be caused by changes in sample viscosity or surface tension,
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by high acid content in a sample, or by high dissolved solids in a sample. These
interferences can be reduced by making sample dilutions.
4.3 Matrix interferences in high solid samples can be overcome by using an internal
standard. Yttrium/Indium mix is used for the Thermo 6500 ICP. The concentration must
be sufficient for optimum precision but not so high as to alter the salt concentration
of the matrix. The element intensity is used by the instrument as an internal
standard to ratio the analyte intensity signals for both calibration and quantitation.
4.4 Chemical interferences are not pronounced with ICP due to the high temperature of
the plasma, however if they are present, they can be reduced by optimizing the
analytical conditions (i.e. power level, torch height, etc.).
5.0 APPARATUS
5.1 Currently there are two solid state ICPs available for use in the lab. Both are Thermo
6500 ICP units. These units have been optimized to obtain lower detection limits
for a wide range of elements. Since they are solid state systems, different lines may
be included for elements to obtain the best analytical results. However, the lines
which are normally included in the normal analysis program are shown in Table 2.
5.2 Instrument auto samplers. For random access during sample analysis.
5.3 Class A volumetric glassware and pipettes.
5.4 Polypropylene auto sampler tubes.
5.5 Eppendorf Pipette (s) - Pipette (s) are checked daily for accuracy and to ensure they
are in good working condition prior to use. Volumes are checked at 100% of
maximum volume (nominal volume). Pipettes are checked within the metals
department and results are stored electronically in the "Pipette Calibration Log".
Refer to SOP QA006, current revision for further information regarding pipette
calibration. BIAS: mean must be within 2% of nominal volume. Precision: RSD
must be < 1 % of nominal volume based on three replicates.
5.6 Fisher Brand 0.45 micron (um) filter or equivalent. Filter lots are checked for
cleanliness through the Method Blank process. All Method Blank analytical results
must be less than one half the reporting limit(LLOQ) to be acceptable, if not, the
contaminated lot must be identified and removed from laboratory use. Samples
filtered through the contaminated filters must be re -filtered through acceptable filters.
5.7 Fisher Brand disposable 10 ml syringes or equivalent. Syringe lots are checked
for cleanliness through the Method Blank process. All Method Blank results must be
less than one half the reporting limit (LLOQ)to be acceptable, if not, the
contaminated lot must be identified and removed from laboratory use. Samples
filtered through the contaminated syringes must be re -filtered through acceptable
syringes.
5.8 Data System
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Microsoft Windows XP Professional Version 2002 Instrument
software SST — Thermo iTEVA version 2.8.0.89
Instrument software SST2 — Thermo iTEVA version 2.7.0.87
5.8.1 A computer system interfaced to the Thermo 6500 ICP that allows for the
continuous acquisition and storage of all data obtained throughout the
duration of the analytical run sequence.
5.8.2 Data is archived to a backup server for longterm storage.
6.0 REAGENTS
All chemicals listed below are trace metal grade unless otherwise specified. Refer to
Acid Certificate of Analysis logbook for Certificates of Analysis and compliance with the
specifications of the grade listed. SGS - Orlando produces DI water to the specifications for
the ASTM Type II standard designation based on the system manufacturer's performance
specifications. The DI water is used exclusively for laboratory purposes. De -ionized (DI)
water should be used whenever water is required. Refer to SOP QA037, current revision
for more information regarding testing and monitoring. Refer to the Metals Department
Standard Prep Logbook for the make-up and concentrations of standards and stock
solutions being used within this SOP. Some of the information included in the logbook is as
follows: standard name, elements in mix, manufacturer, lot number, parent expiration date,
acid matrix, stock concentration, volume of standard added, total volume, final prepared
concentration, prep date, initials, MET number, and prepared standard expiration date.
Standards and prepared reagents must be prepared every 6 months or before stock
standard expiration date, whichever comes first. Refer to tables 3 through 7 of this SOP for
concentration levels of standards used. Unless otherwise approved, the calibration curve
must contain 3 points determined by a blank and a series of standards representing the
elements of interest.
6.1 2.5 ppm Yttrium and 10 ppm Indium internal standard, made from ICP quality standard.
6.2 Hydrochloric acid, trace metals grade.
6.3 Nitric Acid, trace metals grade.
6.4 ICP quality standard stock solutions are available from Inorganic Ventures, Spex,
Plasma Pure, Ultra, Environmental Express, or equivalent.
6.5 Calibration Standards. These can be made up by diluting the stock solutions to the
appropriate concentrations. The calibration standards should be prepared using the
same type of acid (s) and at approximately the same concentration as will result in
the samples following sample preparation.
6.5.1 For calibration and quantitation an internal standard (Yttrium/Indium) is used to
limit nebulization problems. If it is known that the samples contain a significantly
different acid matrix, the samples must be diluted so that they are in a similar
matrix to the curve. All sample results are referenced to the initial calibration
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blank (ICB) Internal Standard counts. The criteria is 60-125 percent of the
initial calibration blank (ICB) counts. If the internal standard counts fall outside
these criteria matrix effects must be suspected and the sample diluted until it
meets the criteria or footnoted in LIMS as suspected matrix interference.
6.5.2 Standards must be prepared so that there is minimal spectral interference between
analytes.
Note: All Ag stock and intermediate solutions must be stored away from
direct sunlight.
6.6 Analytical Quality Control Solutions.
All of the solutions below are prepared by adding either mixed or single element
metals solutions to a solution prepared using the same type of acid (s) and at
approximately the same concentration as will result in the samples following sample
preparation.
6.6.1 Blank (Calibration, ICB, CCB)
This reagent blank contains Nitric Acid at 3 percent and Hydrochloric Acid
at 5 percent.
6.6.2 Initial Calibration Verification solution.
This standard solution must be made from a different source than the
calibration curve. The concentrations for each element must be within the
range of the calibration curve and should be approximately at the midpoint of
the curve. This solution is used to verify the accuracy of the initial
calibration. Levels for the ICV standard are shown in Table 4.
6.6.3 Continuing Calibration Verification solution.
The metals concentrations for this standard should be at approximately the mid-
point of the calibration curve for each element. This standard should be
prepared from the same source that is used for the calibration curve. Levels for
the CCV standard are shown in Table 5.
6.6.4 Spectral Interference Checks (SIC). Two types of SIC checks are used.
Individual element SIC are performed when the instrument is initially set up,
and every six months thereafter. The mixed element SIC solution is used
daily to check that the instrument is free from interference from elements
typically observed in high concentration and to check that interference
corrections (IEC) are still valid.
6.6.4.1 Single element interference checks — At a minimum, single
element SIC checks should be performed for the following elements:
Aluminum 500 mg/I; Barium 4 mg/I; Calcium 500 mg/I; Copper 4 mg/I;
Iron 500 mg/I; Magnesium 500 mg/I; Manganese 4 mg/I; Molybdenum 4
mg/I; Sodium 1000 mg/I; Nickel 4 mg/I; Selenium 4 mg/I; Silicon 50 mg/I;
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Tin 4 mg/l; Vanadium 4 mg/1 and Zn 4 mg/I.
Mixed element SIC solution — The mixed element SIC solution is used
as an ongoing daily check of freedom from spectral interferences. The
mixed element SIC contains the following elements: Aluminum 500 mg/I;
Calcium 500 mg/I; Iron 200 mg/I; Magnesium 500 mg/I.
The absolute value of the concentration observed for any unspiked
analyte in the single element SIC checks must be less than 2 times the
analytes LLOQ. The concentration of the SIC checks are suggested, but
become the highest reportable concentration in the sample analysis
and cannot be higher than the highest established linear range.
Samples with concentrations of elements higher than the SIC check
must be diluted until the concentration is less than the SIC check
solution. Reanalysis of a diluted sample is required even if the high
concentration element is not required to be reported for the specific
sample, since the function of the SIC check is to evaluate spectral
interferences on other elements. The daily mixed element SIC
solution is analyzed daily after calibration. The concentration measured
for any target analytes must be less than +/- the LLOQ. For spiked
elements, the analyzed results must be within 20 percent of the true
value for SIC check and within 10 percent for linear range check. If this
criterion cannot be met then sample analysis may not proceed until the
problem is corrected, or the LLOQ is raised to twice the
concentration observed in the SIC solution. The only exceptions are
those elements that have been demonstrated and documented as
contaminants in the SIC solutions. Levels for the SIC and mixed SIC
can be found on tables 9 and 10.
6.7 CRIA Standard Solution (Also referred to as LLCCV)
The CRIA standard contains the elements of interest at levels equal to SGS -
Orlando quantitation limits (RL). Please refer to Table 6 for list of elements of interest
and concentration levels for the CRIA. If special client reporting limits are requested,
then low checks corresponding to those reporting limits must also be analyzed.
6.8 Matrix Spike, Matrix Spike duplicate, and Spike Blank Solution.
This solution is prepared by adding either mixed or single element metals solutions
to a solution containing 3 percent nitric acid and 5 percent hydrochloric acid and diluting
to a fixed final volume with this acid mixture. Spiking solution (s) must be added to
the spike blank, matrix spike, and the matrix spike duplicate prior to digestion. Levels
for the MS and MSD and Spike Blank standard are shown in Table 7.
6.9 Liquid Argon or Argon Gas. (99.999% purity)
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7.0 ANALYTICAL PROCEDURE
Note: Please refer to section 8 of this SOP for further detail on quality control standards. Please
refer to scheduling sheets and/or project specific QAPP for further information regarding
client specific QC requirements.
7.1 General procedure on how to operate the Thermo 6500 is described below. Refer to
the Thermo 6500 operation manual for further details.
7.2 Before starting up the instrument, make sure that the pump tubing is in good condition,
the torch assembly, the nebulizer, and the spray chamber are clean, the dehumidifier (if
used) is filled with DI water up to the level between Minimum and Maximum, and that
there are no leaks in the torch area.
7.3 Turn on the recirculating cooler. Verify that the argon is turned on and there is enough for
the entire days analytical run.
7.4 Tighten the pump platens and engage the peristaltic pump. Make sure sample and
internal standard solutions are flowing smoothly.
7.5 Put a new solution of acid rinse into the rinse reservoir. The composition of the rinse
solution may be periodically changed to minimize sample introduction problems and
sample carryover. If internal standard is being used, make sure that sufficient
amount of internal standard is prepared for the entire analytical run.
7.6 Start up the instrument following the sequence show below.
7.6.1 Double click the iTEVA Control Center Icon on the desktop. Type
admin in User Name field, and then click OK.
7.6.2 Once the iTEVA Control Center window is opened, click on Plasma Icon at
status bar area. Then click on Instrument Status to check the interlock
indicators (torch compartment, purge gas supply, plasma gas supply, water flow
and exhaust should be in green; drain flow and busy should be in gray) and
the Optics Temperature. (It should be around 380C.) Click on the Close box.
7.6.3 Click Plasma On. When the plasma is on, click close. Let the instrument warm up
for 15 to 20 minutes before starting the analysis. New tubing may take an
hour to stabilize.
7.7 Torch Alignment and Auto Peak
7.7.1 If the torch has been cleaned, then the torch alignment procedure must be
performed.
7.7.2 Open the method and then click on Sequence tab, then click on List View Icon
until you reach rack display.
7.7.3 Go to S-6 position (you can assign any position in the rack for torch alignment),
then right click to select Go to empty sample S:6. (Now, the auto sampler tip
moves from Rinse to this position).
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7.7.4 Click on Analysis tab, then select Torch Alignment from Instrument drop
down menu. There will be a pop up dialog box present. Click Run. Then
there will be another dialog pop up box (This is a reminder for Torch Alignment
Solution (2 ppm Zn)), click Ok. Now, the instrument is initializing an
automated torch alignment. It takes about 7 minutes to complete this step.
Progress is indicated in the progress bar.
7.7.5 After torch alignment is complete, click Close. Click on Sequence tab,
then followed by List View Icon.
7.7.6 Go to Rinse position at rack display, right click to select Go to rinse and let it rinse
for approximately 5 minutes.
7.7.7 Perform Auto Peak
7.7.8 It is recommended that the Auto Peak Adjust procedure be performed daily
prior to calibration. A standard that contains all of the lines of interest is used and
the system automatically makes the appropriate fine adjustments. (High standard
solution should be used for this process.)
7.7.9 Click Sequence tab, then click on List View Icon until the rack is displayed.
7.7.10 Go to S-5 position (you can assign any position in the rack for auto peak adjust),
then right click to select Go to empty sample S:5. (Now, the auto sampler tip
moves from the Rinse position to this position). Click on Analysis tab. All
elements result is shown in the display area. From Instrument drop down
menu, select Perform Auto Peak. There will be a pop up dialog box present.
Highlight "All Elements", and then click Run. Then there will another pop up
dialog box (This is a reminder for Auto Peak Solution), click Ok. Now, the
instrument is performing auto peak adjust. It takes about 5 minutes to complete
this process. The Auto Peak dialog box will show a green check mark in front
of "All Elements", which indicates Auto Peak is complete.
7.8 Open the method and start up the run.
7.8.1 Click on Analyst Icon at the workspace. Go to the method and choose Open
from the drop down menu. Select the method with the latest revision number.
7.8.2 Go to Method tab at the bottom of left hand corner to click on Automated
Output at the workspace area. Type a filename in Filename field in the data
display area (i.e.: SA101010M1, starts with SA, then followed by MM-DD-YY,
then M1; M1 indicates the first analytical run for that day, then followed by M2,
M3 and so on for the second and third runs.) Click on Apply To All Sample
Types.
7.8.3 Click on Sequence tab at the bottom of left hand corner. From Auto Session
drop down menu bar, click on New Auto sampler to create a sequence. This will
pop up a dialog box, then click on New and fill in number of samples (i.e.: 100) in
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the Number of Samples field and the sample I.D. (leave this field empty) in
Sample Name field. Type a sequence name (i.e. : SEQ101010M1, starts with
SEQ, then MM-DD-YY, then M1; M1 indicates the first analytical run for that
day, then followed by M2, M3 and so on for the second and third runs) in the
Sequence Name field. Click Ok, then put in "0" as settle time between sequences,
and click Ok.
7.8.4 Right click on Untitled (Cetac ASX-520 Enviro 5 Named Rack is the rack
that is currently used) at the workspace area, click on Auto -Locate All to locate
all sample positions.
7.8.5 Double click on Untitled again, then click on the sequence name (i.e. :
SEQ101010M1), on the data display area, type the sequence in Samplename
column, dilution factor (if needed) in CorrFact column, check the box in front of
Check column, and select an appropriate check table.
7.8.6 Once done with creating sequence, go to Method drop down menu and save
all changes as Save As. There will be a Save a Method dialog box present,
go to the save option to check on "Overwrite Method and bump revision
number" box, and then click Ok.
7.8.7 Go to Sequence tab, click on List View Icon from tool bar, then click on
Connect Autosampler to PC and Initialize Icon.
7.8.8 See table 8 for a typical run sequence.
7.9 Calibrate the instrument as outlined below. See table 3 for calibration standards
concentrations. This calibration procedure is done a minimum of once every 24 hours.
The calibration standards may be included in the auto sampler program or they may
be run manually from the Calibrate Instrument (graduated cylinder) icon located on
the Analyst tab. The instrument may be calibrated using a single point standard and a
calibration blank or a multipoint calibration. If a multipoint calibration is used a minimum
of three standards are required. All curves must be determined from a linear
calibration prepared in the normal manner using the established analytical procedure
for the instrument. Refer to instrument manual for further detail. Three exposures
will be used with a percent relative standard deviation of less than 5 percent. The
resulting correlation coefficient must be >0.995. If the calibration curves do not meet
these criteria, analysis must be terminated, the problem corrected, and instrument
re -calibrated. Correlation coefficients, slopes, and y-intercepts for each wavelength are
printed and included in each analytical data package.
7.10 Initial Calibration Verification Standard (ICV).
After each calibration, a standard from a different source than the calibration
standard shall be analyzed. For the ICV, all elements to be reported must be within
10 percent of the true value for 6010D. If the ICV is outside these criteria then the
analysis must be terminated, problem corrected, and the instrument re -calibrated.
7.11 After analyzing the ICV, the ICB must be analyzed. The results of the ICB must be
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less than one half the reporting limit (LLOQ). The instrument blank may be failing the
criteria due to contamination or instrument drift. Samples associated with the failing
blank shall be evaluated as to the best corrective action for each particular sample.
This may include reanalyzing the samples bracketed by the failing blank, qualifying the
results with a "B" or "V" qualifier, or raising the reporting limit (LLOQ) for all samples to
greater than two times the background concentration.
7.12 Before analyzing any real world samples the CRIA (also referred to as LLCCV) must
be analyzed. The CRIA contains elements of interest at the reporting limit. The CRIA
will be analyzed at the beginning and end of each analytical run. For all elements the
results must be within 20 percent of the true value. Refer to scheduling sheets and/or
project specific QAPP for further information regarding client specific reporting limits
(CRIA Requirement). If the initial CRIA fails no samples associated with the failing
CRIA can be reported, and the CRIA should be reanalyzed for the failing elements. If
the closing CRIA fails the criteria, the samples associated with the CRIA shall be
evaluated as to the best corrective action for each particular sample. This may include
reanalyzing the samples associated with the CRIA, or qualifying the results in LIMS.
7.13 Before analyzing any real world samples, the Mixed element SIC solution must be
analyzed. The mixed element SIC solution is used as an ongoing daily check of freedom
from spectral interferences. The mixed element SIC contains the following elements:
Aluminum 500 mg/I; Calcium 500 mg/I; Iron 500 mg/I; Magnesium 500 mg/I.
The daily mixed element SIC solution is analyzed daily after calibration. The
concentration measured for any target analytes must be less than +/- the LLOQ. For
spiked elements, the analyzed results must be within 20 percent of the true value for
SIC check and within 10 percent for linear range check. If this criterion cannot be met
then sample analysis may not proceed until the problem is corrected, or the LLOQ is
raised to twice the concentration observed in the SIC solution. The only exceptions
are those elements that have been demonstrated and documented as contaminants
in the SIC solutions. Refer to section 17.0 of this SOP for Interfering Element Correction
(IEC) procedure.
7.14 After the initial analytical quality control has been analyzed, the samples and the
preparation batch matrix quality control shall be analyzed. Each sample analysis must
be a minimum of 3 readings using at least a 5 second integration time. Between
each sample, flush the nebulizer and the solution uptake system with a blank rinse
solution for at least 60 seconds or for the required period of time to ensure that analyte
memory effects are not occurring.
7.15 Analyze the continuing calibration verification solution and the continuing calibration
blank after every tenth sample and at the end of the sample run. If the CCV solution is
not within 10 percent of the true value for method 601 OD, the CCV shall be reanalyzed to
confirm the initial value. If the CCV is not within criteria after the reanalysis, no samples
can be reported in the area bracketed by the failing CCV. Immediately following the
analysis of the CCV the CCB shall be analyzed. The results of the CCB must be
less than one half the reporting limit (LLOQ) for all elements. The instrument blank
may be failing the criteria due to contamination or instrument drift. Samples
associated with the failing blank shall be evaluated as to the best corrective action
for each particular sample. This may include reanalyzing the samples bracketed by
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the failing blank, qualifying the results with a "B" or "V" qualifier, or raising the reporting
limit (LLOQ) for all samples to greater than two times the background concentration.
13.1 One sample per preparation batch, or whenever matrix interferences are suspected
for a batch of samples, a serial dilution (SDL) must be prepared. For the serial
dilution, a 1:5 dilution must be made on the sample. The results of the 1:5 dilution
shall agree within 20 percent of the true value as long as the analyte concentration is
within the linear range of the instrument and sufficiently high (minimally, a factor of 25
times greater than the LLOQ). If the results are outside these criteria then matrix
interference should be suspected and the proper footnote entered into LIMS. A post
digestion spike (PDS) must be performed if the SDL fails. The PDS must recover within
+ 25 percent for method SW846-6010D. If the PDS is outside these limits then matrix
interference must be suspected and the proper footnote entered into LIMS.
13.2 The upper limit of quantitation may exceed the highest concentration calibration point
and can be defined as the "linear range". Sample results above the linear range shall
be diluted under the linear range and reanalyzed. Following calibration, the laboratory
may choose to analyze a standard (or mixed standard solution) at a higher
concentration than the high standard used in the calibration curve. The standard must
recover within 10 percent of the true value, and if successful, establishes the linear
range. The linear range standards must be analyzed in the same instrument run as the
calibration they are associated with, but may be analyzed anywhere in the run.
Samples following a sample with high concentrations of analyte (s) must be examined
for possible carryover. Verification may be done by rinsing the lines with an acid solution
and then reanalyzing the sample. A limit check table is built into the autosampler file so
that samples exceeding the standardization range are flagged on the raw data.
13.3 After the instrument is optimized and all initial QC has been run, click on Run Auto -
Session Icon to start the analytical run sequence.
If you need to add or delete samples once the run is started, follow the steps
shown below.
Click on Sequence tab, then click on List View Icon at the tool bar. There is
the sequence table shown on the display area.
Click on Add Samples Icon. This will pop up a dialog box, and then fill in
number of samples that need to be added. Click Ok. By doing this, samples will
be added to the end of the current sequence without a rack location.
7.15.1 On the Samplename column type in the sample I.D., correction factors,
and check tables. Click on Auto Locate All.
7.15.2 The added samples will be analyzed at the end of the original sequence run
order unless they are assigned a different run order.
7.15.3 Deleting Samples
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7.15.4 Click on Sequence tab, and then click on List View Icon under the
sequence display area.
7.15.5 Highlight all samples that need to be deleted and then click on the Delete
Samples icon.
7.16 When the analysis is completed export the data to LIMS following the procedure
outlined below.
7.16.1 Double click on ePrint Icon on desktop. There will be a LEADTOOLS
ePRINT pop up box, click on Finish Jobs and OK boxes.
7.16.2 Double click the PDF Icon on the desktop; the PDF file will be present as
Document_#. Right click on that file, select rename to change the filename to
an assigned analytical run I.D. (i.e.: MA9000). This is the raw data file for
MA9000.
7.16.3 Drop the raw data to the LIMS Data Drop icon located on the desktop.
7.16.4 By completing the above steps, the raw data (i.e.: MA9000) can be viewed
and/or printed from the Raw Data Search function.
7.16.5 Go to Analysis tab, right click on sample header, and select export all samples.
A pop up dialog box will come up, type in the analytical run I.D. (i.e.:
SA101010M1) and click Ok. Go to Lims Export folder located on the
desktop, right click on analytical run and change extension from .TXT to .ICP.
Open the analytical file and make any necessary changes, such as deleting
any samples that need to be re -run on dilution. Save the file. Drop the data file
to the LIMS Data Drop icon located on the desktop. This will then send the
export file to LIMS for review.
7.17 The data can be evaluated by running an automated data evaluation program, which
will help to generate quality control summary pages. Each run must be evaluated as
quickly as possible to make sure that all required quality control has been analyzed.
With each data package include: cover sheet, copies of all prep sheets, autosampler
run sequence, dilution sheets, and raw data. Label each folder with MA#, instrument run
I.D., instrument used, and date.
7.18 At the end of the analysis day the ICP must be shutdown using the following sequence.
7.18.1 Place the auto sampler tip in the rinse cup and rinse in a mixed solution of
approximately 5 percent nitric acid and 5 percent hydrochloric acid for 10
minutes and then in DI water for 20 minutes.
7.18.2 Turn off the plasma by clicking on the Plasma Icon and then by clicking
Plasma Off.
7.18.3 Close all iTeva programs/windows.
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7.18.4 Release the tension on the sample pump platens.
7.18.5 Turn off recirculating chiller.
8.0 QUALITY CONTROL
This section outlines the QA/QC operations necessary to satisfy the analytical requirements
for method SW846 6010D. Please refer to scheduling sheets and/or project specific QAPP
for further information regarding client specific QC requirements. Check with the area
supervisor or lab manager for any non -compliant quality control for further information.
8.1 Initial Calibration Verification Standard (ICV).
After each calibration, a standard from a different source than the calibration
standard shall be analyzed. For the ICV, all elements to be reported must be within
10 percent of the true value for 6010D. If the ICV is outside these criteria then the
analysis must be terminated, problem corrected, and the instrument re -calibrated.
8.2 Continuing Calibration Blank/Initial Calibration Blank.
Analyze the Initial calibration blank solution at the beginning of each run and the
continuing calibration blank after every tenth sample and at the end of the sample
run. The ICB/CCB must be less than one half the reporting limit (LLOQ) for each
element. The instrument blank may be failing the criteria due to contamination or
instrument drift. Samples associated with the failing blank shall be evaluated as to the
best corrective action for each particular sample. This may include reanalyzing the
samples bracketed by the failing blank, qualifying the results with a "B" or "V" qualifier,
or raising the reporting limit (LLOQ) for all samples to greater than two times the
background concentration.
8.3 Low Standard Check (CRIA or LLCCV).
Before analyzing any real world samples the CRIA (also referred to as LLCCV) must
be analyzed. The CRIA contains elements of interest at the reporting limit. The CRIA
will be analyzed at the beginning and end of each analytical run. For all elements the
results must be within 20 percent of the true value. Refer to scheduling sheets and/or
project specific QAPP for further information regarding client specific reporting limits
(CRIA Requirement). If the initial CRIA fails no samples associated with the failing
CRIA can be reported, and the CRIA should be reanalyzed for the failing elements. If
the closing CRIA fails the criteria, the samples associated with the CRIA shall be
evaluated as to the best corrective action for each particular sample. This may include
reanalyzing the samples associated with the CRIA, or qualifying the results in LIMS.
8.4 ICSA (Mixed SIC Solution)
Before analyzing any real world samples, the Mixed element SIC solution must be
analyzed. The mixed element SIC solution is used as an ongoing daily check of freedom
from spectral interferences. The mixed element SIC contains the following elements:
Aluminum 500 mg/I; Calcium 500 mg/I; Iron 500 mg/I; Magnesium 500 mg/I.
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The daily mixed element SIC solution is analyzed daily after calibration. The
concentration measured for any target analytes must be less than +/- the LLOQ. For
spiked elements, the analyzed results must be within 20 percent of the true value for
SIC check and within 10 percent for linear range check. If this criterion cannot be met
then sample analysis may not proceed until the problem is corrected, or the LLOQ is
raised to twice the concentration observed in the SIC solution. The only exceptions
are those elements that have been demonstrated and documented as contaminants
in the SIC solutions. Refer to section 17.0 of this SOP for Interfering Element Correction
(IEC) procedure.
8.5 Continuing Calibration Verification.
Analyze the continuing calibration verification solution and the continuing calibration
blank after every tenth sample and at the end of the sample run. If the CCV solution is
not within 10 percent of the true value for method 601 OD the CCV must be reanalyzed
to confirm the initial value. If the CCV is not within criteria after reanalysis no samples
can be reported in the area bracketed by the failing CCV.
8.6 Method Blank.
The laboratory must digest and analyze a method blank with each batch of samples.
The method blank must contain elements at less than one half the reporting limit
(LLOQ) for each element. The exception to this rule is when the samples to be
reported contain greater than 10 times the method blank level. In addition, if all the
samples are less than a client required limit and the method blank is also less than
that limit, then the results can be reported as less than that limit. Samples associated
with the contaminated blank shall be evaluated as to the best corrective action for
each particular sample. This may include reanalyzing the samples, re -digesting and
reanalyzing the samples, qualifying the results with a "B" or W" qualifier, or raising the
reporting limit (LLOQ) to greater than two times the background concentration,
8.7 Blank Spike Sample.
The laboratory must digest and analyze a spike blank sample with each batch of
samples. Blank Spikes must be within 20 percent of the true value for method
SW846-6010D. If the lab control is outside of the control limits for a reportable
element, all samples must be re- digested and reanalyzed for that element. The
exception is if the lab control recovery is high and the results of the samples to be
reported are less than the reporting limit (LLOQ). In that case, the sample results may
be reported with no flag. For solid standard reference materials (SRMs) + 20 percent
accuracy may not be achievable and the manufacturer's established acceptance
criterion should be used for all soil SRMs.
8.8 Matrix Spike and Matrix Spike Duplicate Recovery.
The laboratory must digest and analyze a matrix spike and matrix spike duplicate
with each batch of samples. The matrix spike recovery is calculated as shown below
and must be within 20 percent of the true value for method SW846-601 OD. If a matrix
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spike is out of control, then the results must be flagged with the appropriate footnote.
If the matrix spike amount is less than one fourth of the sample amount, then the
sample cannot be assessed against the control limits and must be footnoted to that
effect.
Note: Both the matrix spike amount and the sample amount are calculated to the IDL
for any given element. Any value less than the IDL is treated as zero.
(Spiked Sample Result - Sample Result�x 100 = matrix spike
recovery Amount Spiked
8.9 Matrix Duplicate/Matrix Spike Duplicate Relative Percent Difference.
The laboratory must digest a duplicate with each batch of samples. The relative
percent difference (RPD) between the duplicate and the sample must be assessed
and must be < 20 percent for sample results at or above the reporting limit (LLOQ). If
the RPD is outside the 20 percent criteria the results must be qualified in LIMS.
RPD's are also calculated in LIMS for sample results below the reporting limit
(LLOQ). RPD's outside the 20 percent criteria are not considered failing and LIMS
automatically footnotes these as "RPD acceptable due to low duplicate and sample
concentrations."
Note: Both the duplicate amount and the sample amount are calculated to the IDL for
any given element. Any value less than the IDL is treated as zero.
(/Sample Result - Duplicate Result, x 100 = Duplicate
RPD (Sample Result + Duplicate Result)/2
8.10 Serial Dilution Analysis and Post Digestion Spike.
One sample per preparation batch, or whenever matrix interferences are suspected
for a batch of samples, a serial dilution (SDL) must be prepared. For the serial
dilution, a 1:5 dilution must be made on the sample. The results of the 1:5 dilution
shall agree within 20 percent of the true value as long as the analyte concentration is
within the linear range of the instrument and sufficiently high (minimally, a factor of 25
times greater than the LLOQ). If the results are outside these criteria then matrix
interference should be suspected and the proper footnote entered into LIMS. A post
digestion spike (PDS) must be performed if the SDL fails. The PDS must recover within
+ 25 percent for method SW846-6010D. If the PDS is outside these limits then matrix
interference must be suspected and the proper footnote entered into LIMS.
(Sample Result -Serial DiL Result�x 100 = Serial Dilution
RPD Sample Result
8.11 Linear Calibration ranges.
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The upper limit of quantitation may exceed the highest concentration calibration point
and can be defined as the "linear range". Sample results above the linear range shall
be diluted under the linear range and reanalyzed. Following calibration, the laboratory
may choose to analyze a standard (or mixed standard solution) at a higher
concentration than the high standard used in the calibration curve. The standard must
recover within 10 percent of the true value, and if successful, establishes the linear
range. The linear range standards must be analyzed in the same instrument run as the
calibration they are associated with, but may be analyzed anywhere in the run.
Samples following a sample with high concentrations of analyte (s) must be examined
for possible carryover. Verification may be done by rinsing the lines with an acid solution
and then reanalyzing the sample. A limit check table is built into the autosampler file so
that samples exceeding the standardization range are flagged on the raw data.
8.12 Sample RSD
For samples containing levels of elements greater than five times the reporting
limits (LLOQ), the relative standard deviation for the replicates should be less than
5%. If not, reanalyze the sample. If upon reanalysis, the RSD's are acceptable then
report the data from the reanalysis. If RSD's are not acceptable upon reanalysis,
then the results for that element should be footnoted that there are possible
analytical problems and/or matrix interference indicated by a high RSD between
replicates.
8.13 Interelement Spectral Interference Correction Validity
For the interelement spectral interference corrections to remain valid during sample
analysis, the interferent concentration must not exceed its linear range. If the
interferent concentration exceeds its linear range or its correction factor is big
enough to affect the element of interest even at lower concentrations, sample
dilution with reagent blank and reanalysis is required. In these circumstances,
analyte dilution limits are raised by an amount equivalent to the dilution factor.
8.14 Internal Standard (Yttrium/Indium)
For any readings where the internal standard is outside of the range 60-125 percent of
the internal standard level in the reference standard (Initial Calibration Blank), then
the sample must be diluted until the internal standard is within range and all sample
results must be footnoted in LIMS.
8.15 MSA (Method of Standard Additions)
SGS - Orlando uses the internal standard technique as an alternative to the MSA
per SW846-6010D section 4.4.2. However, in certain circumstances MSA may be
needed by some project specific requirements. SGS - Orlando may perform an
MSA when sample matrix interference is confirmed through the post digestion spike
process or may qualify the results in LIMS. SGS - Orlando will use a single
addition method as described in SW846-7000B.
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9.0 GLASSWARE CLEANING
All glassware must be washed with soap and tap water and then rinsed with 5 percent nitric
acid. It must then be rinsed at least 3 times with DI water. Refer to SOP GN196, current
revision for further information regarding glassware cleaning.
10.0 DOCUMENTATION REQUIREMENTS
Refer to the Laboratory Quality Assurance Manual for documentation requirements. All raw
data is printed to .PDF format and archived to a backup server for long term storage.
11.0 SAFETY
The analyst must follow normal safety procedures as outlined in the SGS -
Orlando Safety Manual which includes the use of safety glasses and lab coats.
In addition, all acids are corrosive and must be handled with care. Flush spills with
plenty of water. If acids contact any part of the body, flush with water and contact
the supervisor. Follow proper safety precautions when working with gas cylinders.
12.0 CALCULATIONS
For water samples, the following calculations must be used. Refer to the QC section for
the calculations to be used for the QC samples.
Original sample concentration of metal (ug/1) =
(conc. in the digestate (ua/I)) x (final digestate volume
(ml)) (initial sample volume (ml))
For soil samples, the following calculations must be used.
Concentration of the metal in the dry sample (mg/kg) =
(conc. in the digestate (mg/1) x final digestate
volume(L)) (sample wt. (kq)) x (% solids/100)
13.0 INSTRUMENT MAINTENANCE
Recommended periodic maintenance includes the items outlined below. All maintenance
must be recorded in the instrument maintenance log.
13.1 Change the pump tubing as needed.
13.2 Clean the filter on the recirculating pump approximately once a month and dust
off the power supply vents as needed.
13.3 Clean or replace the nebulizer, torch assembly, and injector tube as needed.
13.4 Change the sampler tip as needed.
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13.5 Clean the recirculating pump lines and internal sock filter every 3 months or as needed.
13.6 Clean the radial view quartz surface weekly or more often if needed.
14.0 POLLUTION PREVENTION AND WASTE MANAGEMENT
14.1 Pollution Prevention
Users of this method must perform all procedural steps in a manner that controls the
creation and/or escape of wastes or hazardous materials to the environment. The
amounts of standards, reagents and solvents must be limited to the amounts specified
in this SOP. All safety practices designed to limit the escape of vapors, liquids or solids
must be followed. All method users must be familiar with the waste management
practices described in Section 14.2.
14.2 Waste Management
Individuals performing this method must follow established waste management
procedures as described in the Sample and Laboratory Waste Disposal SOP SAM108,
current revision. This document describes the proper disposal of all waste materials
generated during the testing of samples.
15.0 GENERIC DEFINITIONS
15.1 Batch: A group of samples which are similar with respect to matrix and the
testing procedures being employed and which are processed as a unit. A sample
batch is limited to a maximum of 20 samples or 24 hours whichever comes first.
15.2 Blank Spike (BS): An analyte-free matrix spiked with a known amount of
analyte(s), processed simultaneously with the samples through all the steps of the
analytical procedure. Blank Spike Recoveries are used to document laboratory
performance for a given method. This may also be called a Laboratory Control
Sample (LCS).
15.3 Continuing Calibration Verification (CCV): A check standard used to verify
instrument calibration throughout an analytical run. A CCV must be analyzed at the
beginning of the analytical run, after every 10 samples, and at the end of the run.
15.4 Holding Time: The maximum times that samples may be held prior to preparation
and/or analysis and still be considered valid.
15.5 Initial Calibration (ICAL): A series of standards used to establish the working range
of a particular instrument and detector. The low point must be at a level equal to or
below the reporting level.
15.6 Initial Calibration Verification (ICV): A standard from a source different than that used
for the initial calibration. A different vendor must be used whenever possible. The
ICV is used to verify the validity of an Initial Calibration. This may also be called a
QC check standard.
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15.7 Matrix Spike (MS): A sample aliquot spiked with a known amount of analyte(s),
processed simultaneously with the samples through all the steps of the analytical
procedure. The matrix spike recoveries are used to document the performance of
a method in a given sample matrix.
15.8 Matrix Spike Duplicate (MSD): A replicate sample aliquot spiked with a known
amount of analyte(s), processed simultaneously with the samples through all the
steps of the analytical procedure. The matrix spike recoveries are used to document
the precision and performance of a method in a given sample matrix.
15.9 Method Blank (MB): An analyte-free matrix to which all reagents are added in the
same volumes or proportions as used in sample processing. The method blank is
processed simultaneously with the samples through all the steps of the analytical
procedure. The method blank is used to document contamination resulting from the
analytical process.
15.10 Sample Duplicate (DUP): A replicate sample which is used to document the precision
of a method in a given sample matrix.
15.11 Preservation: Refrigeration and/or reagents added at the time of sample collection
(or later) to maintain the chemical integrity of the sample.
16.0 METHOD PERFORMANCE
Method performance is monitored through the routine analysis of negative and positive
control samples. These control samples include method blanks (MB), blank spikes (BS),
matrix spikes (MS), and matrix spike duplicates (MSD). The MB and BS are used to
monitor overall method performance, while the MS and MSD are used to evaluate the
method performance in a specific sample matrix.
Blank spike, matrix spike, and matrix spike duplicate samples are compared to method
defined control limits. Statistical control limits are stored in the LIMS for QA purposes
only. Additionally, blank spike accuracy is regularly evaluated for statistical trends that
may be indicative of systematic analytical errors.
17.0 GENERATION OF INTERFERING ELEMENT CORRECTION
FACTORS
17.1 It is recommended that all IEC's be verified and updated approximately every 6
months or whenever instrument conditions change significantly. It is also recommended
that elements with frequent high concentrations or with large IEC's should be checked
more frequently.
17.2 Calculate the IEC correction factors and enter them into the method (refer to Thermo
6500 instrument manual). Calculate the correction factor using the equation shown
below. This correction factor must be added to the correction factor already in place in
the method for a given element.
IEC = Concentration Result of the element with the interference Concentration
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result of the interfering element
17.3 Verify the new correction factors by reanalyzing the ICSA/ICSAB solutions and/or the
SIC solutions or by reloading and recalculating the previously stored results. If the
reanalysis is not within QC limits, make additional changes to the IEC factors and then
re -verify both the individual and combined solution values.
17.4 Save and update the method.
17.5 Interfering element correction factors are saved as raw data along with the run
printouts on a daily basis so that the IEC's for a given run are traceable.
TABLE 1: REPORTING LIMIT BY ELEMENT
Analyte
Water
Reporting
Limit(LLOQ) (ug/L)
Soil TCLP
Reporting Reporting
Limit(LLOQ)(mg/kg) Limit(LLOQ) (mg/L)/MCL
Tin
50
5
Aluminum
200
20
Antimony
5
1
Arsenic
10
0.5
0.10 / 5.0
Barium
200
20
10 / 100
Beryllium
4
0.5
Cadmium
5
0.4
0.05 / 1.0
Calcium
1000
500
Chromium
10
1
0.10 / 5.0
Cobalt
50
5
Copper
25
2.5
Iron
300
10
Lead
5
1
0.5 / 5.0
Magnesium
5000
500
Manganese
15
1.5
Nickel
40
4.0
Potassium
5000
500
Selenium
10
1
0.5 / 1.0
Silver
10
1
0.10 / 5.0
Sodium
5000
500
Thallium
10
1
Vanadium
50
5
Zinc
20
2
Molybdenum
50
2.5
Strontium
10
0.5
Titanium
10
0.5
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TABLE 2. THERMO 6500 ANALYSIS LINES
Element
Wavelength
Al
396.1
As
189.042
Ca
317.933
Fe
259.9
Mg
279.078
Mn
257.610
Pb
220.353
Se
196.026
TI
190.864
V
292.402
Ag
328.068
Ba
455.4
Be
313.042
Cd
226.502
Co
228.616
Cr
267.716
Cu
324.753
K
766.491
Na
589.5
Ni
231.604
Sb
206.838
Zn
206.2
MO
202.030
Sn
189.900
Sr
407.7
Ti
334.9
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TABLE 3: LOW, MID AND HIGH STANDARD LEVELS
Single Point Calibration (blank and high standard) may be
used
Element
Low
Mid
High
ug/1
ug/1
ug/1
Al
10000
40000
80000
As
500
2000
4000
Ca
10000
40000
80000
Fe
10000
40000
80000
Mg
10000
40000
80000
Mn
500
2000
4000
Pb
500
2000
4000
Se
500
2000
4000
TI
500
2000
4000
V
500
2000
4000
Ag
62.5
250
500
Ba
500
2000
4000
Be
500
2000
4000
Cd
500
2000
4000
Co
500
2000
4000
Cr
500
2000
4000
Cu
500
2000
4000
K
10000
40000
80000
Na
10000
40000
80000
Ni
500
2000
4000
Sb
500
2000
4000
Zn
500
2000
4000
MO
500
2000
4000
Sn
500
2000
4000
Sr
500
2000
4000
Ti
500
2000
4000
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

TABLE 4: ICV STANDARD LEVELS
Element
Concentration
ug/1
Al
40000
As
2000
Ca
40000
Fe
40000
Mg
40000
Mn
2000
Pb
2000
Se
2000
TI
2000
V
2000
Ag
250
Ba
2000
Be
2000
Cd
2000
Co
2000
Cr
2000
Cu
2000
K
40000
Na
40000
Ni
2000
Sb
2000
Zn
2000
MO
2000
Sn
2000
Sr
2000
Ti
2000
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TABLE 5: CCV STANDARD LEVELS
Element
Concentration
ug/1
Al
40000
As
2000
Ca
40000
Fe
40000
Mg
40000
Mn
2000
Pb
2000
Se
2000
TI
2000
V
2000
Ag
250
Ba
2000
Be
2000
Cd
2000
Co
2000
Cr
2000
Cu
2000
K
40000
Na
40000
Ni
2000
Sb
2000
Zn
2000
MO
2000
Sn
2000
Sr
2000
Ti
2000
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

TABLE 6: CRIA(LLCCV) STANDARD LEVELS
Element
CRIA
ug/1
Al
200
As
10
Ca
1000
Fe
300
Mg
5000
Mn
15
Pb
5
Se
5
TI
10
V
50
Ag
10
Ba
200
Be
5
Cd
5
Co
50
Cr
10
Cu
25
K
5000
Na
5000
Ni
40
Sb
5
Zn
20
MO
50
Sn
50
Sr
10
Ti
10
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TABLE 7: BLANK SPIKE, MATRIX SPIKE AND MATRIX SPIKE DUPLICATE LEVELS
Element
Concentration
ug/1
Al
27000
As
2000
Ca
25000
Fe
26000
Mg
25000
Mn
500
Pb
500
Se
2000
TI
2000
V
500
Ag
50
Ba
2000
Be
50
Cd
50
Co
500
Cr
200
Cu
250
K
25000
Na
25000
Ni
500
Sb
500
Zn
500
MO
500
Sn
500
Sr
500
Ti
500
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TABLE 8: TYPICAL RUN SEQUENCE
BLANK
LOW
MID
HIGH
HIGH STD
ICV
ICB
CRIA
I CSA
I CSAB
CCV
CCB
MB
SIB
SAMPLE1
DUPLICATE
SERIAL DILUTION
MATRIX SPIKE
MATRIX SPIKE DUPLICATE
POST DIGESTION SPIKE
SAMPLE2
SAMPLE3
CCV
CCB
SAMPLE4
SAMPLE5
SAMPLE6
SAMPLE7
SAMPLE8
SAMPLE9
SAMPLE10
SAMPLE11
SAMPLE12
SAMPLE13
CRIA CLOSING
ICSACLOSING
ICSAB CLOSING
CCV
CCB
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

TABLE 9: ICSA (Mixed SIC) SOLUTION LEVELS
Element
Concentration
mg/I
Al
500
As
0
Ca
500
Fe
500
Mg
500
Mn
0
Pb
0
Se
0
TI
0
V
0
Ag
0
Ba
0
Be
0
Cd
0
Co
0
Cr
0
Cu
0
K
0
Na
0
Ni
0
Sb
0
Zn
0
MO
0
Sn
0
Sr
0
Ti
0
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

TABLE 10: SINGLE ELEMENT INTERFERENCE CHECK SOLUTION (SIC) LEVELS
Element
Concentration
Mg/1
Al
500
As
0
Ca
500
Fe
500
Mg
500
Mn
4
Pb
0
Se
4
TI
0
V
4
Ag
0
Ba
4
Be
0
Cd
0
Co
0
Cr
0
Cu
4
K
0
Na
1000
Ni
4
Sb
0
Zn
4
MO
4
Sn
4
Si
50
Sr
0
Ti
0
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

SGS - ORLANDO STANDARD OPERATING PROCEDURE
FN: MET 107.02
Rev. Date: 02/2018
Pages F-106 - F-126
TITLE: METALS BY INDUCTIVELY COUPLED PLASMA —
MASS SPECTROMETRY (ICP-MS)
REFERENCES: SW846 6020A, Revision 1, February 2007
SW846 6020B, Revision 2, July 2014.
EPA200.8, Revision 5.4, 1994.
REVISED SECTIONS: added WV state -specific requirements (Sec. 4); added WV 47CSR32 to
references (Sec. 16); added removed all references to Accutest
Note: Many clients require special reporting limit (LLOQ) and analytical criteria. Refer to
scheduling sheets and/or project specific QAPP for further information regarding client specific QC
requirements. Also check with metals supervisor for additional information.
Main Instrument: Agilent 7700x, serial # JP12151709
Auto -sampler: CETAC ASX500, serial # US09132OA520
1.0 SCOPE AND APPLICATION
1.1 This method is applicable for the
samples and in waste extracts o
1 for a list of reportable elements
2.0 SUMMARY
determination of total and dissolved metals in water
r in solid or aqueous digests. Please refer to table
2.1 Samples are prepared for analysis by digestion. The prepared samples are introduced
into a radiofrequency plasma by pneumatic nebulization. There the energy transfer
processes cause desolvation, atomization, and ionization. The ions are extracted
from the plasma through a differentially pumped vacuum interface and separated on
the basis of their mass to charge ratio by a quadrupole mass spectrometer. The ions
transmitted through the quadrupole are detected by an electron multiplier and the ion
information is processed by a data handling system.
3.0 REPORTING LIMIT(RL), Lower Limit of Quantitation (LLOQ)
AND METHOD DETECTION LIMIT
3.1 Reporting Limit (LLOQ). Current reporting limits (LLOQ) for this method have been
established at the levels listed in Table 1. The reporting limits (LLOQ) are dependent
upon the metal being analyzed and are in all cases greater than the IDL and the
MDL for each element. Refer to scheduling sheets and/or project specific QAPP for
further onformation regarding client specific reporting limits.
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3.2 Method Detection Limit. Experimentally determine MDLs using the procedure specified in
40 CFR, Part 136, Appendix B. This value represents the lowest reportable concentration
of an individual compound that meets the method qualitative identification criteria.
3.2.1 Experimental MDLs must be determined annually for this method.
4.0 DEFINITIONS
BATCH. A group of 20 samples or less that behaves similarly with respect to the sampling or
the testing procedures being employed and which are processed as a unit within a 24 hour
period. For QC purposes, if the number of samples in a group is greater than 20, then
each group of 20 samples or less will all be handled as a separate batch.
CALIBRATION CHECK STANDARD. (CCV) calibration check standard is a mid -range
calibration standard. It is recommended that the calibration check standard be run at a
frequency of 10 percent or every 2 hours during an analysis run, whichever is more frequent,
and at the end of the analysis sequence. For this method, the mid -level calibration check
standard criteria is ± 10 percent of the true value and the relative standard deviation for the
replicates that are greater than 5 times the reporting limit (LLOQ) is less than 5 percent.
The exception to this rule is if the recovery on the calibration check standard is high and the
samples to be reported are less than the reporting limit (LLOQ).
EXTERNAL CHECK STANDARD. (ICV) The external check standard is a standard from a
separate source than the calibration curve that is used to verify the accuracy of the calibration
standards. It must be run after each calibration. The external check standard criteria is ± 10%
of the true value and the replicates that are greater than 5 times the reporting limit (LLOQ)
should have a relative standard deviation of less than 5 percent. If the external check is
outside of the control limits for a given parameter, all samples must be reanalyzed for that
parameter after the problem has been resolved.
SPIKE BLANK OR LAB CONTROL SAMPLE. Digest and analyze a laboratory control
sample or spike blank with each set of samples. A minimum of one lab control sample or
spike blank is required for every 20 sample batch. A sample batch is defined as a maximum
of 20 field samples in a preparation batch over a time period of 24 hours. Assess laboratory
performance against the control limits of 80 to 120 percent for method SW846-6020A and
6020B. Recovery of 85 to 115 percent for method EPA 200.8. In house limits should also
be generated once sufficient data is available to support the default limits. If the lab control or
spike blank is outside of the control limits for a parameter, all samples must be redigested and
reanalyzed for that parameter. The exception is if the lab control or spike blank recovery is
high and the results of the samples to be reported are less than the reporting limit (LLOQ). In
that case, the sample results can be reported with no flag.
MATRIX: The component or substrate (e.g., water, soil) which contains the analyte of interest.
MATRIX DUPLICATE: A duplicate sample is digested at a minimum of 1 in 20 samples.
The relative percent difference (RPD) between the duplicate and the sample should be
assessed. The duplicate RPD is calculated as shown below. Assess laboratory
performance against method limits. If the sample and the duplicate are less than 5 times
the reporting limits (LLOQ) and are within a range of ± the reporting limit (LLOQ), then the
duplicate is considered to be in control.
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(ISample Result- Duplicate Resultl) x 100 = Duplicate RPD
(Sample Result + Duplicate Result)/2
MATRIX SPIKE: The laboratory must add a known amount of each analyte to a minimum of 1
in 20 samples. The matrix spike recovery is calculated as shown below. Assess laboratory
performance against default limits of 80 to 120 % recovery for method 6020A and 6020B.
Recovery criteria of 70 to 130 % recovery for method 200.8. In house limits should be
generated once sufficient data is available. If a matrix spike is out of control, then the results
should be flagged with the appropriate footnote. If the matrix spike amount is less than one
fourth of the sample amount, then the sample cannot be assessed against the control limits and
should be footnoted to that effect.
(Spiked Sample Result - Sample Result) x 100 = Matrix Spike
Recovery (Amount Spiked)
MATRIX SPIKE DUPLICATES: Intralaboratory split samples spiked with identical
concentrations of target analyte(s). The spiking occurs prior to sample preparation and
analysis. They are used to document the precision and bias of a method in a given sample
matrix.
METHOD BLANK. The laboratory must digest and analyze a method blank with each set
of samples. A minimum of one method blank is required for every 20 sample batch. If no
digestion step is required, then the method blank is equivalent to the reagent blank. The
method blank must contain the parameter of interest at levels of less than 1/2 the
reporting limit (LLOQ) for that parameter. The exception to this rule is when the samples to
be reported contain greater than 10 times the method blank level. In addition, if all the
samples are less than a client required limit and the method blank is also less than that limit,
then the results can be reported as less than that limit. Samples associated with the
contaminated blank shall be evaluated as to the best corrective action for each particular
sample. This may include reanalyzing the samples, re -digesting and reanalyzing the samples,
qualifying the results with a "B" or "V" qualifier, or raising the reporting limit (LLOQ) to greater
than two times the background concentration. Note: 200.8 Method Blanks associated with
samples originated in West Virginia are evaluated to MDL (WV 47CSR32)
METHOD DETECTION LIMITS (MDLS). The minimum concentration of a substance that can
be measured and reported with 99% confidence that the analyte concentration is greater than
zero and is determined from analysis of a sample in a given matrix containing the analyte.
MDLs should be determined approximately once per year for frequently analyzed parameters.
REAGENT BLANK. The reagent blank is a blank that has the same matrix as the samples,
i.e., all added reagents, but did not go through sample preparation procedures. The reagent
blank is an indicator for contamination introduced during the analytical procedure. (Note: for
methods requiring no preparation step, the reagent blank is equivalent to the method
blank.) Either a reagent blank or a method blank must be analyzed with each batch of 20
samples or less. The concentration of the analyte of interest in the reagent blank must be less
than 1/2 the reporting limit (LLOQ) for that analyte. If the reagent blank contains levels over
the reporting limits (LLOQ), the samples must be reanalyzed. The exception to this rule is
when the samples to be reported contain greater than 10 times the reagent blank level. In
addition, if all the samples are less than a client required limit and the reagent blank is also
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less than that limit, then the results can be reported as less than that limit.
REAGENT GRADE. Analytical reagent (AR) grade, ACS reagent grade, and reagent grade
are synonymous terms for reagents which conform to the current specifications of the
Committee on Analytical Reagents of the American Chemical Society.
REAGENT WATER. Water that has been generated by any method which would achieve
the performance specifications for ASTM Type I I water.
STANDARD ADDITION. The practice of adding a known amount of an analyte to a
sample immediately prior to analysis. It is typically used to evaluate interferences.
STANDARD CURVE: A plot of concentrations of known analyte standards versus the
instrument response to the analyte. Calibration standards are prepared by successively
diluting a standard solution to produce working standards which cover the working range of the
instrument. Standards should be prepared at the frequency specified in the appropriate
section. The calibration standards should be prepared using the same type of acid or solvent
and at the same concentration as will result in the samples following sample preparation.
This is applicable to organic and inorganic chemical analyses.
5.0 HEALTH & SAFETY
5.1 The analyst must follow normal safety procedures as outlined in the SGS - Orlando
Health and Safety Program, which include the use of safety glasses and lab coats. In
addition, all acids are corrosive and must be handled with care. Flush spills with plenty of
water. If acids contact any part of the body, flush with water and contact the supervisor.
5.2 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely determined; however, each chemical must be treated as a potential health
hazard. Exposure to these reagents must be reduced to the lowest possible level. The
laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference
file of data handling sheets must be made available to all personnel involved in these
analyses.
6.0 SAMPLE COLLECTION, PRESERVATION & HOLDING TIME
6.1 Aqueous samples are collected in 500ml or 1000 ml HDPP bottles. All water samples
should be preserved with nitric acid to a pH of 2 or less. Solid samples are collected in
glass jars (4 oz or 8 oz) with PTFE lined lid. All solid samples should be stored in
a refrigerator at 4 degrees C until digestion.
6.2 All samples should be analyzed within 6 months of the date of collection.
7.0 INTERFERENCES
7.1 Several types of interferences can cause inaccuracies in trace metals determinations by
ICP- MS. These interferences are discussed below.
7.2 Isobaric elemental interferences are caused by isotopes of different elements which
form singly or doubly charged ions of the same nominal mass -to -charge ratio and which
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cannot be resolved by the mass spectrometer in use. If isobaric interferences are
present in the ion being analyzed, then the data must be corrected by measuring
the signal from another isotope of the interfering element and subtracting the
appropriate signal ratio from the element of interest.
7.3 Abundance sensitivity is a property that defines the degree to which the wings of a
mass peak contribute to adjacent masses and is affected by ion energy and
quadrupole operating pressure. Wing overlap interferences may result when a small
ion peak is being measured next to a large one. Spectrometer resolution should be
adjusted to minimize these interferences.
7.4 Isobaric polyatomic ion interferences are caused by ions consisting of more than one
atom which have the same nominal mass -to -charge ratio as the isotope of interest,
and which cannot be resolved by the mass spectrometer in use. Refer to method 200.8
and 6020A for lists of common interferences and correction equations to be applied. If
these interferences cannot be avoided by the use of different isotopes, then
correction equations should be applied to the data. Alternatively, collision/reaction cell
technology can be applied to physically and chemically remove interferences.
7.5 Physical interferences can occur during the transfer of the solution to the nebulizer
(viscosity effects).
7.6 Memory interferences can be caused by build up on the sampler and skimmer
cones, and from buildup of sample material in the torch and spray chamber. Some
elements, such as mercury, can suffer from severe memory effects. In that case,
gold is added to the rise solution to decrease the Hg rinse out time.
7.7 Interference correction equation procedure.
Interference correction equations are used to correct interference with target elements
due to other elements or formation of polyatomic ions. Specify the elements related to
the interference to be corrected. Isotope masses and isotope ratios are displayed in
Mass table. Select the check boxes for the masses for which correction equations are
set. Equations are displayed in the Equation table. Select the elements for which the
correction equations are set. Select the masses for which the correction equations are
set. Select positive or negative sign for the factor, enter masses in the Mass field and
enter the factors of the correction equations in the Multiplier field. "OK" applies to
settings and the specified interference correction equation is displayed in the "Select
Elements on Periodic Table" dialog box.
8.0 APPARATUS
8.1 Currently in use is an Aglilent 7700x ICP-MS with collision/reaction cell capacity and
HMI (High matrix interface) and the associated autosampler.
8.2 Data system
8.2.1 Microsoft Windows 7 Professional Version 2009
8.2.2 Agilent Masshunter, Version B.01.03, Build 393.17, Patch 2, 2014
8.2.3 Computer system interfaced with Agilent ICP-MS that allows continuous
data acquisition and storage of all data obtained throughout the duration of
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the analytical run sequence. Data is backed up and archived for long-term
storage
8.3 Class A volumetric glassware as needed and instrument autosampler tubes.
8.3.1 All glassware must be washed with soap and tap water and then soaked in a
10% nitric acid bath for several hours. It must then be rinsed at least 3 times
with distilled, deionized water.
8.4 Polypropylene bottles for standard storage. These bottles must also be cleaned as
outlined above.
9.0 REAGENTS
9.1 All chemicals listed below are reagent grade unless otherwise specified. Deionized
water must be used whenever water is required. Note: All reagents can be scaled
up or down proportionately if different final volumes are required.
9.2 Hydrochloric acid, trace metals grade.
9.3 Nitric acid, trace metals grade. Note — ultra trace grade may be required if lower
detection limits than normal are needed.
9.4 Standard stock solutions available from Inorganic Ventures, Ultra Scientific, VHG
Laboratories or equivalent. Note: All standards must be ICP-MS quality standards or
must be demonstrated to be free of interferences at the levels of use. Standards
should come labeled with an expiration date and certificate of concentrations from
the manufacturer. If both of these items are not received, then the manufacturer
should be contacted before use of the standard.
9.5 Calibration Standards: These can be made up by diluting the stock solutions to the
appropriate concentrations. Fresh calibration standards should be prepared a
minimum of every two weeks. They must be monitored weekly for stability.
9.5.1 Standards should be made in a low acid matrix. Concentrations of 1 to 2
percent nitric acid and 0 to 0.5 percent hydrochloric acid are suggested,
although any acid concentration that provides good analytical results may
be used. High chloride concentrations may cause interferences so chloride
concentrations should be limited. HCI may be omitted if silver and antimony are
not elements of interest.
9.5.2 Refer to the standard prep logbook for the make-up and concentrations of
standards and stock solutions being used to calibrate the ICP-MS. Suggested
standard levels are shown in Table 2.
9.6 Aglient P/A Factor and Tuning/Performance Check Solution. Mix 1.0 ml of PA
Tuning 1 solution and 1.0 ml of PA Tuning 2 solution (available from Aglient, part
number 5188-6524) and bring to 100 ml final volume with a solution of 1 % nitric acid
and 0.5% HCI. This final solution contains 200 ppb of As, Be, Cd, Zn; 100 ppb of Mg,
Ni, and Pb; 50 ppb of Al, Ba, Bi, Co, Cr, Cu, In, Lib, Lu, Mn, Na, Sc, Sr, Th, TI, U, and
V; and 25 ppb of Y and Yb; 100 ppb of Ge, Mo, Pd, Ru, Sb, Sn ; and 50 ppb of Ir and
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Ti.
9.7 Tuning Standard, Agilent ICP-MS. This solution is used to verify mass calibration
and thermal stability and must contain a mix of elements representing all of the mass
regions of interest. Elements include 1 ppb Ce, Co, Li, Mg, TI, and Y.
9.8 Internal Standards. Internal standards are added to all calibration standards, quality
control, and samples during analysis, normally using a second channel of the
peristaltic pump and a mixing manifold. The internal standard solution is recommended
to contain Sc, Y, In, Tb, and Bi.
9.8.1 For the Aglient instrument, a solution containing 1 ppm of Li, Sc, Lu, In, Tb,
Bi, Te and Ge in 1 % nitric is recommended. Refer to Table 3.
9.9 Calibration /Rinse Blank. The calibration and rinse blanks are prepared by diluting
acids to the same concentrations found in the standards. The calibration blank is
used to establish the analytical calibration curve and the rinse blank is used to flush
the instrument between samples in order to reduce memory interferences.
9.10 Continuing Calibration Verification Check (CCV). This solution is prepared by adding
either mixed or single element metals solutions to a solution containing the same acid
matrix as the calibration standards. The metals should be at concentrations near
the middle of the calibration curve. (Note: This check is run after the calibration,
after every 10 samples or every 2 hours during an analysis run, whichever is more
frequent, and at the end of the sample run.) CCV should be prepared from the
same source as the calibration standards. Refer to Table 2 for suggested
concentrations for the CCV.
9.11 Matrix Spike and Spike Blank Solution. Suggested levels for the final concentrations
of the spike are shown in Table 4. This solution is prepared by adding either
mixed or single element metals solutions to a solution containing 1 percent nitric acid
and 0 to 0.5 % HCl and diluting to a fixed final volume with this acid mixture.
9.12 Lab Control Solution. This solution is prepared by adding either mixed or single element
metal solutions to a solution containing 1 percent nitric acid and 0 to 0.5 % HCI and
diluting to a fixed final volume with this acid mixture.
9.13 Interference Element Check Solutions or spectral interference check solutions (SIC).
The purpose of the ICSA and ICSAB solutions is to demonstrate the magnitude of
interferences and provide an adequate test of any corrections. It is recommended that the
following solutions be purchased commercially.
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9.13.1 ICSA Solution. The ICSA solution contains only the interfering elements. The
recommended concentrations are shown below. The ICSA solution must be made
fresh weekly.
Al
100 mg/L
Ca
100 mg/L
Fe
100 m /L
Mg
100 mg/L
Na
100 mg/L
P
100 mg/L
K
100 mg/L
S
100 mg/L
C
200 m /L
Cl
1000 mg/L
Mo
2.00 mg/L
Ti
2.00mg/L
9.13.2 ICSAB Solution. The ICSAB solution contains both the interferents and the analytes
of interest. The recommended concentrations are shown below. The ICSAB
solution must be made fresh weekly.
Al
100 mg/L
Ca
100 m /L
Fe
100 mg/L
Mg
100 mg/L
Na
100 mg/L
P
100 mg/L
K
100 mg/L
S
100 m /L
C
200 mg/L
Cl
1000 mg/L
Mo
2.00 mg/L
Ti
2.00mg/L
As
0.020 mg/1
Cd
0.020 mg/1
Cr
0.020 mg/1
Co
0.020 mg/1
Cu
0.020 mg/I
Mn
0.020 mg/1
Ni
0.020 mg/1
Ag
0.020 mg/I
Zn
0.020 mg/1
9.14 Initial Calibration Verification (ICV) or Quality Control Sample (QCS). The metals in
this solution should be at final concentrations that are at the mid -point of the
calibration curve. This solution is prepared by adding either mixed or single
element metals solutions to a solution containing 1 percent nitric acid and 0 to 0.5
percent hydrochloric acid and diluting to a fixed final volume with this acid mixture.
Please see Table 2 for suggested levels. The ICV sample must be from an
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independent source from the calibration standards.
9.15 CRI Standards (also referred to as LLCCV). The CRI standard must contain the
elements of interest at (or below) the reporting limit (LLOQ) for each element. The
CRI level is at the reporting limit (LLOQ) as shown in Table 1. This should be
prepared by diluting calibration standard(s) to the reporting limit (LLOQ) level for each
element. They should be made in the same matrix as the calibration standards
9.16 Liquid Argon or Argon Gas. Argon is provided by Air Products in the large outdoor tank.
No lab monitoring of the tank is normally necessary.
9.17 Helium Gas. Required for running the reaction cell on the Agilent 7700X.
10.0 INITIAL INSTRUMENT SET-UP PROCEDURE FOR THE
AGILENT 770OX ICP-MS
3.3 A general procedure on how to operate the Agilent 770OX ICP-MS is given below.
Refer to the operation manual for further details.
3.4 Before bringing up the instrument, make sure that the lines, the torch, the nebulizer,
and the spray chamber are clean, and that there are no leaks in the torch area.
3.5 Turn the vacuum pump and the heat exchanger on and verify that the liquid argon is
turned on and the helium gas is turned on.
3.6 Connect the pump tubing and engage the peristaltic pump.
3.7 Put a new solution of acid rinse into the rinse reservoir. (Note: the composition of the
rinse solution may be periodically changed to minimize sample introduction problems
and sample carryover.) Make sure that sufficient internal standard solution is present.
10.1 Open the ICP-MS Mass Hunter Top software. Click on the instrument and open the
instrument control panel. Click the plasma on. The instrument will automatically go
through the start-up cycle. Then let the instrument warm up for at least 30 minutes.
3.8 Tune the instrument on a daily basis. Tuning must always be done after moving the
position of the torch or the cones. Tuning can be done either manually or by
following autotune procedures. It is recommended that autotune procedures be
followed initially and then manual tuning be done as a second step. The purpose of
tuning is to optimize the instrument for the highest sensitivity while obtaining low levels
of oxides and doubly charged species. After the tune is complete, make sure to save
the optimized parameters.
10.1.1 Open the ICP-MS top software, click on the instrument, and open the ICP-MS
tuning page.
10.1.2 Click file and open the 6020AB_200.8 Method .b file. Keep the internal standard
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line in a solution of 1 % nitric acid and 0.5% hydrochloric acid. Place the
carrier line into the 1 ppb tuning solution. (see 9.7). On the tuning page, click
start under the tune window to see the counts and RSD values. Do not start
the tune process until the count and mean have similar readings and the RSD
is < 5%. The counts per second values should be > 40000 for all masses. Click
stop under tune window.
10.1.3 Before starting auto tune and printing tune report, create a new batch folder
from existing method 6020AB_200.8.b. Save new batch using format
11xaMMDDm1".
10.1.4 On the tuning page, click Autotune, type the date (MMDDYYM1) on the
popup window and click OK. This will perform the tuning of the instrument.
Verify acceptable mass calibration by monitoring the peak width measurement
at 5% of peak maximum for Co_59, Y_89, and TI_205. If the peak widths are
outside of the range of 0.65 to
0.85 and the masses are off by more than 0.1 amu, then redo the mass
calibration. After all criterion is met, print the report and include with raw data.
The tune report is automatically stored in the batch folder.
10.2 Before calibrating, run and print out a performance test. This must include the
following items.
10.2.1 Relative standard deviations of the absolute signals must be less than 5
percent for all monitored masses. This includes Li_7, Y_89, and TI_205. If
these criteria are not met, correct the problem and then repeat the stability test.
Print the results of this test and store with the raw data for the run.
10.3 Before starting sample analysis, set up the internal standards. Internal standards are
added to all calibration standards, quality control, and samples during analysis,
normally using a second channel of the peristaltic pump and a mixing manifold. Refer
to Table 3 and Section 9.8 for additional information.
10.4 To start running samples, add samples to sequence and click "Add to Queue".
Unpause once ready to start analysis.
10.5 Calibrate the instrument using a minimum of a calibration blank and three non -zero
standards that bracket the desired sample concentration range. The lowest non -zero
standard must be at or lower than the RL or LOQ levels for all the elements. (Note:
The calibration standards may be included in the autosampler program or they
may be run separately.) A correlation coefficient of 0.998 or better must be obtained
using a first order (linear) curve fit. A minimum of three replicate integrations are
required for all data acquisitions.
10.5.1 In between each analysis of a separate standard or sample, a rinse blank
must be run through the lines of the sample introduction system. Each
sample or standard should be aspirated for a minimum of 30 seconds prior to
the acquisition of data to allow equilibrium to be established.
10.6 After the instrument is properly calibrated, begin by analyzing the ICV solution. The ICV
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must be run after each calibration. For the ICV, all elements to be reported must be
within 10 percent of the true value and the replicates that are greater than 5 times
the reporting limit (LLOQ) should have a relative standard deviation of less than 5
percent. If the ICV is outside of criteria, then the problem must be identified and
corrected before samples can be run and reported for the element(s) that are outside
of criteria. Correction of the problem can be verified by rerunning the check
standard(s) and showing that they meet QC criteria.
10.6.1 An ICB may be run after the ICV, but is not required for this method. If it is run,
then all elements must be less than '/2 the reporting limit (LLOQ) for each
element.
10.6.2 Run the CRI (LLCCV) solution right after the ICV and ICB, (or any other place
at the beginning of the run after the ICV, ICB and before any real samples are
analyzed). For the CRI, all elements of interest must be within 30% of the true
value, 20% for 6020B, or within client specified limits.
10.7 Then analyze the CCV and CCB check standards. For the CCV, all elements to be
reported must be within 10 percent of the true value and the replicates that are
greater than 5 times the reporting limit (LLOQ) should have a relative standard
deviation of less than 5 percent. For the CCB, all elements to be reported must
normally be less than 1/2 the reporting limit (LLOQ). If either the CCV or CCB do not
meet criteria, then elements failing this criteria must not reported in the area bracketed
by this QC.
10.7.1 The internal standard levels in the CCV and CCB must also be within 30%
of the internal standard level for the initial calibration. If they are outside of
these levels, then no samples can be reported in the area bracketed by this QC.
10.8 After the initial QC is completed and before any samples are analyzed, the ICSA and
ICSAB solutions (SIC solutions) must be analyzed. The method does not list specific
criteria for the ICSA and ICSAB, but in house criteria will be applied. For all the
spiked elements, the analyzed results must be within 20 percent of the true results.
For unspiked elements, the interfering element solution should contain less than the
absolute value of 2 times the reporting limit (LLOQ) for each element. If these criteria
are not met, then samples with significant interferences cannot be reported until the
instrument is optimized and the ICSA and ICSAB are within specifications.
10.8.1 If the run is longer than 12 hours, a second ICSA, ICSAB pair must be analyzed
before the next 12 hours is started.
10.8.2 If mass changes are made for the analysis of an element, all QC criteria must be
met for the new mass and it must be verified that appropriate correction factors are
in place.
10.8.3 The Agilent 770OX includes collision/reaction cell technology. The instrument is
tuned in regular (non -cell) mode and in helium (collision/reaction) cell mode.
This technology is used to minimize interferences during analysis. If this
technology is not applied, then correction factors for interferences must be added
into the method.
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10.9 After the initial analytical quality control has been analyzed, the samples and the
preparation batch quality control should be analyzed. Depending on the type of
digestion and the sample matrix, samples and the associated QC should normally be
diluted by a factor of from 2 to 5 before analysis. This dilution factor should be indicated
in the sample ID file on the instrument.
10.9.1 Each sample analysis must be a minimum of 3 integrations. For samples
containing levels of elements greater than approximately 5 times the reporting
limits (LLOQ), the relative standard deviations for the replicates should be
less than 10%. If not, reanalyze the sample. If, upon reanalysis, the RSDs
are acceptable, then report the data from the reanalysis. If RSD's are not
acceptable on reanalysis, then the results for that element may, on the
reviewer's discretion, be footnoted that there are possible analytical problems
indicated by a high RSD between replicates. In some cases, an additional
dilution analysis may be needed. Check with the area supervisor or manager for
additional information.
10.9.2 The internal standard levels must be monitored for all samples and quality
control. If the internal standard is not within 70%-120% of the internal
standard level for the initial calibration blank, then the sample must be diluted
by a factor of 5 to bring the internal standard to within the correct range. If the
internal standard is still outside of the range after the initial 1:5 dilution, then
additional dilutions must be done until the internal standard is within the
appropriate range.
10.15.2.1 If an internal standard is present in a sample, then do not use that internal
standard. For example, Y is sometimes seen in real samples. If the Y
recoveries are high relative to the other internal standards, then do not use the Y
internal standard.
10.9.3 For readings that exceed the linear range for a given element, a dilution is
required. For method 6020B, after calibration the laboratory may choose to
analyze a standard at a higher concentration than the high standard used in the
calibration curve. The standard must recover within 10 percent of the true
value, and if successful, establishes the linear range. The linear range
standards must be analyzed in the same instrument run as the calibration they
are associated with, but may be analyzed anywhere in the run .After a high
reading, the following samples must be examined for possible carryover. A
verification may be necessary by rinsing the lines with an acid solution and then
re -reading the sample.
10.9.4 Indicate dilution factors for samples using "df" followed by the dilution factor after
the sample ID. There should be a space between the sample number and the df.
10.10 Between each sample, flush the nebulizer and solution uptake system with a blank
rinse solution for a minimum of 30 seconds or for the required period of time to ensure
that analyte memory effects are not occurring. (60 seconds is recommended for
normal methods excluding Hg and B. Longer times may be needed when Hg and B are
being analyzed.)
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10.11 Analyze the continuing calibration verification solution and the continuing calibration
blank after every ten samples and at the end of the sample run.
10.11.1 For the CCV, all elements to be reported must be within 10 percent of the true
value and the replicates that are greater than 5 times the reporting limit
(LLOQ) should have a relative standard deviation of less than 5 percent. If
the CCV solution is not within 10 percent of the true value, no samples can be
reported in the area bracketed by the failing CCV for the failing element.
10.11.2 For the CCB, all elements to be reported must be less than 1/2 the reporting
limit (LLOQ).
10.12 The CRI (LLCCV) must be analyzed at the end of each calibration (analysis) batch.
The acceptance criterion for the CRI check is 70 to 130% recovery, and 80 to 120%
for method 6020B. If an element does not meet this criterion, then all samples for
that element in the concentration range between the CRI and the CCV must be
reanalyzed. Samples containing concentrations higher than the CCV may be reported
as long as CCV criteria are met.
10.12.1 More frequent CRI (LLCCV) checks may be analyzed during the course of the
run if system stability at the low end of the calibration is questionable or if the
lab wants to ensure that fewer samples will have to be submitted for reanalysis
if there is a failed CRI at the end of a run.
10.12.2 It is recommended that the CRI check be run bracketing every 4 to 8 hour
period of analysis. It may be run as frequently as every 10 samples if the
supervisory staff deems that this is necessary.
10.13 After the run is completed, convert the data file to a CSV format using the option
on the results screen. First save the file on the local drive using the file naming
system described below. Update the run in the LIMS and enter the run name into the
workgroup using lower case characters. Then copy the data from the local drive to the
LIMS drive.
10.13.1 The file should be named as followed- initial instrument indicator (xa), date
(MMDD), year, and sequential run number for that day (M1). For example, the
first run from 12/17/02 would be designated xa121702m1.csv.
10.14 Calculations are done in the LIMS using the calculations shown below.
10.14.1 Calculation for aqueous samples.
original sample concentration of metal (µg/I) _
(conc. in the digestate (ug/I)) x (final digestate volume (ml))
(Initial sample volume (ml))
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10.14.2 Calculation for solid samples.
original sample concentration of metal (mg/kg) =
(conc. in the digestate (uq/I)) x (final digestate volume (ml))
(Initial sample weight (g)) x (%sol/100)
10.21 At the end of the analysis day the ICP-MS must be brought down using the
following sequence.
10.21.1 Rinse the tip in a solution of 1 percent nitric acid and 0.5 percent hydrochloric
acid for 10 minutes and in DI water for 20 minutes. (Note: a stronger acid
solution may be needed depending on the matrix of the samples that were
analyzed.)
10.21.2 Turn off the plasma using off button.
10.21.3 Release the tension on the pump tubing.
10.21.4 Turn off the cool flow and the printer.
11.0 QC REQUIREMENTS
11.1 This section outlines the QA/QC requirements necessary to meet the method 6020A.
11.2 Instrument Detection Limits (IDLs). IDLs must be established for all analytes. Please
refer to specific method for instructions on performing IDL studies.
11.3 Lower Limit of Quantitation (LLOQ) check standard. LLOQ is the lowest point of
quantitation. The LLOQ is initially verified by the analysis of 7 replicate samples,
spiked at the LLOQ and processed through all preparation and analysis steps of the
method. The mean recovery should be within +/- 35 percent of the true value with an
RSD < 20 percent.
Ongoing Lower limit of quantitation (LLOQ) check sample. The lower limit of
quantitation check sample should be analyzed on a quarterly basis to demonstrate the
desired detection capability. The LLOQ sample is carried through the entire
preparation and analytical procedure.
11.4 LLQC (Lower Limit of Quantitation Check Sample) or LOQ Verification sample. A
sample must be digested and analyzed initially and on an as needed basis to verify
the quantitation limits for the method. Recoveries of this check must be within 70 to
130% of the true value, 80 to 120% for method 6020B. If recoveries are outside of this
level, then the reporting limit (LLOQ) must be increased to a level that can be verified.
11.5 Method Detection Limits (MDLs). MDLs should be established for all analytes,
using a solution spiked at approximately 2 to 5 times the estimated detection limit. To
determine the MDL values, take seven replicate aliquots of the spiked sample and
process through the entire analytical method. The MDL is calculated by multiplying the
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standard deviation of the replicate analyses by 3.14, which is the student's t value for
a 99% confidence level. MDLs should be determined approximately once per year or
whenever there is a significant change in the background or instrument response. In
general, if the amount spiked for the MDL is greater than 10 times the actual MDL,
then the MDL should be redone with a lower spike level. Please refer to specific
method for more information regarding MDL studies.
11.6 Linear Calibration ranges. The upper limit of the linear dynamic range needs to be
established for each wavelength used by determining the signal responses from a
minimum of three, preferably five, different concentration standards across the linear
range. The linear calibration range which may be used for the analysis of samples
should be judged by the analyst from the resulting data. The data, calculations and
rationale for the choice of range made must be documented and kept on file. A
standard at the upper limit must be prepared, analyzed and quantitated against the
normal calibration curve. The calculated value should be within ±10% of the true
value. Linear calibration ranges should be determined whenever there is a significant
change in instrument response. They must be done at least every six months. For
any readings that exceed the linear range for a given element, a dilution is
required. In addition, if there are significant interferences generated from elements
above the linear range, than these elements must also be diluted so that
accurate interfering element corrections can be applied. For method 6020B, after
calibration the laboratory may choose to analyze a standard at a higher concentration
than the high standard used in the calibration curve. The standard must recover
within 10 percent of the true value, and if successful, establishes the linear range.
The linear range standards must be analyzed in the same instrument run as the
calibration they are associated with, but may be analyzed anywhere in the run.
Normal linear range values by element are shown in Table 2.
11.7 Initial Calibration Verification (ICV) or Quality Control Sample (QCS) and Initial
Calibration Blank (ICB). After every new calibration, an ICV must be analyzed. The
analysis of the ICV may be followed by the analysis of the ICB, although this is not
required by the method.
11.7.1 For the ICV, all elements to be reported must be within 10 percent of the true
value and the replicates that exceed 5 times the reporting limit (LLOQ)
should have a relative standard deviation of less than 5 percent. The ICV
must be from a different source than the calibration standards and must be
near the mid -point of the calibration curve. If the ICV does not meet
criteria, then the problem must be identified and corrected before samples
can be run and reported for the element(s) that are outside of criteria.
Correction of the problem can be verified by rerunning the check standard and
showing that it meets QC criteria.
11.7.2 For the ICB, all elements to be reported must be less than 1/2 the RL (LLOQ).
If the ICB is outside of criteria, then the problem must be identified and
corrected before samples can be run and reported for the element(s) that
are outside of criteria. Correction of the problem can be verified by
rerunning the check standard and showing that it meets QC criteria. Analysis
of a CCB before running any reportable samples can be used to verify that the
system meets calibration blank requirements.
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11.8 Continuing Calibration Verification (CCV) and Continuing Calibration Blank (CCB).
Analyze the continuing calibration verification solution and the continuing calibration
blank after every tenth sample and at the end of the sample run.
11.8.1 For the CCV, all elements to be reported must be within 10 percent of the true
value and the replicates that are greater than 5 times the reporting limit
(LLOQ) should have a relative standard deviation of less than 5 percent. The
CCV should be made from the same source as the calibration standards at a
concentration near the mid- level of the calibration curve. If an element does
not meet the recovery criteria of the CCV, than no samples can be reported for
that element in the area bracketed by the CCV.
11.8.2 For the CCB, all elements to be reported must be less than '/2 the RL (LLOQ).
If an element does not meet this criterion, then no samples can be
reported for that element in the area bracketed by the CCB.
11.9 Interference Check Standards. After the initial QC is completed and before any
samples are analyzed, the ICSA and ICSAB solutions (SIC solutions) must be
analyzed. The method does not give specific criteria for the ICSA and ICSAB, but
in house criteria should be applied. For all the spiked elements, the analyzed results
must be within 20 percent of the true results. For unspiked elements, the interfering
element solution should contain less than the absolute value of 2 times the reporting
limit (LLOQ) for each element. If these criteria are not met, then samples with significant
interferences cannot be reported until the correction factors are optimized and the ICSA
and ICSAB are within specifications.
11.9.1 If the run is longer than 12 hours, a second ICSA, ICSAB pair must be analyzed
before the next 12 hours is started.
11.9.2 If mass changes are made for the analysis of an element, all QC criteria must be
met for the new mass and it must be verified that appropriate correction factors are
in place.
11.10 Low Level Calibration Verification (CRI or LLCCV). The CRI standard containing the
elements of interest at (or below) the reporting level for each element. The CRI
(LLCCV) must be analyzed at the beginning and end of each calibration (analysis)
batch. The acceptance criterion for the CRI check is 70 to 130% recovery and 80-
120% for method 6020B. If an element does not meet this criterion, then all
bracketed samples for that element in the concentration range between the CRI and
the CCV must be reanalyzed. Samples containing concentrations higher than the CCV
may be reported as long as CCV criteria are met.
11.10.1 More frequent CRI (LLCCV) checks may be analyzed during the course of the
run if system stability at the low end of the calibration is questionable or if the
lab wants to ensure that fewer samples will have to be submitted for reanalysis
if there is a failed CRI at the end of a run.
11.10.2 It is recommended that the CRI check be run bracketing every 4 to 8 hour
period of analysis. It may be run as frequently as every 10 samples if the
supervisory staff deems that this is necessary.
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11.11 Method Blank. The laboratory must digest and analyze a method blank with each
set of samples. A minimum of one method blank is required for every 20 sample batch.
11.11.1 The default SOP limit for the method blank is that is must be less than one half
of the reporting limit (LLOQ).
11.11.2 In addition, the blank is considered acceptable if it is less than 10% of the
regulatory limit, or less than 10% of the lowest sample concentration for each
analyte in a given preparation batch, whichever is greater. Samples associated
with the contaminated blank shall be evaluated as to the best corrective action
for each particular sample. This may include reanalyzing the samples, re -
digesting and reanalyzing the samples, qualifying the results with a "B" or "V"
qualifier, or raising the reporting limit (LLOQ) to greater than two times the
background concentration.
11.12 Lab Control Sample or Spike Blank. The laboratory must digest and analyze a
laboratory control sample or spike blank with each set of samples. A minimum of
one lab control sample or spike blank is required for every 20 sample batch. The
laboratory should assess laboratory performance of the lab control and spike blank
against recovery limits of 80 to 120 percent for method 6020A and 6020B. Recovery
must be within 85 to 115 percent for method 200.8. In house lab control and spike
blank limits may also be generated to support these default limits. If the lab control or
spike blank is outside of the control limits for a given element, all samples must be
redigested and reanalyzed for that element.
11.12.1 If solid lab controls are used, then the manufacturer's limits should be applied.
11.13 Matrix Spike. The laboratory must add a known amount of each analyte to a minimum
of 1 in 20 samples. The matrix spike recovery is calculated as shown below.
Recoveries should be assessed against default limits of 80 to 120 percent for method
6020A and 6020B. Recovery must be within 70 to 130 percent for 200.8. In house
limits may be generated for this method for informational purposes only. If a matrix
spike is out of control, then the results should be flagged with the appropriate footnote
and it is recommended that a post -digest spike be analyzed for the out of control
element(s). If the matrix spike amount is less than one fourth of the sample amount,
then the sample cannot be assessed against the control limits and should be
footnoted to that effect. Note: Both the matrix spike amount and the sample amount
are calculated to the IDL for any given element. Any value less than the IDL is
treated as zero.
((Spiked Sample Result - Sample Result) / Amount Spiked) x 100 = matrix spike recovery
11.13.1If a post -digest spike is required, the sample should be spiked with
approximately 2 times the sample level or two times the reporting limits
(LLOQ), whichever is greater. Limits of 80 to 120 percent are normally
applied. The serial dilution is used to confirm any matrix effects. The post -
digest spike recovery must be footnoted on the matrix spike recovery or
otherwise noted in the quality control summary report.
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11.14 Matrix Spike Duplicate (MSD) or Matrix duplicate (DUP). The laboratory must digest a
matrix spike duplicate or matrix duplicate sample for a minimum of 1 in 20 samples.
The relative percent difference (rpd) between the MSD and the MS or between the
DUP and the sample should be assessed. The rpd is calculated as shown below.
The control limit for the duplicate rpd is method defined as 20%. If the sample and the
duplicate are less than 5 times the reporting limits (LLOQ) and are within a range of ±
the reporting limit (LLOQ), then the duplicate is considered to be in control. Note:
Both the duplicate amount and the sample amount are calculated to the IDL for any
given element. Any value less than the IDL is treated as zero.
11.14.1 If a MSD or duplicate is out of control, then the data should be checked
carefully to confirm that the high rpd for a given element is not a result of an
analytical problem. If an analytical problem is suspected, the MSD or duplicate
must be reanalyzed for confirmation. If the initial and reanalysis are in
agreement (within 20%), then the high rpd is a result of preparation or
sample issues and further analysis of the initial preparation is not required.
If the initial and reanalysis are not in agreement due to an analytical problem,
then any affected samples in the associated batch should also be reanalyzed
for that element.
11.14.2 If more than 50% of the elements in a sample (that have levels of at least 5
times the reporting limit) (LLOQ) have a high RPD, then the MSD or
duplicate should be redigested for confirmation, unless the sample matrix is
such that the non- homogeneity of the sample is visually apparent. If the
results confirm, the results from the original MSD or duplicate should be
flagged as indicative of possible sample non -homogeneity. If the results do
not confirm, then the whole batch should be digested and reanalyzed.
11.14.3If 50% or less of the elements in a sample (that have levels of at least 5
times the reporting limit)(LLOQ) have a high rpd, then the high rpd(s) should
be footnoted as indicating possible sample non -homogeneity unless other
problems are suspected. If problems are suspected, the reviewer will initiate
redigestion and reanalysis of the batch.
11.14.4 The calculations used to calculate RPD are shown below.
(IMS Result- MSD Resultl) x 100 = MSD RPD
(MS Result + MSD Result)/2
(ISample Result - Duplicate Resultl) x 100 = Duplicate RPD (Sample
Result + Duplicate Result)/2
11.15 Serial Dilution. A serial dilution is required on a frequency of one in 20 samples. It
is normally done on the same sample as is used for the matrix spike. If the analyte
concentration is within the linear dynamic range of the instrument and sufficiently
high (minimally a factor of at least 100 times greater than the concentration in the
reagent blank), then an analysis of a fivefold (1+4) dilution must agree to within
+10% of the original determination. If not, an interference effect must be suspected
and the serial dilution result for the element with the suspected interference must be
footnoted. The serial dilution is calculated as shown below.
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100 x ((Sample result — Serial dilution result)) = Serial dilution percent difference
Sample result
11.15.1 Results of less than the IDL are treated as 0. The concentration in the reagent
blank is normally < 3 times the IDL, so the factor of 100 times the
concentration in the reagent blank (listed above) so the limits should be
applied to sample concentrations of greater than 300 times the IDL.
11.16 Lower Limit of Quantitation check sample (LLQC). The LLQC is a sample at the
reporting limit (LLOQ) that is taken through the entire preparation and analytical
process. This standard must be analyzed when reporting limits (LLOQ) are initially
established and on an as needed basis after that. The limits of quantitation are
verified when all analytes in the LLQC sample are detected within 20% of their true
value. If the limits cannot be verified at the spiked level, then the quantitation limit
must be adjusted to a level where verification is successful.
12.0 DOCUMENTATION REQUIREMENTS
12.1 If samples or QC checks require reanalysis, a brief explanation of the reason
must be documented on the run log. All instrument data should be exported to the
LIMS system.
12.2 The Standard Preparation Logbook must be completed for all standard
preparations. All information requested must be completed. The SGS - Orlando Lot
Number must be cross- referenced on the standard vial.
12.3 The Instrument Maintenance Logbook must be completed when any type of
maintenance is performed on the instrument. Each instrument has a separate log.
12.4 The correction factors from each method must be printed out each time a change is
made and stored in a notebook in the lab. Each time the correction factors are
modified, a new printout must be obtained.
12.5 Any corrections to laboratory data must be done using a single line through the error. The
initials of the person and date of correction must appear next to the correction.
12.6 Supervisory (or peer) personnel must routinely review (approximately once per
month) all laboratory logbooks to ensure that information is being recorded properly.
Additionally, the maintenance of the logbooks and the accuracy of the recorded
information should also be verified during this review.
13.0 INSTRUMENT MAINTENANCE and TROUBLESHOOTING
Recommended periodic maintenance includes the items outlined below.
13.4 Change the pump tubing weekly or as needed.
13.5 Clean the nebulizer, torch, and injector tube every two to four weeks or more
often as needed.
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13.6 Change the sampler tip as needed (every one to two months).
13.7 Clean the recirculating pump lines as needed.
Record all maintenance in the Maintenance logbook. Repairs by manufacturer representative
and outside contractors must be documented in this logbook as well.
14.0 POLLUTION PREVENTION & WASTE MANAGEMENT
14.1 Users of this method must perform all procedural steps in a manner that controls the
creation and/or escape of wastes or hazardous materials to the environment. The
amounts of standards, reagents, and solvents must be limited to the amounts
specified in this SOP. All safety practices designed to limit the escape of vapors,
liquids or solids to the environment must be followed. All method users must be
familiar with the waste management practices described in section 14.2
14.2 Waste Management. Individuals performing this method must follow established
waste management procedures as described in the waste management SOP,
SAM108, current revision. This document describes the proper disposal of all
waste materials generated during the testing of samples as follows:
14.2.1 Non hazardous aqueous wastes.
14.2.2 Hazardous aqueous wastes
14.2.3 Chlorinated organic solvents
14.2.4 Non -chlorinated organic solvents
14.2.5 Hazardous solid wastes
14.2.6 Non -hazardous solid wastes
15.0 METHOD PERFORMANCE
Method performance (accuracy and precision) is monitored through the routine analysis of
negative and positive control samples. These control samples include method blanks (MB),
blank spikes (BS), matrix spikes (MS), and matrix spike duplicates (MSD). The MB and BS are
used to monitor overall method performance, while the MS and MSD are used to evaluate
the method performance in a specific sample matrix.
Blank spike, matrix spike, and matrix spike duplicate samples are compared to method
defined control limits. Control limits are stored in the LIMS. Additionally, blank spike accuracy
is regularly evaluated for statistical trends that may be indicative of systematic analytical errors.
Filtered method blanks and blank spikes to act as QC check of the filters. Unfiltered
method blanks and blank spikes are used to monitor overall method performance.
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16.0 ADDITIONAL REFERENCES
16.1 Refer to other SOP's for ICP-MS analysis (EPA 200.8).
16.2 MUR 2007 and 2012
16.3 TNI Standards, 2009
16.4 DoD QSM ver. 5.0, 2013
16.5 WV 47CSR32
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GNSOP: GN226.6
Rev. Date: 09/2015
Pages F-127 - F-142
TITLE: TOTAL CARBON and TOTAL ORGANIC CARBON IN SOIL SAMPLES
METHOD REFERENCES: SW846 Method 9060A, Revision 1, November 2004
Revised Sections: software revisions and procedure for update added (Sec. 8.1); multiple CCV
procedure incorporated in compliance with DoD QSM 5.0 (Secs. 10.6, 10.8); Deletions in 10.3
and after 10.5.1; removed low and high standard requirements (Sec. 12.2); corrected MS/MSD
frequency per method (Secs. 12.5, 12.6); Sec. 12.7 and 12.8 edited to include ICV/CCV
reanalysis; footnotes and qualifications of results associated with high —biased QC (Sec 12.10);
DoD QSM rev. 5.0 added, SM 19th and NELAC 2003 removed (Sec.16.0) ; added QC frequency
to Table 1; Instrument -specific start-up procedures are discussed in Attachments I and 11;
1.0 SCOPE AND APPLICATION
1.1 This method can be used to determine total organic carbon in solid matrix. It
may also be used for liquid matrices containing a high level of total organic
carbon. Samples that are primarily aqueous may also be analyzed using this
method, but sample sizes should be limited to < 0.10 g to reduce splattering.
1.2 Determination of Total Carbon is performed as modification of the method 9060A.
1.3 The product code for total organic carbon is TOC. Total Carbon product code
is TCAR
2.0 SUMMARY OF METHOD
2.1 Total organic carbon is determined by combusting an acidified and dried
sample, and converting the carbon dioxide CO2 into methane CH4, which is
measured by FID. Amount of CH4 is directly proportionate to the amount of
carbonaceous material in the sample. The quantitation is done by comparison to
a linear calibration curve. Total Carbon is determined by skipping the acidification
step.
2.2 Method has been modified to use Dextrose (instead of Potassium Hydrogen
Phthalate) as calibration standard per instrument manufacturer's specifications.
3.0 REPORTING LIMIT AND METHOD DETECTION LIMIT
3.1 The normal reporting limit for TOC in soils is 1000 mg/kg. This is based on a 0.1 g
sample size. A minimum reporting limit of 100 mg/kg can be obtained by using a 1.0
g sample size.
3.2 Method Detection Limit. Experimentally determine MDLs using the procedure
specified in 40 CFR, Part 136, Appendix B. This value represents the lowest
reportable concentration of an individual compound that meets the method
qualitative identification criteria. Experimental MDLs must be determined annually for
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this method.
4.0 DEFINITIONS
4.1 Batch: A group of samples which are similar with respect to matrix and the
testing procedures being employed and which are processed as a unit. A
sample batch is limited to a maximum of 20 samples or samples loaded on an
instrument within the same 12-hour shift, which ever comes first.
4.2 Blank Spike (BS): An analyte-free matrix spiked with a known amount of
analyte(s), processed simultaneously with the samples through all the steps of
the analytical procedure. Blank Spike Recoveries are used to document
laboratory performance for a given method. This may also be called a
Laboratory Control Sample (LCS).
4.3 Continuing Calibration Verification (CCV): A check standard used to verify
instrument calibration throughout an analytical run. CCV must be analyzed at the
beginning of the analytical run, after every 10 samples, and at the end of the run.
4.4 Holding Time: The maximum times that samples may be held prior to preparation
and/or analysis and still be considered valid.
4.5 Initial Calibration (ICAL): A series of standards used to establish the working
range of a particular instrument and detector. The low point should be at a level
equal to or below the reporting level.
4.6 Initial Calibration Verification (ICV): A standard from a source different than that
used for the initial calibration. A different vendor should be used whenever
possible. The ICV is used to verify the validity of an Initial Calibration. This may
also be called a QC check standard.
4.7 Matrix Spike (MS): A sample aliquot spiked with a known amount of analyte(s),
processed simultaneously with the samples through all the steps of the analytical
procedure. The matrix spike recoveries are used to document the bias of a
method in a given sample matrix.
4.8 Matrix Spike Duplicate (MSD): A replicate sample aliquot spiked with a known
amount of analyte(s), processed simultaneously with the samples through all the
steps of the analytical procedure. The matrix spike duplicate recoveries are used
to document the precision and bias of a method in a given sample matrix.
4.9 Method Blank (MB): An analyte-free matrix to which all reagents are added in the
same volumes or proportions as used in sample processing. The method blank is
processed simultaneously with the samples through all the steps of the analytical
procedure. The method blank is used to document contamination resulting
from the analytical process.
4.10 Method Detection Limits (MDLs) MDL is defined as the minimum concentration of a
substance that can be measured and reported with 99% confidence that the
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analyte concentration is greater than zero and is determined from analysis of a
sample in a given matrix containing the analyte. This definition is qualitative in
nature and does not evaluate an acceptable quantitative limit for method
performance. MDLs should be determined annually for every matrix and method.
Refer to SOP QA020, current revision.
4.11 Reagent Blank: The reagent blank is a blank that has the same matrix as the
samples, i.e., all added reagents, but did not go through sample preparation
procedures. The reagent blank is an indicator for contamination introduced during
the analytical procedure. For methods requiring no preparation step, the reagent
blank is equivalent to the method blank.
4.12 Reagent Grade: Analytical reagent (AR) grade, ACS reagent grade, and reagent
grade are synonymous terms for reagents, which conform to the current
specifications of the Committee on Analytical Reagents of the American
Chemical Society.
4.13 Reagent Water: Water that has been generated by any method, which would
achieve the performance specifications for ASTM Type II water.
4.14 Reference Material: A material containing known quantities of target analytes in
solution or in a homogeneous matrix. It is used to document the bias of the
analytical process.
4.15 Preservation: Refrigeration and/or reagents added at the time of sample
collection (or later) to maintain the chemical integrity of the sample.
5.0 HEALTH & SAFETY
5.1 The analyst should follow normal safety procedures as outlined in the Accutest
Laboratory Safety Manual which includes the use of safety glasses and lab coats. In
addition, all acids are corrosive and should be handled with care. Flush spills with
plenty of water. If acids contact any part of the body, flush with water and contact
the supervisor.
5.2 The instrument furnace operates at high temperature and the furnace should be
allowed to cool down before doing any system maintenance or troubleshooting. If
there are any signs of a system blockage, open the sample introduction port and
turn off the furnace to prevent build up of back pressure.
5.3 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely determined; however, each chemical should be treated as a potential
health hazard. Exposure to these reagents should be reduced to the lowest
possible level. The laboratory is responsible for maintaining a current awareness
file of OSHA regulations regarding the safe handling of the chemicals specified in
this method. A reference file of data handling sheets should be made available to
all personnel involved in these analyses.
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6.0 SAMPLE COLLECTION, PRESERVATION & HOLDING TIME
6.1 Soil samples are collected in glass jars, PTFE-lined lids with minimum head
space and should be kept under refrigeration at 4° C until they are analyzed.
6.2 No holding time for soils is outlined in SW846 9060A. 40 CFR part 136 specifies
a holding time of 28 days for aqueous samples. Unless otherwise specified, this
28-day holding time will also be applied to solid samples analyzed using this
SOP.
7.0 INTERFERENCES
7.1 High results may be obtained if the inorganic carbon is not completely removed
from the sample before analysis. To ensure that all of the inorganic carbon is
removed, heat an acidified sample at least 10 minutes at 750C before starting the
analysis. Some volatile organics may be lost in this heating step, resulting in a
low bias in the TOC result.
8.0 APPARATUS
The following items are needed for the analysis of samples following the method outlined below:
8.1 Shimadzu 5000 TOC analyzer with soil analysis module or equivalent.
At the time of SOP revision the software versions were TOC Control V
Ver.1.06.00 (TOC 1) and TOC Control V Ver. 2.0 (TOC 2). Data systems software
versions will be updated at the scheduled SOP revision. For interim software
changes refer to Maintenance Log assigned to the individual unit.
8.1.1 Maintenance and Troubleshooting
8.1.1.1 Each day of analysis, the humidifier should be checked to ensure
that the water level is within 1 inch of the top of the humidifier.
8.1.1.2 Each day of analysis, the baseline should be checked to make
sure that it is stable and near zero.
8.1.1.3 Whenever calibration check recoveries or blanks are out of
compliance, the flow and the condition of the catalyst should be
checked. If the catalyst appears bad (contains many small fines), it
should be cleaned and replaced. Refer to the instrument manuals
for additional information on system maintenance.
8.1.1.4 Use a cotton swab to remove any residual acid or any other
corrosive substance from TC or IC ports, boat holder and push -
rod included, or on internal parts of combustion tube connector.
Wash off with water, if necessary.
8.2 Syringes, 100 ul size.
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8.3 Analytical balance, capable or weighing to 0.1 mg. The calibration of the
analytical balance should be verified each day before use with Class 1 weights.
Calibrated and serviced annually by outside contractor.
8.4 Volumetric glassware, class A, for standards preparation.
8.5 Eppendorf pipettes. For maintenance and calibration refer to SOP QA006,
current revision.
8.6 Ceramic boats. For cleaning refer to SSM-5000A operator's manual.
8.7 Clean tweezers for handling ceramic boats. For cleaning procedure refer to SSM-
5000A operator's manual.
8.8 Drying oven, capable of being set to 75°C, used in acidification of the samples.
Monitored daily with NIST-traceable thermometer.
9.0 REAGENTS
All chemicals listed below are reagent grade unless otherwise specified. Deionized water
taken from the DI tap with the carbon filter should be used whenever water is required.
Make sure to properly label all reagents and record the reagent preparation in the reagent
logbook.
Commercially available standards should be used whenever possible. These standards
and reagents must be accompanied by the Certificate of Analysis (CoA). CoA is
examined for accuracy and completeness of the information, including verification of the
standard concentration
9.1 Dextrose Calibration Stock Solution, 200000 mg C/I (40% C from dextrose): Dry
dextrose in desiccator. Weigh out 46.5 grams into a 100-ml volumetric flask
containing approximately 80 ml of DI water. Add concentrated hydrochloric acid to
bring the pH < 2. Mix well and bring to a final volume of 100 ml. Note: This
stock should be replaced whenever crystallization of the dextrose is apparent. It
can be held for a maximum of 3 months. Refrigeration is not required.
9.2 Dextrose Calibration Standard Solutions: Dilute the above stock solution (200000
mg C/1) as shown below to make the suggested calibration standards. Add
concentrated hydrochloric acid to bring the pH to less than 2 before diluting each
standard to the final volume.
9.2.1 Different standards may be used, but a minimum of 5 standards and a
blank are required for the initial calibration. The top standards shown
below are close to the top of the linear range of the instrument and
sometimes will not work at these levels. Standard solutions must be held
for no longer than one month.
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Standard concentration, mYtock
Standard volume, ml
Final volume, ml
C/I
100000
25
50
75000
18.75
50
50000
12.5
50
30000
7.5
50
20000
10.0
100
10000
5.0
100
5000
2.5
100
2000
1.00
100
1000
0.5
100
Blank
0.00
100
9.3 Dextrose Second Source Stock solution, 200000 mg C/I. Dry dextrose in dessicator.
Weigh out 46.5 grams into a 100-ml volumetric flask containing approximately 80
ml of DI water. Add concentrated hydrochloric acid to bring the pH < 2. Mix well and
bring to a final volume of 100 ml. Note: This stock should be replaced whenever
crystallization of the dextrose is apparent. It can be held for a maximum of 3 months.
Refrigeration is not required.
9.4 Dextrose Second Source Independent Check Solution (ICV and Blank Spike),
200000 mg C/I Dilute 10.0 ml of the dextrose stock solution (200000 mg C/1) to
approximately 80 ml with DI water. Add concentrated hydrochloric acid to bring the pH
to less than 2 and then dilute to a final volume of 100 ml with DI water. This solution
should be made up monthly.
9.5 Hydrochloric acid, concentrated reagent grade. Used for acidifying samples to
remove inorganic carbon.
9.5.1 Dohrman Instruments has compared various acids used for this purpose and
recommends that phosphoric acid not be used. Dohrman Instruments found
that the phosphoric acid tended to coat both the boat and the catalyst in the
furnace with a layer of polyphosphoric acid and that, possibly as a
consequence of this, the release of inorganic carbon as carbon dioxide was
slower than with nitric acid and possibly incomplete.
9.6 Oxygen Gas, high purity.
9.7 Pre -cleaned glass wool.
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10.0 PROCEDURE
Below is the procedure to be followed for the analysis of soil samples for total organic carbon using
the Shimadzu TOC soil analyzer.
10.1 Turn on the oxygen. The pressure in the soil module should be set at 2 and the carrier
gas should be set as marked on the dial (0.5 I/min). The oxygen pressure at the tank
must be at least 50 psi to maintain sufficient pressure at the instrument. Check to
make sure that the humidifier contains sufficient water. It should be filled to within
approximately 1 inch of the top of the humidifier. (The humidifier is located under the
magnetic plate on the right side of the instrument.)
10.2 If the power is off, then turn on the power at the side of the soil and water
modules and for the computer.
10.3 Due to different software versions (see 8.1) refer to Attachment I and II for
instrument -specific start up procedures.
10.4 Go to instrument and click on the background monitor. A graph will appear on screen
showing the position of the baseline and the status of the furnace temperature. Wait
for the baseline to stabilize and for the furnace temperature to indicate that it is OK.
Make sure that the hatch on the boat sampler is tightly closed and that there are no
leaks in the system. If the baseline is not within + 10 of zero, then the zero of the
instrument may need to be adjusted. Check with the lab supervisor or manager for
further instructions.
10.5 If the instrument has not been calibrated within the last month, then it is recommended
that it be calibrated at this point. (A new calibration is required at least once per
quarter.)
10.5.1 Select a new file and insert standards. A minimum of 2 injections must be
used for each standard. Five standards and a blank are required for the
calibration. The lowest standard should be at 1000 mgC/I or lower. A 100 ul
injection size should be used for all standards.
10.5.2 Press "start" in the software and follow the prompts. Place a clean boat filled
with a small tuft of glass wool in the boat sampler. When indicated by the
software, inject 100 ul of standard into the boat. Close the hatch and push the
boat all the way forward. Enter OK at the software.
10.5.3 After the sample has finished running, the software will prompt you to pull the
boat back to the cool position. Pull the boat only to the cool line at this point.
When indicated by the software, then pull the boat back to the starting position.
10.5.4 Select the option to repeat the injection and repeat the steps outlined above.
10.5.5 When all of the standards have been completed, then review the curve using the
view, calibration option. Make sure "zero shift" box is unchecked, as it forces
curve through 0,0 point (forces through origin).
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10.5.5.1 If a correlation coefficient of greater than 0.995 is obtained
and intercept is <1/2 RL (see 12.2), then save the curve using the "file
save" option.
10.5.5.2 If either the correlation coefficient or the intercept does not meet
the above criteria, than recalibrate before proceeding with the samples.
10.6 If a previous calibration is being used, then it must be verified with a CCV and a
calibration blank before proceeding on each analysis day (see 10.8). Note: the CCV
must be made from a different source than the initial calibration standards. CCV must
be within 10% of the true value. If the CCV solution is not within 10% of the true
value, analyst must demonstrate acceptable performance with two CCVs analyzed
immediately (started within 1 hour), with no samples between failing CCV and the
two additional CCVs. If they are still not in control, then all bracketed samples for that
analyte must be reanalyzed. The blank must contain <1/2RL for TOC. Make sure to
use duplicate injections for all analyses. Note: The method blank may be used as the
calibration blank check.
10.7 After the calibration or calibration checks are completed, then analyze the external
check standard made from dextrose. This standard must agree within 10% of the
true value. If it is not within this range, determine the source of the problem before
proceeding. Note: The spike blank may be used as the external calibration check, but
then the results must be within 10% of the true value.
10.8 Verify Initial calibration curve every 10 samples with a continuing calibration check
made from external QC sample (ICV) stock. The continuing calibration check
should be a standard near the mid -range of the curve and recover within 10% of true
value. Procedure discussed in sec. 10.6 applies. If the check standard is not within
10% of the true value, then no samples can be reported in the area bracketed by this
calibration check unless the check is biased high (110 to 150%) and the sample results
to be reported are less than the reporting limit.
10.9 Begin analyzing the samples following the procedure outlined below.
10.9.1 Weigh out from 100 to 1000 mg of sample (wet weight) into a ceramic boat
using a 4-place analytical balance. For samples that contain high levels of TOC
smaller sample sizes may be needed. For unknown samples, start with a
sample size of 100 mg. (All method blanks and spike blanks should be
calculated assuming a 100 mg sample size and should be set up using pre -
cleaned glass wool.) Samples that contain non -homogeneous particulates
should be homogenized with a mortar and pestle before weighing out the
sample aliquot.
10.9.2 If samples are analyzed for Total Carbon, proceed without acidification step.
When Total Organic Carbon analysis is requested, sample is pre-treated to
remove inorganic carbon:
10.9.2.1 A few drops of concentrated Hydrochloric acid is carefully
added on top of the sample. Inorganic carbon (carbonates and
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bicarbonates) effervesces under acidic conditions in the form of CO2.
Sample boat is placed into the oven at 75 °C for a minimum of 10
minutes to dry the sample, drive off CO2 and reduce amount of acid
introduced into the detector.
It is important to use volatile acid, such ad HCI, for pretreatment.
Non-volatile acid may remain in the sample and hinder the analysis
or damage the detector.
10.9.3 If a client is requiring a detection limit lower than 1000 mg/kg, then larger
sample sizes are required. A detection limit of 100 mg/kg requires a weight of
1 gram. A smaller sample size may be used only to bring the sample to
within the range of the calibration curve.
10.9.3.1 Analyze samples in quadruplicates. Report average and the
range.
10.9.4 Matrix spike and a matrix spike duplicate should be analyzed at 10%
frequency. Method blank and spike blank must be analyzed every batch. All of
these quality control points must be prepared and analyzed the same way as
samples.
10.9.5 Prepare the method blank by treating a small amount (approximately 100
mg) of pre -cleaned glass wool with hydrochloric acid and heating at 75 ° C
for a minimum of 10 minutes. Skip acid treatment and heating for Total
Carbon test.
10.9.6 Prepare the spike blank in the same manner as the method blank, but spike it
with 100 ul of a 20000 mg C/I standard or external solution before adding
the acid. Note: The spike blank can be used in place of the external check,
but then must be prepared from the external source and must meet the 10 %
check criterion.
10.9.7 Prepare the matrix spike by adding 100 ul of a 20000 mg C/I standard or
external solution to a sample aliquot before adding the acid and heating the
sample. Prepare the matrix spike duplicate in the same manner as the matrix
spike.
10.9.8 At the end of the analysis, a continuing calibration check must be analyzed. If
the calibration check is not within 10% of the true value then all samples
bracketed by the out of compliance CCV must be reanalyzed. (If the CCV is
within 110 to 150%, then samples with results <RDL may be reported.)
10.9.9 If required, a CCB should be analyzed after the final CCV check of the analysis.
10.10 The final sample results are calculated using the equation shown below. The
calculation is done automatically in the Shimadzu TOC software except for the
percent solids correction. The percent solids correction is added when the data is
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transferred in the LIMS system. See area supervisor or manager for further details.
Organic Carbon, Total (mg/kg) = Conc. from curve (uq)
Sample weight in g x %sol/100
11.0 METHOD PERFORMANCE
Method performance is monitored through the routine analysis of negative and positive
control samples. These control samples include method blanks (MB), blank spikes (BS), matrix
spikes (MS), and matrix spike duplicates (MSD). The MB and BS are used to monitor
overall method performance, while the MS and MSD are used to evaluate the method
performance in a specific sample matrix.
Blank spike, matrix spike, and matrix spike duplicate samples are compared to method
defined control limits. Control limits are stored in the LIMS. Additionally, blank spike
accuracy is regularly evaluated for statistical trends that may be indicative of systematic
analytical errors.
12.0 QUALITY CONTROL
Below is a summary of the quality control requirements for this method. Make sure to check with
the laboratory supervisor or manager for any additional client specific quality control
requirements. All calculations are to be performed according to SOP QA042, current
revision.
12.1 Method Detection Limits (MDLs). MDLs should be established using a blank sample
spiked at approximately 3 times the estimated detection limit. To determine the MDL
values, take seven replicate aliquots of the spiked sample and process through the
entire analytical method. The MDL is calculated by multiplying the standard deviation
of the replicate analyses by 3.14, which is the student's t value for a 99% confidence
level. MDLs should be determined approximately once per year.
12.2 Calibration Curve. The instrument must be calibrated a minimum of once per quarter.
It is recommended that the instrument be calibrated at least once per month. The
calibration curve must have a correlation coefficient of at least 0.995 and the intercept
must be <1/2RL. Check standards must be within 10% of the true value. The calibration
blank must not contain TOC at >1/2RL.
12.3 Method Blank. The laboratory must prepare and analyze a method blank with each batch.
MB must be analyzed on clean matrix similar to the samples — pre -cleaned glass wool.
The method blank must not contain TOC at >1/2RL.
12.4 Spike Blank. The laboratory must prepare and analyze a spike blank with each batch.
LSC must be analyzed on clean matrix similar to the samples — precleaned glass wool.
The laboratory should assess laboratory performance of the spike blank against in-house
recovery limits.
12.5 Matrix Spike. The laboratory must add a known amount of each analyte to a
minimum of 1 in 10 samples.
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12.5.1 The spike recovery should be assessed using in house limits. Until these limits
can be generated, then default limits of 75 - 125% recovery should be applied. If a
matrix spike is out of control, then the results should be flagged with the
appropriate footnote. If the matrix spike amount is less than one fourth of the
sample amount, then the sample cannot be assessed against the control limits
and should be footnoted to that effect.
12.5.2 The matrix spike recovery should be calculated as shown below.
(Spiked Sample Result - Sample Result) x 100 = MS Recovery
(Amount Spiked)
12.6 Matrix Spike Duplicate. The laboratory must run an MSD for a minimum of 1 in 10
samples. The relative percent difference (RPD) between the duplicate and the
sample must be assessed. The duplicate RPD is calculated as shown below.
12.6.1 The duplicate RPD should be assessed using in house limits. Until these limits
can be generated, then default limits of 20%RPD must be applied. If a
duplicate is out of control, then the results must be flagged with the
appropriate footnote. If the sample and the duplicate are less than 5 times
the reporting limits and are within a range of + RL, then the duplicate is
considered to be in control.
12.6.2 The duplicate RPD must be calculated as shown below.
(MS Result- MSD Result) x 100 = % RPD
(MS Result + MSD Result) x 0.5
12.7 Quality Control Sample (also referred to as Initial Calibration Verification Standard,
(ICV)). A standard from a separate source than the calibration should be run at the
beginning of each run and every 10 samples — see 10.8. If the ICV is not within
acceptance criteria, a second ICV should be prepared and analyzed. If the ICV is still
outside of the limits, sample analysis must be discontinued and the cause
determined (preparation of ICV from third source, instrument recalibration, etc).
Note: The spike blank may be used in place of the ICV as long as a separate source
standard is used and the 10% criterion is met.
12.8 Continuing Calibration Verification (CCV). Analyze the continuing calibration
verification solution after every tenth sample and at the end of the sample run. The
CCV concentration should be at or near the mid -range of the calibration curve. If
the CCV solution is not within 10% of the true value, analyst must demonstrate
acceptable performance with two CCVs analyzed immediately (started within 1 hour),
with no samples between failing CCV and the two additional CCVs. If they are still not
in control, then all bracketed samples for that analyte must be reanalyzed. (Note:
Also see section "Contingencies for handling out -of -control QC" below) See also
12.9.
12.9 Continuing Calibration Blank (CCB). For some clients, a continuing calibration
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blank (CCB) may be required. This is not required as part of the normal TOC
protocol. If it is required, than it should be run after each CCV check. The results of the
CCB must be <1/2 RL for TOC. If the CCB is not <1/2 RL, then no samples can
be reported in the area bracketed by this CCB unless the sample results to be
reported are <1/2 RL.
12.10 Contingencies for handling out -of -control QC. Upon certain circumstances data
can be reported from batches with QC non -conformances. Such samples are to be
qualified accordingly. Examples include:
• If the MB is contaminated but the samples are non -detect, then the source
of contamination should be investigated and documented. The sample results
can be reported with qualification and footnotes why data is acceptable. If the
MB is contaminated but the samples results are > 10 times the contamination
level, the source of the contamination should be investigated and documented.
The samples results may be reported with the appropriate "B" or "V" qualifier.
This must be approved by the department supervisor. Samples with hits <10
times contamination are reprepped and reanalyzed. If there is insufficient
sample to reanalyze, or if the sample is re -analyzed beyond hold time, the
appropriate footnote and qualifiers should be added to the results. This must be
approved by the department supervisor
• Similarly, if the recovery of LCS or CCV is high and the associated sample
is non -detect, the data may be reportable with appropriate footnotes and
qualifiers. If the recovery of LCS or CCV is below lower acceptance limit, the
department supervisor shall review the data and determine what further
corrective action is best for each particular sample. That may include
reanalyzing the samples, reprepping and/or reanalyzing the samples, or
qualifying the results as estimated. This must be approved by the
department supervisor. If there is insufficient sample to reanalyze, or if the
sample is re -analyzed beyond hold time, the appropriate footnote and
qualifiers should be added to the results. This must be approved by the
department supervisor.
• If the matrix spike recoveries are not within the established control limits,
compare the recoveries to those of the LCS to assess method performance
in clean QC matrix. Matrix spike recovery failures are not grounds for reanalysis
but are an indication of the sample matrix effects.
13.0 DOCUMENTATION REQUIREMENTS
13.1 Each analyst should review all data and assemble a data package consisting of the
following information. This data package should be turned into the supervisor for review
after the analysts complete their LIMS review (see 13.3 below).
• Results report, showing dilutions, replicate injection results, and CCV or RPD
results.
• Preparation/run log showing weights taken at the balance for each injection.
• QC Summary sheet with calculations
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13.2 In addition, all reagent information such as lot numbers should also be recorded in the
reagent logbook. Any unusual characteristics of the samples should be noted on the
raw data or on the preparation log. Make sure that all sample ID's and dilutions are
labeled on the data.
13.3 An ASCI format file should be generated and copied over to the LIMS. The analyst is
responsible for reviewing the data in the LIMS and adding appropriate spike amounts
and true values before sending the data for supervisor approval.
14.0 POLLUTION PREVENTION & WASTE MANAGEMENT
14.1 Users of this method must perform all procedural steps in a manner that controls the
creation and/or escape of wastes or hazardous materials to the environment. The
amounts of standards, reagents, and solvents must be limited to the amounts
specified in this SOP. All safety practices designed to limit the escape of vapors,
liquids or solids to the environment must be followed. All method users must be familiar
with the waste management practices described in section 14.2.
14.2 Waste Management. Individuals performing this method must follow established waste
management procedures as described in the waste management SOP, SAM108,
current revision.
15.0 GLASSWARE CLEANING
All glassware should be washed with soap and tap water and then rinsed with de -ionized water
as described in SOP GN196, current revision.
Special procedure for cleaning the ceramic boats and tweezers is described in Sec 3.1 of
SSM-5000A operator's manual, pp. 16 and 17. Manual is on file with GenChem
Department.
16.0 ADDITIONAL REFERENCES
Shimadzu Instrument Manual, SSM-5000A; TNI
Standards, 2009 revision
DoD QSM 5.0, 2013 revision
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Table 1 QC Criteria
Quality Control
Frequency
Acceptance Criteria
Corrective Action
Rerun calibration
Initial Calibration:
>_0.995
standards, and/or prepare
At least once
new calibration standards
r =coefficient of
per quarter
and recalibrate the
correlation
instrument, or document
why the data are
acceptable.
Initial Calibration
90 - 110% of the
Rerun standard, and/or
Verification
standard's true value
prepare new standard,
standard (ICV)
One per
and/or recalibrate
calibration
instrument, or document
why the data are
acceptable.
Continuing
90 - 110% of the
Rerun standard, and/or
Calibration
standard's true value
recalibrate instrument and
Verification
Every tenth
reanalyze all samples run
standard (CCV)
sample
since the last acceptable
CCV, or document why the
data are acceptable.
Reanalyze, and/or stop the
Method blank (MB)
run and determine the
and Calibration
One per batch
< 1/2RL
source of contamination, or
Blank (CB)
document why the data are
acceptable.
Determine and correct the
Blank Spike
90-110%
problem, reanalyze
(BS or LCS)
One per batch
samples, if necessary, or
document why data are
acceptable.
Determine and correct the
90-110%
problem, reanalyze
MS/MSD
10% of matrix
samples and MS/MSD, or
document why data are
acce table
Determine and correct
MSD
10% of matrix
<20% RPD
cause of the poor
reproducibility
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Attachment
7.z01 ff
Go to TOC software (TOC-Control V)
TOC-Control V window will pop-up, select sample table editor icon.
1.1 To start up the instrument go to File -New
1.2 Calibration Curve (if instrument is calibrated proceed to 1.2)
1.2.1 In System, select SSM-COPY 2
1.2.2 Make sure that Edit calibration manually box is checked
1.2.3 In analysis, select SSM-TC, uncheck zero shift box, and in file name, enter the
TOC 1 so cal -curve mmddyyyy
1.2.4 In unit, select mg and check weight box
1.2.5 Press add to enter the calibration curve point starting with the higher
concentration to the lower
1.2.6 Check use default setting box
1.2.7 Make sure that the enable history log box is uncheck.
1.3 Go to File -New
1.3.1 Sample Run
1.3.2 In System, select SSM Copy 2
1.3.3 In File Name, enter the name of the run using the following template TOC 1 SO
mmddyyyy, save.
1.4 Connect
1.4.1 Go to Instrument — Connect and check use setting on computer box.
1.4.1.1 A pop-up window will appear; once the connection is complete, this
window will close.
1.5 To turn on the furnace
1.5.1 Go to Instrument — Properties
1.5.1.1 Under TOC tab: TC Furnace (Deg. C) select 680
1.5.1.2 Under SSM tab: Check the TC Furnace On box
1.6 To schedule calibration and/or samples
1.6.1 To run the calibration, go to Insert — Calibration Curve, select the calibration that
it is going to perform.
1.6.1.1 To update the method with the new calibration, go to File -Open
1.6.1.1.1 In File of type select Method
1.6.1.1.2 Select Copy 2 of SSM
1.6.1.1.3 SSM-TC, calibration curve browse for the last calibration that
passed.
1.6.2 To run a sample sequence go to Insert — Auto Generate
1.6.2.1 In Method, select Copy 2 of SSM
1.6.2.2 In Sample Number: enter the number of sample to be analyzed including
the standards. Press next until finished.
2.1 Before start the run go to Instrument - Maintenance and select the following option in order
2.2 Zero point detection
2.3 Regeneration of TC catalyst
2.4 Residual Removal
2.5 Washing
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Attachment II
TOC 2
Go to TOC software (TOC-V Sample Table Editor)
1.1 To start up the instrument, go to File -New
1.2 Calibration Curve (if instrument is calibrated proceed to 1 .2)
1.2.1 Calibration Wizard window will pop-up
1.2.2 In System, select TOC-2 Solid
1.2.3 Make sure that Edit calibration manually box is checked
1.2.4 In analysis, select SSM-TC, uncheck zero shift box, and in file name enter the
TOC 2 so cal -curve mmddyyyy
1.2.5 In unit, select mg and check weight box
1.2.6 Press add to enter the calibration curve point starting with the lower
concentration to the higher
1.2.7 Check use default setting box and Correlation Coefficient box
1.3 Go to File -New
1.3.1 A Select H/W Setting will pop-up
1.3.2 In System selected TOC 2 -Solid
1.3.3 In File Name enter the name of the run using the following template TOC 2 SO
mmddyyyy, save.
1.4 Connect
1.4.1 Select Connect icon.
1.4.1.1 A pop-up window will appear once the connection is complete; this
window will close.
1.5 To turn on the furnace
1.5.1 Go to Instrument — H/W Setting
1.5.1.1 Under TOC tab: TC Furnace (Deg. C) select 680
1.5.1.2 Under SSM tab: Check the SSM TC Furnace On box
1.6 To schedule calibration and/or samples
1.6.1 To run the calibration, go to Insert — Calibration Curve, select the calibration that it is
going to perform.
1.6.1.1 Press start, in file name enter the date MMDDYYYY and start.
1.6.1.2 To update the method with the new calibration, go to File -Open
1.6.1.2.1 In File of type select Method
1.6.1.2.2 Select SSMI-COPY
1.6.1.2.3 SSM-TC, calibration curve browse for the last calibration that
passed.
1.6.2 To run a sample sequence go to Insert — Auto Generate
1.6.2.1 In Method, select SSMI-COPY
1.6.2.2 In Sample Number: enter the number of sample to be analyzed including the
standards. Press next until finished.
2.1 Before start the run go to Instrument - Maintenance and select the following option in order
2.2 Zero point detection
2.3 Regeneration of TC catalyst
2.4 Residual Removal
2.5 Washing
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BULK DENSITY METHOD
Pages F-143 - F-148
Designation: C 29/C 29M — 97 (Reapproved 2003) American Association of State
Highway and Transportation Officials Standard
*U1 AASHTO No.: T19/T19M
nrrnnrfrrwuc
Standard Test Method for Bulk Density ("Unit Weight") and Voids in Aggregate
This standard is issued under the fixed designation C 29/C 29M; the number immediately following the designation indicates
the year of op-iginal adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year
of last reapproval. A superscript epsilon ( ) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the Department of Defense.
1. Scope
1.1 This test method covers the determination of bulk density ("unit weight") of aggregate in a compacted or
loose condition, and calculated voids between particles in fine, coarse, or mixed aggregates based on the same
determination. This test method is applicable to aggregates not exceeding 5 in. [125 mm] in nominal maximum size.
NOTE 1—Unit weight is the traditional terminology used to describe the property determined by this test
method, which is weight per unit volume (more correctly, mass per unit volume or density).
1.2 The values stated in either inch -pound units or SI units are to be regarded separately as standard, as
appropriate for a specification with which this test method is used. An exception is with regard to sieve sizes and
nominal size of aggregate, in which the SI values are the standard as stated in Specification E 11. Within the text,
SI units are shown in brackets. The values stated in each system may not be exact equivalents; therefore each
system must be used independently of the other, without combining values in any way.
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is
the responsibility of the user of this standard to establish appropriate safety and health practices and determine
the applicability of regulatory limitations prior to use.
2. Referenced Documents
2.1 ASTMStandards:
C 125 Terminology Relating to Concrete and Concrete Aggregates2
C 127 Test Method for Density, Relative Density (Specific Gravity), and Absorption of Coarse Aggregate2
C 128 Test Method for Density, Relative Density (Specific Gravity), and Absorption of Fine Aggregate2
C 138/C 138M Test Method for Density (Unit Weight), Yield, and Air Content (Gravimetric) of Concrete2
C 670 Practice for Preparing Precision and Bias Statements for Test Methods for Construction Materials2
C 702 Practice for Reducing Samples of Aggregate to Testing Size2
D 75 Practice for Sampling Aggregates3 D 123 Terminology Relating to Textiles4
E 11 Specification for Wire Cloth and Sieves for Testing Purposes5
2.2 AASHTO Standard:
T19/T19M Method of Test for Unit Weight and Voids in Aggregate6
' This test method is under the jurisdiction of ASTM Committee C09 on Concrete and Concrete Aggregates and is the direct responsibility of Subcommittee
C09.20 on Normal Weight Aggregates. Current edition approved July 10, 1997. Published September 1997. Originally approved in 1920. Last previous edition
approved in 1991 as C 29/C 29M — 91 a.
2 Annual Book of ASTM Standards, Vol 04.02.
3 Annual Book of ASTM Standards, Vol 04.03.
4 Annual Book of ASTM Standards, Vol 07.01.
5 Annual Book of ASTM Standards, Vol 14.02.
6 Available from American Association of State Highway and Transportation Officials, 444 N. Capitol St. NW, Suite 225, Washington, DC 20001.
C 29/C 29M — 97 (2003)
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3. Terminology
3.1 Definitions -Definitions are in accordance with Terminology C 125 unless otherwise indicated.
3.1.1 bulk density, n-of aggregate, the mass of a unit volume of bulk aggregate material, in which the
volume includes the volume of the individual particles and the volume of the voids between the
particles. Expressed in lb/ft3 [kg/m3].
3.1.2 unit weight, n-weight (mass) per unit volume. (Dep- recated term used -preferred term bulk
density.)
3.1.2.1 Discussion -Weight is equal to the mass of the body multiplied by the acceleration due to
gravity. Weight may be expressed in absolute units (newtons, poundals) or in
gravitational units (kgf, lbf), for example: on the surface of the earth, a body with a mass
of 1 kg has a weight of 1 kgf (approximately 9.81 N), or a body with a mass of 1 lb has a
weight of 1 lbf (approximately 4.45 N or 32.2 poundals). Since weight is equal to mass
times the acceleration due to gravity, the weight of a body will vary with the location
where the weight is determined, while the mass of the body remains constant. On the
surface of the earth, the force of gravity imparts to a body that is free to fall an
acceleration of approximately 9.81 m/s2 (32.2 ft/s2). D 123.
3.2 Definitions of Terms Specific to This Standard.•
3.2.1 voids, n-in unit volume of aggregate, the space between particles in an aggregate mass not occupied
by solid mineral matter.
3.2.1.1 Discussion -Voids within particles, either permeable or impermeable, are not included in
voids as determined by this test method.
4. Significance and Use
4.1 This test method is often used to determine bulk density values that are necessary for use for many methods of
selecting proportions for concrete mixtures.
4.2 The bulk density also may be used for determining mass/volume relationships for conversions in
purchase agreements. However, the relationship between degree of compaction of aggregates in a hauling
unit or stockpile and that achieved in this test method is unknown. Further, aggregates in hauling units and
stockpiles usually contain absorbed and surface moisture (the latter affecting bulking), while this test
method determines the bulk density on a dry basis.
4.3 A procedure is included for computing the percentage of voids between the aggregate particles based on the
bulk density determined by this test method.
5. Apparatus
5.1 Balance -A balance or scale accurate within 0.1 % of the test load at any point within the range of use,
graduated to at least 0.1 lb [0.05 kg]. The range of use shall be considered to extend from the mass of the
measure empty to the mass of the measure plus its contents at 120 lb/ft3 [1920 kg/m3].
5.2 Tamping Rod -A round, straight steel rod, 5/8 in. [16 mm] in diameter and approximately 24 in. [600
mm.] in length, having the tamping end, or both ends, rounded to a hemispherical tip, the diameter of which
is 5/8 in. (16 mm).
5.3 Measure -A cylindrical metal measure, preferably pro- vided with handles. It shall be watertight, with
the top and bottom true and even, and sufficiently rigid to retain its form under rough usage. The
measure shall have a height approximately equal to the diameter, but in no case shall the height be less
than 80 % nor more than 150 % of the diameter. The capacity of the measure shall conform to the
limits in Table 1 for the aggregate size to be tested. The thickness of the metal in the measure shall be as
described in Table 2. The top rim shall be smooth and plane within 0.01 in. (0.25mm) and shall be parallel
to the bottom within 0.5° (Note 2). The interior wall of the measure shall be a smooth and continuous
surface.
NOTE 2 -The top rim is satisfactorily plane if a 0.01-in (0.25mm) feeler gage cannot be
inserted between the rim and a piece of '/4-in (6-mm) or thicker plate glass laid over the
measure. The top and bottom are satisfactorily parallel if the slope between pieces of plate glass
in contact with the top and bottom does not exceed 0.87 % in any direction.
5.3.1 If the measure is to also be used for testing for bulk density of freshly -mixed concrete
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according to Test Method C 138, the measure shall be made of steel or other suitable metal
not readily subject to attack by cement paste. Reactive materials, such as aluminum alloys are
permitted, where as a consequence of an initial reaction, a surface film is formed which
protects the metal against further corrosion.
5.3.2 Measures larger than nominal 1 ft3 (28 L) capacity shall be made of steel for rigidity, or the
minimum thicknesses of metal listed in Table 2 shall be suitably increased.
5.4 Shovel or Scoop -A shovel or scoop of convenient size for filling the measure with aggregate.
5.5 Calibration Equipment -A piece of plate glass, prefer- ably at least 1/4 in. [6 mm] thick and at least 1
in. [25 mm] larger than the diameter of the measure to be calibrated. A supply of water -pump or
chassis grease that can be placed on the rim of the container to prevent leakage.
TABLE 1 Capacity of Measures
Nominal Maximum Size of Aggregate
Capacity of Measure
In. mm.
ft 3
L (m3)
'/z 12.5
1/10
2.8 (0.0028)
1 25.0
1/3
9.3 (0.0093)
1 '/z 37.5
'h
14 (0.014)
3 75
1
28 (0.028)
4 100
2'/2
70 (0.070)
5 125
3'h
100 (0.100)
The indicated size of measure shall be used to test aggregates of a nominal maximum size equal to or smaller than that
listed. The actual volume of the measure shall be at least 95 % of the nominal volume listed.
TABLE 2 Requirements for Measures
Thickness of Metal, min
Capacity of Measure
Bottom
Upper 1 '/2 in
Remainder of wall
or 38 mm
of wall
Less than 0.4 ft 3
0.20 in.
0.10 in.
0.10 in.
0.4 ft 3 to 1.5 ft 3, incl
0.20 in.
0.20 in.
0.12 in.
over 1.5 to 2.8 ft 3, incl
0.40 in.
0.25 in.
0.15 in.
over 2.8 to 4.0 ft 3, incl
0.50 in.
0.30 in.
0.20 in.
Less than 11 L
5.0 mm
2.5 mm
2.5 mm
11 to 42 L, incl
5.0 mm
5.0 mm
3.0 mm
over 42 to 80 L, incl
10.0 mm
6.4 mm
3.8 mm
over 80 to 133 L
13.0 mm
7.6 mm
5.0 mm
The added thickness in the upper portion of the wall may be obtained by placing a reinforcing band around the top of the
measure.
6. Sampling
6.1 Obtain the sample in accordance with Practice D 75, and reduce to test sample size in
accordance with Practice C 702.
7. Test Sample
7.1 The size of the sample shall be approximately 125 to 200 % of the quantity required to fill the measure,
and shall be handled in a manner to avoid segregation. Dry the aggregate sample to essentially constant
mass, preferably in an oven at 230 f 9°F [110 ± 5°C].
8. Calibration of Measure
8.1 Fill the measure with water at room temperature and cover with a piece of plate glass in such a way as
to eliminate bubbles and excess water.
8.2 Determine the mass of the water in the measure using the balance described in 5.1.
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8.3 Measure the temperature of the water and determine its density from Table 3, interpolating if necessary.
8.4 Calculate the volume, V, of the measure by dividing the mass of the water required to fill the measure by
its density. Alternatively, calculate the factor for the measure (1/17) by dividing the density of the water
by the mass required to fill the measure.
TABLE 3 Density of Water
Temperature lb/ft3 Kg/m3
F° Co
60
15.6
62.366
999.01
65
18.3
62.336
998.54
70
21.1
62.301
997.97
73.4
23.0
62.274
997.54
75
23.9
62.261
997.32
80
26.7
62.216
996.59
85
29.4
62.166
995.83
NOTE 3- For the calculation of bulk density, the volume of the measure in SI units should be expressed in cubic metres, or
the factor as 1 /m3. However, for convenience the size of the measure may be expressed in litres.
8.5 Measures shall be recalibrated at least once a year or whenever there is reason to question the accuracy
of the calibration.
9. Selection of Procedure
9.1 The shoveling procedure for loose bulk density shall be used only when specifically stipulated. Otherwise,
the compact bulk density shall be determined by the rodding procedure for aggregates having a nominal
maximum size of 112 in. [37.5 mm] or less, or by the jigging procedure for aggregates having a nominal
maximum size greater than 112 in. [37.5 mm] and not exceeding 5 in. [125 mm].
10. Rodding Procedure
10.1 Fill the measure one-third full and level the surface with the fingers. Rod the layer of aggregate with 25
strokes of the tamping rod evenly distributed over the surface. Fill the measure two-thirds full and
again level and rod as above. Finally, fill the measure to overflowing and rod again in the manner
previously mentioned. Level the surface of the aggregate with the fingers or a straightedge in such a way
that any slight projections of the larger pieces of the coarse aggregate approximately balance the larger
voids in the surface below the top of the measure.
10.2 In rodding the first layer, do not allow the rod to strike the bottom of the measure forcibly. In rodding the
second and third layers, use vigorous effort, but not more force than to cause the tamping rod to
penetrate to the previous layer of aggregate.
NOTE 4-In rodding the larger sizes of coarse aggregate, it may not be possible to penetrate the layer
being consolidated, especially with angular aggregates. The intent of the procedure will be
accomplished if vigorous effort is used.
10.3 Determine the mass of the measure plus its contents, and the mass of the measure alone, and record the
values to the nearest 0.1 lb [0.05 kg].
11. Jigging Procedure
11.1 Fill the measure in three approximately equal layers as described in 10.1, compacting each layer by placing
the measure on a firm base, such as a cement -concrete floor, raising the opposite sides alternately about 2
in. [50 mm], and allowing the measure to drop in such a manner as to hit with a sharp, slapping blow. The
aggregate particles, by this procedure, will arrange themselves in a densely compacted condition. Compact
each layer by dropping the measure 50 times in the manner described, 25 times on each side. Level the
surface of the aggregate with the fingers or a straightedge in such a way that any slight projections of the
larger pieces of the coarse aggregate approximately balance the larger voids in the surface below the top of
the measure.
11.2 Determine the mass of the measure plus its contents, and the mass of the measure alone, and record the
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February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report

values to the nearest 0.1 lb [0.05 kg].
12. Shoveling Procedure
12.1 Fill the measure to overflowing by means of a shovel or scoop, discharging the aggregate from a height not
to exceed 2 in. [50 mm] above the top of the measure. Exercise care to prevent, so far as possible,
segregation of the particle sizes of which the sample is composed. Level the surface of the aggregate with
the fingers or a straightedge in such a way that any slight projections of the larger pieces of the coarse
aggregate approximately balance the larger voids in the surface below the top of the measure.
12.2 Determine the mass of the measure plus its contents, and the mass of the measure alone, and record the
values to the nearest 0.1 lb [0.05 kg].
13. Calculation
13.1 Bulk Density —Calculate the bulk density for the rodding, jigging, or shoveling procedure as follows:
M= (G—T)/V (1)
or
M = (G —T) x F (2)
where:
M = bulk density of the aggregate, lb/ft3 [kg/m3],
G = mass of the aggregate plus the measure, lb [kg],
T = mass of the measure, lb [kg],
V = volume of the measure, ft3 [m3], and
F = factor for measure, f —3 [m 3]
13.1.1 The bulk density determined by this test method is for aggregate in an oven -dry condition. If the
bulk density in terms of saturated -surface -dry (SSD) condition is desired, use the exact
procedure in this test method, and then calculate the SSD bulk density using the following
formula:
Mssd = M[1 + (All00)] (3)
where:
MssD = bulk density in SSD condition, lb/ft3 [kg/m3], and
A = % absorption, determined in accordance with Test Method C 127 or Test Method C 128.
13.2 Void Content —Calculate the void content in the aggregate using the bulk density determined by either the
rodding, jigging, or shoveling procedure, as follows:
%Voids = 100[(S x W) — M]I(S x W) (4)
where:
M = bulk density of the aggregate, lb/ft3 [ kg/m3],
S = bulk specific gravity (dry basis) as determined in accordance with Test Method C 127 or Test Method C 128, and
W = density of water, 62.3 lb/ft3 [998 kg/m3].
14. Report
14.1 Report the results for the bulk density to the nearest 1 lb/ft3 [10 kg/m3] as follows:
14.1.1 Bulk density by rodding, or
14.1.2 Bulk density by jigging, or
14.1.3 Loose bulk density.
14.2 Report the results for the void content to the nearest 1 % as follows:
14.2.1 Voids in aggregate compacted by rodding, %, or
14.2.2 Voids in aggregate compacted by jigging, %, or
14.2.3 Voids in loose aggregate, %.
15. Precision and Bias
15.1 The following estimates of precision for this test method are based on results from the AASHTO
Materials Reference Laboratory (AMRL) Proficiency Sample Program, with testing conducted using this
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test method and AASHTO Method T 19/T19M. There are no significant differences between the two test
methods. The data are based on the analyses of more than 100 paired test results from 40 to 100
laboratories.
15.2 Coarse Aggregate (bulk density):
15.2.1 Single -Operator Precision —The single -operator standard deviation has been found to be 0.88
lb/ft3 [14 kg/m3] (Is). Therefore, results of two properly conducted tests by the same operator on
similar material should not differ by more than 2.5 lb/ft3 [40 kg/m3] (d2s).
15.2.2 Multilaboratory Precision —The multilaboratory standard deviation has been found to be 1.87
lb/ft3 [30 kg/m3] (Is). Therefore, results of two properly conducted tests from two different
laboratories on similar material should not differ by more than 5.3 lb/ft3 [85 kg/m3] (d2s).
15.2.3 These numbers represent, respectively, the (Is) and (d2s) limits as described in Practice C 670.
The precision estimates were obtained from the analysis of AMRL proficiency sample data for
bulk density by rodding of normal weight aggregates having a nominal maximum aggregate
size of 1 in. [25.0 mm], and using a 12-ft3 [14- L] measure.
15.3 Fine Aggregate (bulk density):
15.3.1 Single -Operator Precision —The single -operator standard deviation has been found to be 0.88
lb/113 [14 kg/m3] (Is). Therefore, results of two properly conducted tests by the same operator on
similar material should not differ by more than 2.5 lb/113 [40 kg/m3] (d2s).
15.3.2 Multilaboratory Precision —The multilaboratory standard deviation has been found to be 2.76
lb/ft3 [44 kg/m3] (Is). Therefore, results of two properly conducted tests from two different
laboratories on similar materials should not differ by more than 7.8 lb/ft3 (125kg/m3) (d2s).
15.3.3 These numbers represent, respectively, the (Is) and (d2s) limits as described in Practice C 670. The
precision estimates were obtained from the analysis of AMRL proficiency sample data for
loose bulk density from laboratories using a IAA-113 [2.8-L] measure.
15.4 No precision data on void content are available. However, as the void content in aggregate is calculated
from bulk density and bulk specific gravity, the precision of the voids content reflects the precision of
these measured parameters given in 15.2 and 15.3 of this test method and in Test Methods C 127 and C
128.
15.5 Bias —The procedure in this test method for measuring bulk density and void content has no bias because
the values for bulk density and void content can be defined only in terms of a test method.
16. Keywords
16.1 aggregates; bulk density; coarse aggregate; density; fine aggregate; unit weight; voids in aggregates
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C 29/C 29M — 97 (2003)
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PCS Phosphate Company, Inc.
February 2011-revised September 2012; Appendix F updated August 2018 for inclusion in the 2018 creeks report