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Home My WebLink AboutNC0021709_Report_19870331NPDES DOCW'IENT !;CANNING COVER SHEET NPDES Permit: NC0021709 Jefferson WWTP Document Type: Permit Issuance Wasteload Allocation Authorization to Construct (AtC) Permit Modification Complete File - Historical Engineering Alternatives (EAA) Correspondence Owner Name Change Report Instream Assessment (67b) Speculative Limits Environmental Assessment (EA) Document Date: March 31, 1987 This document is printed on reuse paper - ignore any content on the resrerse side DIVISION OF ENVIRONMENTAL MANAGEMENT March 31, 1987 MEMORANDUM TO: Larry Coble Dennis Ramsey FROM: St W. Tedder Steve e erf / 4 SUBJECT: Toxicological Evaluation - Jefferson WWTP Attached is the final report concerning an intensive toxicological evaluation of the Jefferson WWTP in Ashe County. If there are any questions, please contact myself or Ken Eagleson at (919)733-5083. SWT:ps cc: Ken Eagleson Larry Ausley Bob DeWeese Trevor Clements Jay Sauber Jim Overton Jefferson WWTP Toxicity Examination NPDES#NC0021709 111111111111 li1111hILllL 111111111111 North Carolina Department of Natural Resources & Community Development MOB/LE id & Bioassay and Biomonitoring LABORATORY 0 oia 0 NORTH CAROLINA DEPARTMENT OF NATURAL RESOURCES AND COMMUNITY DEVELOPMENT WATER QUALITY SECTION March, 1987 TOXICITY EXAMINATION OF JEFFERSON WASTEWATER TREATMENT PLANT NC0021709 ASHE COUNTY NORTH CAROLINA DEPARTMENT OF NATURAL RESOURCES AND COMMUNITY DEVELOPMENT DIVISION OF ENVIRONMENTAL MANAGEMENT WATER OUALITY SECTION TECHNICAL SERVICES BRANCH TABLE OF CONTENTS Page List of Figures and Tables Introduction 1 Toxicity Examination 3 Chemical Sampling 9 Benthic Macroinvertebrate Analysis 17 Conclusions 26 Recommendations 27 Footnotes 28 Appendix 29 Daphnia pulex Test Procedure 30 96 Hour Flow -Through Test Procedure 31 Ceriodaphnia Reproduction Test Procedure 34 Benthic Macroinvertebrate Procedure 35 List of Definitions 36 LIST OF FIGURES Page Figure 1. Schematic of Jefferson WWTP 4 Figure 2. Seven Day Ceriodaahnia Mortality 6 Figure 3. Jefferson WWTP Study Area 11 LIST OF TABLES Page Table 1. Jefferson WWTP Self -Monitoring Bioassay Results 2 Table 2. Industrial Contributors to Jefferson WWTP 2 Table 3. 96 Hour Fathead Minnow Mortality 5 Table 4. Industrial Contributor Bioassay Results 8 Table 5. Sampling Site Descriptions 9 Table 6. Results of Inorganic Chemical Analyses 12 Table 7. Results of Industrial Contributor Metals Analyses 14 Table 8. Results of Organic Chemical Analyses 15 Table 9. Taxa richness by Group, Naked Creek, Ashe Co., July 1986 19 Table 10. Species List, Naked Creek, Ashe Co., July 1986 20 INTRODUCTION An on -site toxicity examination was conducted at the Jefferson Wastewater Treatment Plant (NC0021709) from July 21 through July 26, 1986. The Jefferson WWTP, located in central Ashe County, serves the Town of Jefferson and three major industries. This document details findings of chemical, biological and toxicological evaluations, including the following: 1.) 48 hour static bioassays employing Daphnia pulex in effluent and influent samples from the WWTP, as well as discharges from several industrial contributors, to determine acute toxicity. 2.) 96 hour flow -through bioassay using Pimephales promelas (fathead minnows) performed on effluent collected prior to chlorination. 3.) Seven day Ceriodaphnia reproduction bioassay to assess chronic toxic- ity. 4.) Chemical analyses of samples collected from the plant influent, efflu- ent, receiving stream and industrial contributors. 5.) Collection and analysis of benthic macroinvertebrates to determine the impact of the effluent on receiving stream populations. The Jefferson WWTP discharges into Naked Creek (Class C-trout) in the New Lao River basin. The 7010 (7 day, 10 year low flow) for Naked Creek is /4 cubic feet per second (cfs). At maximum permitted flow (0.15 MGD) and 7010 stream flow 46 conditions, the WWTP produces an instream waste concentration (IWC) of 8 3%. In August of 1986 the Division of Environmental Management required the Jefferson WWTP to begin 48 hour Daphnia pulex`static self -monitoring bioassays on a monthly basis. Table 1 shows available results for these bioassays. The tar- get acute toxicity value (LC60) for these tests is -90%. The LC60 value is the concentration of effluent lethal to fifty percent of the test organisms. Table 1. Jefferson WWTP Self —Monitoring Bioassay Results Month �.CB,Q August 86 13.6 September 86 None October 86 7.1 November 86 None December 86 None January 87 None The waste treatment processes at the Jefferson WWTP include a bar screen, aeration basin, clarifier, chlorine contact chamber, aerated digester and sand sludge drying. A schematic of the WWTP appears in Figure 1. The major industrial contributors to the Jefferson WWTP and their flows are presented in Table 2. During the period surveyed, Gates Rubber Co. was the dominant contributor, making up 20% of the WWTP's flow. Table 2. Industrial Contributors to Jefferson WWTP % of Total Industry Flow(MGD)* Influent to WWTP Ashe County Memorial Hospital .008 7.3 Hanes Knitting .004 3.7 Gates Rubber Co. .022 20.2 * Flows are averages from August 1985 to July 1986. Average flow from WWTP during this period was .109 MGD TOXICITY EXAMINATION An on -site toxicity examination was conducted at the Jefferson WWTP as a result of a 48 hour Daphnia pulex static bioassay conducted on a composite effluent sample collected February 5, 1986 with a resulting LC60 of 8.2%. Previ- ous metals analysis of effluent samples had also revealed elevated levels of zinc and copper. On -site bioassays were performed from July 16-21, 1986 and included a 96 hour flow -through bioassay using fathead minnows (Pimephales promelas) as the test species, Daphnia pulex 48 hour static bioassays, and a seven-day Ceriodaph- nia test determining chronic lethality. Dilution water for these on -site tests was obtained from Naked Creek approximately 15 meters upstream of the Jefferson WWTP discharge. This water was tested prior to use at the Aquatic Toxicology Laboratory using the Ceriodaphnia reproduction test. Reproduction in this dilu- tion water was similar to that of laboratory culture water. The 96 hour flow -through bioassay was performed on effluent collected from a drop -box prior to chlorination. The test organisms (fathead minnows) were 45 days old at the beginning of the test. They were obtained from cultures at the Aquatic Toxicology Laboratory. These minnows were acclimated to the dilution water approximately 48 hours prior to test initiation. At approximately 19 hours prior to test initiation, the minnows were randomly transferred to each of 14 test chambers. The percentages of effluent to which the minnows were exposed were 0 (dilution water), 5, 10, 25, 50, 75 and 100%. These dilutions of effluent were tested in replicate. Each test chamber contained 10 minnows. The bioassay was initiated at 9:15 AM on July 22 and terminated at 9:30 AM on July 26, 1986. The toxicant delivery system cycled 372 times over the 96 hour test period, yielding a 90% replacement volume of test solutions every 5 hours. Mortality in 4- Figure 1. Schematic of Jefferson WWTP Laboratory Building Chlorination Aeration Basi n Clarifier Aerobic Sludge Digester Sludge Drying Beds Bar Screen Grit Chamber To Heatherly Creek Bioassay Sampling Point -4- Influent Sampling Point this bioassay is summarized in Table 3. Probit analysis of this data results in an LCso of 6.76%. Table 3. 96 Hour Fathead Minnow Mortality Effluent Concentration t%) Mortality (%) 0 0 5 60 10 40 25 90 50 100 75 100 100 100 Daphnia pulex 48 hour static bioassays were conducted while on -site using a 24 hour composite sample of the effluent and an instantaneous grab sample of the influent The resulting LCso's were 55% and 6.6% respectively. Mortality was also recorded at the 24 hour mark in the effluent bioassay, resulting in a 24 hour LCso of 74%. A seven-day Ceriodaphnia static replacement bioassay was performed on dilu- tions of effluent to assess lethal chronic toxicity. This test was initiated on -site on July 21, 1986 and terminated at the Aquatic Toxicology Laboratory on July 28, 1986. A 168 hour chronic LCso value of 3.6% was calculated based on mortality results. The log-probit graph used to calculate this value is depicted in Figure 2. Before proceeding with a discussion of the 7-day Ceriodaphnia test results, it must be noted that the neonates produced on Day 5 were inadvertently destroyed in all test rows. This loss of data in no way affected determination of the 168 hour LCso. At the conclusion of the seven-day test period a significant decrease —5— Figure 2. Seven Day Ceriodaphnia Mortality 100 T 0 X C A N T 10 V 0 L u M E LOG -CONCENTRATION VS R MORTALITY 0 10 20 30 40 50 60 70 80 90 100 R MORTALITY LC 50 =3.6% Jefferson WWTP -6- in reproductive success was observed in the 10% test concentration. Reproduction similar to that of the controls was recorded in the 1%, 0.1%, and 0.01% effluent concentrations. In this test, 10% effluent represents the Lowest Observed Effect Concentration (LOEC), while 1% effluent represents the No Observed Effect Concen- tration (NOEC). There was no mortality noted in any effluent concentrations less than 10% nor in the controls. Ninety percent mortality was recorded in the 10% concentration, while complete organism mortality was observed in effluent concen- trations of 25, 50, 75, and 100% after 168 hours. The calculated Chronic Value (ChV) for the test, which is the geometric mean of the LOEC and NOEC, is 3.16%. All of these observations are based only on the available reproduction data which does not include data destroyed on the fifth day of the bioassay. Therefore the NOEC, LOEC and chronic value are being presented only as observations and not reportable results. It should be noted that in this situation the level of sen- sitivity is essentially the same for chronic mortality and what appears to be the level of reproduction suppression. Additionally, two series of 48 hour Daphnia pulex static bioassays were conducted on grab samples from industrial contributors and area wells. The first series of samples was collected July 10, prior to the on -site investigation. The second series was collected on July 24 during the on -site investigation. The results of these bioassays are summarized in Table 4. All three industrial con- tributors displayed acute toxicity. Gates Rubber Co. showed the highest levels with two 48 hour LCso's of <5%. Two of the three wells tested displayed no toxicity. LCso's in one of Gates Rubber Co.'s wells ranged from 80 to 84%. Copper, zinc and aluminum were found in concentrations which may have contributed in part of the observed toxicity, although at the measured concentrations only chronic effects would be expected. These constitutents are quite variable in effect based on chemical form. Variations in chemical form significantly influences bioavailability. —7— Table 4. Industrial Contributor Bioassay Results Source Sample Date* _CBQ+ Ashe Memorial Hospital 860709 34 Ashe Memorial Hospital 860724 None Hanes Knitting 860724 28 Gates Rubber Co. 860709 (5 Gates Rubber Co. 860724 (5 Gates Rubber Co. Well *3 860709 None Gates Rubber Co. Well *3 860724 None Gates Rubber Co. Well *4 860709 80 Gates Rubber Co. Well *4 860724 84 Jefferson City Well 860724 None * All samples are instantaneous grabs. + All bioassays are 48 hour Daphnia pulex statics CHEMICAL SAMPLING Two series of chemical samples collected during the evaluation were analyzed at the Division of Environmental Management Chemistry Laboratory. Table 5 lists descriptions of the sampling stations. All samples were collected as instanta- neous grabs, with the exception of Station 02 (effluent bioassay sampling point), which were taken as 24 hour composites. Figure 3, a map of the study area, illustrates sampling site locations. Results and summary of chemical analyses are documented in Tables 6-8. Table 5. Sampling Site Descriptions Station 01 - Naked Creek at NC 88/16 approximately 15 meters upstream of the Jefferson WWTP discharge. At this point the creek is about 3 meters wide with a mean depth of 0.1 meter. The substrate was evenly distributed between boulder, rubble, gravel and sand. This was the dilution water source for all on -site bioassays. Station 02 - Jefferson WWTP effluent at the drop box just prior to chlorination. This is the bioassay sampling point. Station 02A - Jefferson influent from influent trough to aeration chamber. Station 02B - Chlorinated Jefferson effluent from basin just prior to effluent discharge pipe. Station 03 - Naked Creek at SR-1585 approximately 2000 meters below the Jeffer- son WWTP discharge. At this point the creek is about 3 meters wide with a mean depth of 0.1 meter. The substrate type is again evenly divided between boulder, rubble, gravel and sand, although there was an excessive amount of sediment. Station 05 - Jefferson City well. Station 06 - Gates Rubber Co. effluent at manhole just outside main building. Station 07 - Ashe County Memorial Hospital effluent at lower manhole by drive- wa Y• Station 08 - Hanes Knitting effluent at manhole just outside parking lot. Station 09 - Gates well water from well *3. Station 10 - Gates well water from well *4. Metals analyses revealed copper concentrations of 36 ppb and 62 ppb in the effluent (02) samples of July 24 and 26 respectively. At these concentrations and 7010 conditions, the concentration in the receiving stream would average 4.3 ppb, assuming receiving stream concentration of zero. This level is well below the N.C. Water Duality Action Level of 15 ppb for copper. Copper was not detected (lower detection limit (10 ppb), in the upstream, downstream or dilution water (same as the upstream site). Zinc was detected at elevated concentrations in the effluent on both sampling dates, with 440 ppb on July 24 and 890 ppb on July 26 (average value = 665 ppb). At these concentrations and 7010 conditions, the concentration in the receiving stream would average 58.7 ppb, a value that represents an exceedence of the North Carolina Water Quality Action Level of 50 ppb. Zinc was detected in the influent (02A) at levels of 1700 ppb and 730 ppb on July 24 and 26, respectively. Small amounts of zinc were detected at down- stream sites with values of 19 ppb on July 24 and 28 ppb on July 26 being recorded. Upstream zinc levels on these same dates measured 22 and (10 ug/liter. Copper LC60's have been reported as low as 9.8 ppb for 48 hour Daphnia manna bioassays' in water of 45 ppm hardness and 17 ppb in 48 hour Ceriodaphnia reticulate bioassays2. Zinc LC60's have been reported as low as 107 ppb in D. pulex bioassays and 762 ppb in Ceriodaphnia reticulate 48 hour bioassays. Fathead minnows are less sensitive to these metals than cladoceran species, with 96 hour LC6o's of 490 ppba and 600 ppb' for copper and zinc, respectively. Mercury was detected in the effluent at 0.4 ug/I on both sampling dates. Available data indicate this level of mercury will not cause acute toxicity. A Daphnia manna LC60 of 5 ug/I' and a fathead minnow seven day flow -through LC60 of 74 ug/I6 have been reported for mercury. However, fathead minnow juveniles have shown significant growth retardation when exposed to levels of mercury as low as 0.58 ug/I6. Also of concern is mercury's tendency to bioaccumulate. 8ioaccumu- Figure 3. Jefferson WWTP Study Area NC 88/16 N ew River .=:1 Mile -11- Table 6. Results of Inorganic Chemical Analyses Permitted Flow (MOD) 0.15 7Q10 (CFS) 2.4 Chemical/Physical Units Water Quality Sta 01 Sta 02 Sta 02A Sta 02B Sta 03 Analyses Standards 860724 860724 860724 660724 660724 BOD PPM 0.6 51 78 3.6 COD PPM <5 600 770 20 Coliform: MF Fecal 0/ 100m1 moo8000 10~ Residue TOTAL , PPM 83 720 1100 88 volatile PPM 32 320 420 22 fixed PPM 51 390 670 66 Residue SUSPENDED PPM , 1 110 140 1 volatile PPM 1 96 130 1 fixed PPM <1 18 14 <1 pH (standard units) _ 6.0-9.0 7.1, 7 7.1 7.1 Acidity PPM _ 2 38 35 5 Alkalinity PPM 33 140 160 32 Arsenic PPB <10 <10 Chromium Hex. , PPB __ <50 Cyanide PPM <.01 <.01 Fluoride PPM 0.1 Formaldehyde PPM <0.1 <0.1 Grease and 011s PPM _ 24 66 Hardness PPM 34 110 110_ 36 MBAS PPM 0.5 6.2 Phenols PPB 50 19 <2 Specific Conductance uMhos/cm _ 86 770 1200 110 NH3 PPM 0.04 15 16 0.46 TKN PPM 0.2 22 42 0.7 NO2.NO3 PPM 0.47 0.03 0.31 0.51 P • total PPM 0.03 0.05 0.04, 0.03 Silver PPB <25 <25 <25 <25 Aluminum PPB 100 250 400 50 Cadmium PPB 0.4 <10 <10 <10 <10 Chromium (Total) PPB 50 <25 <25 <25 <25 Copper PPB 15(AL )' <10 36 110 <10 Iron PPB 1000 170 1400 1200 440 Mercury PPB 0.2 <0.2 0.4 <0.2 <0.2 Manganese PPB 30 95 75 120 Nickel PPB 50 <50 <50 <50 <50 Lead PPB 25 <50 <50 <50 <50 Zinc _ PPB 50(AL) 22 440 1700 19 ' Values represent action levels,as specified in .0211(bX4) Fresh Water Classifications Standards Table 6. fewlts of Inorganic Chemical Analyses -continued Permitted Flow (MGD) 0.15 7Q10 (CFS) 2.4 Chemical/Physical Units Sta 01 Ste 02 Sta 02A Sta 03 Predicted stream** Analyses 860726 660726 860726 860726 conc. at 7Q10 B0D PPM COD PPM 9 660 490 21 Coliform: MF Fecal +/ 100m1 Residue TOTAL PPM 64 680 670 84 volatile PPM 7 360 370 25 fixed PPM 57 320 4 300 59 Residue SUSPENDED PPM , 13 170 170 8 volatile PPM 5 140 150 3 fixed pH (standard units) r PPM 8 7 _27 7.1 _ 20 7.1 5 6.7 Acidity � PPM 4 39 32 10 Alkalinity PPM _ 29 160 170 28 Arsenic PPB < 10 <10 Chromium Hex. PPB Cyanide PPM <.01 <.01 Fluoride PPM <0.1 Formaldehyde PPM <0.1 <0.1 Grease and Oils PPM 37 Hardness . PPM 34 87 77 34 MBAS PPM 4.1 0.455 Phenols PPB 170 21 2 9.713 Specific Conductance uMhos/cm 88 660 540 110 NH3 PPM 0.04 19 8.7 0.57 1.501 TKN PPM 0.3 30 20 0.9 2.296 NO2.NO3 PPM 0.51 0.02 0.22 0.62 0.002 P. total _ PPM 0.23 1.9 6.2 0.05 0.086 Silver PPB <25 <25 <25 <25 <2.207 Aluminum PPB 400 350 200 150 26.490 Cadmium PPB c10 <10 <10 <10 <0.883 Chromium (Total) PPB <25 <25 <25 <25 <2.207 Copper PPB <10 62 61 <10 4.327 Iron PPB 800 1700 1200 600 136.865 Mercury PPB <0.2 0.4 <0.2 _ • <0.2 0.035 Manganese PPB 50 70 70 140 7.285 Nickel PPB <50 <50 <50' <50 <4.415 Lead PPB <50 <50 <50 <50 <4.415 Zinc PPB <10 890 730 28 58.720 * 41 Values represent predicted Instream concentrations using average effluent concentrations of 0. concentrations. maximum permitted flow and_assuming_upstream Table 7. Results of Industrial Contributor Metals Analyses Chemical/Physical Units Water Quality Sta 05 Sta 06 Ste 07 Sta 08 Ste 09 Sta 10 Analyses Standards 860724 860724 860724 860724 860724 860724 Silver PPB <25 <25 <25 <25 <25 <25 Aluminum PPB <50 100 100 750 100 50 Cadmium PPB 2 <10 <10 <10 <10 <10 <10 Chromium PPB 50 <25 <25 (25 (25 (25 <25 Copper PPB 15(AL)' 41 120 140 120 14 24 Iron PPB 1000 <50 430 470 1600 <50 <50 .Mercury PPB 0.2 <0.2 2.9 18 <0.2 <0.2 <0.2 Manganese PPB _ <25 1300 55 90 250 30 Nickel _ PPB 50 <50 <50 <50 <50 <50 <50 Lead PPB 25 50 75 <50 <50 <50 <50 Zinc PPB 50(AL) 52 6700 140 3300 75 170 ' Values represent action levels _as specified in .O211(bX4) fresh Water Classifications Standards 1 Table 8. Results of Organic Chemical Analyses Permitted Flow (MGD) 0.15 7Q10 (CFS) 2.4 Organic Analyses Units Sta 02 Sta 02A Sta 02 Sta 02A Toxicity Value Test Type 860724 860724 860726 860726 (PPB) Methyl propyl benzene PPB 3 NO DATA AVAILABLE Phenol PPB 16 1050=28.800 96 hr Fathead Minnow 6 Trimethyl bicycloheptanone PPB 7 LC50=110.000 96 hr Fathead Minnow 6 Biphenyl-o1 PPB 14_ LC50=4700 48 hr Daphnia magna 7 Methyl phenol PPB 45 26 35 LC50=9600 48 hr Daphnia pulex 8 Tetramethyl thiourea PPB 73 LC50=770,000 48 hr Daphnia magna 9 Benzene acetic acid PPB 7 NO DATA AVAILABLE ,Propenyi methoxy phenol PPB 7 LC50=24000 96 hr Fathead Minnow 10 Nonyl phenol PPB 12 10 LC50=140 96 hr Fathead Minnow 6 Pyrene' PPB 9 NO DATA AVAILABLE Phenothiazine PPB 18 NO DATA AVAILABLE Aldrin' PPB 1 _ 1.1 _ LC50=5.6 96 hr Fathead Minnow 11 LC50=8.2 96 hr Bluegill 12 Pentanoic acid PPB 32 LC50-77,000 96 hr Fathead Minnow 10 Tetraethyl butyl phenol PPB 10 NO DATA AVAILABLE Unidentified peaks * 28 23 7 2 * Footnote I ' Included in N.C. Water Quality Standards list of 'Chemicals Requiring Special Attention.' ' N.C. Water Quality Standard for Aldrin is 0.002 pg/I 1 ] lation factors from 4,380 to 5,680 have been determined for mercury in fathead minnow juveniles'. The Ashe County Memorial Hospital appears to be the largest contributor of mercury according to available data with lesser amounts contrib- uted by Gates Rubber (Table 7). The BOD level in the July 24 effluent was 51 ppm. This value would indicate a possible exceedence of the permitted effluent limitation (45 ppm weekly aver- age). If concentrations of TSS residue remain at encountered levels (110 ppm on July 24 and 170 ppm on July 26) then it is predicted the effluent will exceed the permitted effluent limitation of 45 ppm (weekly average). Also of note is the level of MBAS found in the effluent. On July 24 and 26, 6.2 and 4.1 ppm MBAS, respectively were detected in 24 hour composite samples of the effluent. The average of these data would produce 0.455 ppm MBAS instream during low stream flows, only .045 ppm below the N.C. Water Ouality Standard of 0.5 ppm. It appears that the high level of zinc detected on July 26 (890 ppb), at least in part, contributed to the acute toxicity exhibited by the fathead minnows in the flow -through test, although it is likely that other constituents contrib- uted to the observed 96 hour LC60. Both copper and zinc were present in the effluent in sufficient quantities to account for portions of the toxicity observed in the seven day Ceriodaphnia life cycle bioassay and the 48 hour Daphnia pulex static bioassay. Metals analyses performed on grab samples collected from the three indus- trial contributors and several associated water wells (see Table 7.), indicate that the effluent from Gates Rubber and Hanes Hosiery Mill contain elevated lev- els of zinc. These zinc levels could contribute significantly to the observed toxicity of aliquots of the same samples. The Gates Rubber Co. effluent, was the most toxic with a 48 hour Daphnia ➢ulex LC60 of <5%, followed by Hanes Knitting (LCao = 28%). Ashe County Hospital's effluent did not display any appreciable acute toxicity. Results of analyses for organic chemicals in the Jefferson WWTP influent and effluent are shown in Table 8 with available toxicity data. Although there are a number of toxic organic chemicals present, none appear to be in amounts which would cause acute toxicity. However, the organic component of the Jefferson WWTP effluent should not be ruled out as a contributing source of either acute or chronic toxicity due to the numbers of different chemicals present, the unidenti- fied compounds present, and the fact that toxicity data is not available for all chemicals detected. BENTHIC MACROINVERTEBRATE ANALYSIS Benthic macroinvertebrates were collected at 2 sites on Naked Creek on July 21, 1986. These sites are: Station 01, Naked Creek at US 88/16 bridge, Ashe County, 15 meters above the Jefferson WWTP discharge point. The creek at this site was about 3.0 meters wide with a mean depth of 0.1 meters. The substrate was a mixture of boulder, rubble, gravel and sand, although there was an excessive amount of sediment. A layer of Aufwuchs covered the substrate. This site was adjacent to a cow pasture. Station 03, Naked Creek at SR-1585, Ashe County, 2000 meters below the Jef- ferson WWTP discharge. The creek at this site was about 5 meters wide with a mean depth of 0.3 meters. The substrate was similar to Station 01 except there was an abundance of filamen- tous algae. This site was also adjacent to the cow pasture. The water was dark in color and had a sewage odor. These stations correspond to chemical sampling sites. Station 01 was also used as the dilution water site for all bioassays. -17- Benthic macroinvertebrates were collected using a standardized qualitative collection technique (DEM 1983). The primary output from this collection tech- nique is a tabulation of taxa richness, i.e., the number of different kinds of animals present. Unstressed streams and rivers always have high taxa richness. Various types of pollution will reduce or eliminate the more pollution intolerant species, producing lower taxa richness values. In-house criteria have been developed to relate taxa richness to five water quality ratings or bioclassifi- cations: Excellent, Good, Good -Fair, Fair, and Poor. Taxa richness values are calculated both for all species (ST) and for the more intolerant groups (Ephem- eroptera, Plecoptera, and Trichoptera - SEpT). The distribution can be utilized to deduce changes in water quality. Taxa richness by group for this survey are given in Table 9, while a list of all taxa collected is presented in Table 10. Station 01 received a bioclassification of Good -Fair, with total taxa richness of 76 and an EPT (pollution intolerant) taxa richness of 31. Station 03 received a bioclassification of Poor, with total and EPT taxa richness values of 38 and 6, respectively, indicating a severe decline in water quality below the Jefferson WWTP discharge. Particularly significant was the complete absence of Plecoptera at Station 03. Station 01 had 3 Plecopteran species, 2 of which were abundant. Ephemeroptera declined from 17 taxa at Sta- tion 01 to 3 at Station 03, while Trichoptera declined from 11 to 3. Declines in taxa richness among the more pollution tolerant groups such as Coleoptera and Diptera were also noted. Additionally, species dominance within groups shifted to more pollution tolerant taxa. Within the diptera, the dominant taxa at Stations 03, were Cri- cotopus bicinctus (c/0 sp. 1), C. infuscatus gr. (C/O sp 5), and Polydedilum illinoense. These organisms are often found together as the dominant taxa in areas of toxic stresses such as zinc and copper (Simpson and Bode, 1980). TABLE 9. TAXA RICHNESS♦ BY GROUP, NAKED CR., ASHE C0.,-1986. TABLE OF ORDER BY STATION ORDER STATION 101 1 03 1 + + + EPHEMEROPTERA 1 17 1 3 1 + + + PLECOPTERA 1 3 1 0 1 --M-_-N_-+ + + TRICHOPTERA 1 11 1 3 1 + + + C OL EOPT ER A 1 10 1 4 1 --- + + + OnONATA 1 4 1 5 1 MEGALOPTERA 1 0 1 1 1 + + + DIPTERA-CHIRON 1 14 1 9 1 + + + MISC. DIPTERA 1 5 1 5 1 + + + OLIGOCHAETA 1 5 1 3 1 + + + C RUSTAC EA 1 2 1 1 i + + + MOLLUSCA 1 3 1 3 1 + + + OTHER 1 2 1 1 1 + + + TOTAL TAXA RICHNESS 76 38 EPT TAXA RICHNESS 31 6 PIOCLASSIFICATION G000/FAIR POOR TABLE 10. SPECIES LIST NAKED CR., ASHE CO., JULY, 1986. R=RARE, C=COMMON, A=ABUNDANT STATION ORDER EPHEMEROPTERA !SPECIES + 5AETIS FLAVISTRIGA +---+ BAFTIS 1 1 INTERCALARIS 1 A 1 +---♦ BAETIS 1 1 PROPINRUUS 1 C 1 +---+-- f3AETISCA 1 1 BERNERI 1 R 1 CAENIS SPP 1 C 1 C +---+ CENTROPTILUM 1 1 SPP 1 R 1 ♦---+ EURYLOPHELLA 1 1 BICOLOR 1 R 1 +---+ HEPTAGENIA SPP 1 C 1 +---+ ISONYCHIA SPP 1 A 1 +---+ PARALEPTOPHLEB-1 1 IA SPP I A 1 +---♦ PSEUDOCLOEON 1 1 SPP I A 1 STENACRON 1 1 INTERPUNCTATUM 1 C 1 +---+ STENACRON 1 PALLIDUM 1 C 1 + -- -+ STENONEMA 1 1 ITHACA 1 A 1 (CONTINUED) TABLE 10. SPECIES LIST NAKED CR., ASHE CO., JULY, 1986. R=RARE, C=COMMON, A=ABUNDANT ORDER EPHEMEROPTERA PLECOPTERA TRICHOPTFRA 'SPECIES + STATION 01 103 1 1 1 1 1 1 STENONEMA 1 1 MODESTUM 1 AIC STENONEMA 1 1 PUOICUM 1 C 1 ACRONEURIA 1 1 ABNORMIS 1 C 1 ALLOCAPNIA SPP 1 C 1 PELTOPERLA SPP 1 R 1 CHEUMATO°SYCHE 1 1 SPP 1 A 1 +---+-- CHIMARRA SPP 1 R 1 +---+-- GLOSSOSOMA SPP 1 R 1 HYDROPSYCHE 1 1 BRONTA 1 AIC +-_-+-- HYOROPSYCHE 1 i MOROSA 1 C I C HYOROPSYCHE 1 1 SPARNA 1 A 1 A LEUCOTRICHIA 1 1 PICTIPES 1 R 1 + ---+ -- LYPE DIVERSA 1 C 1 NEOPHYLAX 1 1 OLIGIUS 1 A 1 +---+-- POLYCENTROPUS 1 1 SPP 1 R 1 (CONTINUED) TABLE 10. SPECIES LIST NAKED CR.♦ ASHE CO., JULY, 1986. R=RARE • C=COMMON, A=ABUNDANT ORDER 1SPECIES TRICHOPTERA 1PYCNOPSYCHE 1 GENTILIS + COLEOPTERA OD ON AT A STATION 1 1 1 1 1 1 1 R I + ---+ 9EQ0SUS SPP 1 R 1 +---+ DINEUTES SPP 1 C 1 DUBIRAPHIA 1 1 VITTATA 1 C 1 +---+-- ECTOPRIA 1 1 NERVOSA 1 1 R. +---+-- ENCCHRUS S PP I R 1 +---+-- GYRIMUS SPP 1 R 1 HELICHUS SPP 1 C 1 LACCOPHILUS SPP) 1 A MACRONYCHUS 1 1 GLABRATUS 1 R 1 PSEPHENUS 1 1 HERRICKI 1 A 1 R +---+ STENELMIS SPP 1 C 1 +__r+_-- TRC?ISTERNUS 1 1 SPP 1 R 1 R + +---}-_- IAESCHNA UMBROSAI R 1 C 1 +---+--- IBOYERIA VINOSA 1 R 1 A 1 +---+--- 1CALOPTERYX SPP 1 A 1 I ENALLAGMA SPP 1 1 R (CONTINUED) TABLE 10. SPECIES LIST NAKED CR.• ASHE CO., JULY• 1986. R=RARE, C=COMMON, A=ABUNDANT ORDER ODONATA STATION 01 103 1 1 SPECIES 1 1 + 1 1 IGOMPHUS SPP IISCHNURA SPP + I R I R 1 I A MEGALOPTERA 1CORYJALUS 1 1 1CORNUTUS + DI PTFRA:CHIR0N 1 1 R +---+-- ARILLIA SPP 1 A 1 CARO!OCLADIUS 1 1 SPP 1 C 1 R CHIRINOMUS SPP 1 1 A CONCHAPELOPIA 1 1 GROUP IAIA CRICOTOPUS/ORT-1 1 HOCLAD1US SP1 1 1 A +---+-- CRIC9TPPUS/ORT-1 I HOCLADIUS SP5 1 C 1 A CRYPTOCHIRONOM-1 1 US FULVUS 1 C 1 °AGASTIA SPP 1 R 1 PARAPHAENOCLAO-1 1 IUS SPECIES 1 1 C 1 PHAENOPSECTRA 1 1 SP? 1 R 1 POLYPEDILUM 1 1 EALLAX 1 R 1 POLYPEDILUM 1 1 ILLINOENSE 1 C 1 A (CONTINUED) TABLE 10. SPECIES LIST NAKED CR., ASHE CO.,•JULY, 1986. R=RARE, C=COMMON, A=ABUNDANT ORDER OIPTERA:CHIRON DI PTERA: MI SC !SPECIES + !STATION 101 103 1---+--- 1 1 1 1 + 1 1 1 1 POLYPEDILUM 1 1 SCALAE ! A 1 R PS=CTROTAVYPUS 1 1 DYARI 1 1 A SYNORTHOCLADIUS1 1 SPP 1 1 A +---+-- TANYTARSUS S°2 1 R 1 TANYTARSUS S°6 1 R 1 +---+-- THIENEMANIELLA 1 1 SPP ANTOCHA SPP DIXA SPP HEXATOMA SPP PALP1MYIA 1 1 (COMPLEX) 1 1 A. SEPEDON 1 R 1 4. SIMULIUM SPP 1 1 C SIMULIUM 1 1 VITTATUM 1 C 1 A TIPULA SPP 1 R 1 + +---+-- !'LIGOCHAETA 1 BRANCHIURA 1 1 ISOWERBYI 1 C 1 lILYODRILUS 1 1 1 TEMPLETONI 1 1 A (CONTINUED) TABLE 10. SPECIES LIST NAKED CR., ASHE CO., JULY, 1986. R=RARE, C=COMMON, A=ABUNDANT 1 STATION 1 101 103 1 1 1 1 +---+-- ORDER 1SPECIES 1 1 + 1 1 OLIGOCHAETA 1 LIMN')DRILUS 1 1 1 HOFEMEISTERI 1 A 1 1 +---+ I LUMBRI CUL I OAE ICIA 1NAIS SPP 1 C 1 +---+__ 10PISTHOPORA SPP1 C 1 R + +___+ CRUSTACEA I CAMBARUS SPP 1 R 1 R MOLLUSCA OTHER 1 1ORCONECTES SPP 1 R 1 FLIMIA SP 1 A 1 'ERRISSIA 1 1 PIVULARIS 1 A 1 HELISOMA SPP 1 j C MENETUS DILATUSI 1 R +---+-- PHYSFLLA SPP 1 A 1 A -+ ---+-- 1DUGESIA TIGRINAI 1 R IHYDRACARINA 1 R 1 1 +__-+ 1 SIGARA SPP 1 R 1 Although C/O sp. 5 and P. illinoense were collected at Station 01. they were not dominant and C/O sp. 1 was absent. Although streams in North Carolina had been stressed last spring and summer by extremely low flows, the greatly reduced taxa richness and the shift to dom- inance by tolerant organisms at Station 03 clearly associates instream impacts in water quality from the effluent of Jefferson WWTP. CONCLUSIONS On -site toxicity tests conducted on the effluent of the Jefferson WWTP resulted in a 96 hour fathead minnow LC6* of 6.76%, a Daphnia, pulex 48 hour LC60 of 55%, and a Ceriodaphnia dubia seven-day LC6o of 3.6%. Given these toxicity results and the facility's instream waste concentration of 8.83% during low stream flow conditions, the effluent would be expected to cause significant acute toxicity in the receiving stream. Analyses of chemical samples show elevated effluent concentrations of copper and zinc. Both of these metals were present in sufficient concentrations to have caused acutely toxic responses in the cladoceran test species. Zinc most likely contributed to the observed fathead minnow toxicity. None of the organic con- stituents detected in the effluent were present in amounts sufficient to cause acute toxicity. Mercury is of concern due to its bioaccumulation tendencies. Given available data, it appears that there are no organic components pre- sent in large enough quantities to cause chronic toxicity. A chronically toxic effect from the organic portion of the effluent should not be ruled out, however, due to the numbers of different compounds present, the unidentified compounds present, and because toxicity data is not available for all chemicals detected. Analysis of benthic macroinvertebrate communities in Naked Creek upstream and downstream of the Jefferson WWTP clearly indicates a negative impact on water quality by the WWTP effluent. Bioassay data from the industrial contributors tested indicates Gates Rubber Co. to be the most toxic with two 48 hour Daphnia,pulex, static LCgo's of <5%. Chemical analyses indicate Gates to be the heaviest contributor of zinc (6700 ppb) among the industries analyzed on the date sampled. Zinc appears to be the major toxic constituent of the Jefferson effluent. That Gates Rubber Co. efflu- ent typically makes up approximately one-third of the influent wastes to the Jef- ferson WWTP compounds its effect on the makeup of the WWTP's effluent. It should be noted that Hanes Knitting and the Ashe Co. Memorial Hospital also contribute toxicity to the Jefferson WWTP. However, these contributions are present at lower flows and toxicity levels. RECOMMENDATIONS 1. The Jefferson WWTP should continue performance of the 48 hour Daphnia,pulex acute bioassays until the test has achieved the target level of >90% consis- tantly. At such time the facility should initiate Pass/Fail Ceriodaphnia survival and reproduction tests at a test concentration equal to the facil- ity's instream waste concentration (IWC) of 8.83%. The subject permit, upon re -issuance, should have toxicity limits included as Ceriodaphnia survival and reproduction bioassays. The final chronic value achieved as a toxicity limit should equal or exceed the facility's IWC of 8.83%. 2. The facility should develop a written toxicity reduction plan with a sched- ule of attaining compliance as will be set forth. 3. Due to effluent levels of copper, zinc and mercury on -site, the sources of major contribution of these compounds should be further investigated and efforts made to minimize discharge, and thus reduce observed acute toxicity. 4. The facility may wish to consider performing chemical fractionation proce- dures in order to determine if any toxicity is contributed by the organic chemical components of the WWTP's effluent. FOOTNOTES 1 Biesinger, K.E. and G.M. Christensen. 1972. Effects of various metals on survival, growth, reproduction and metabolism on Daphnia manna. J. Fish Res. Board. Con. 29:1691. 2 Mount, D.I. and T.J. Norberg. A Seven -Day Life -Cycle Cladoceran Toxicity Test. Environmental Toxicology and Chemistry. Vol. 3. pp 433. 1984. 8 Pickering, 0., W. Brungs, and M. Gast. 1977. Effect of exposure time and copper concentration on reproduction of the fathead minnow (Pimephales prome- las. Water Res., 11:1079-83. • Benoit, D.A. and G.W. Holcombe. 1978. Toxic Effects of zinc on fathead min- nows Pimephales promelas in soft water. J. Fish Biol. 13:701-708. 6 Snarski, V.M. and G.F. Olson. Chronic Toxicity and Bioaccumulation of Mercuric Chloride in the Fathead Minnow (Pimephales promelas. Aquatic Toxicology, 2 (1982) 143-156. e Geiger, D.L. et. al. 1985. Acute Toxicities of Organic Compounds to Fathead Minnows (Pimephales promelas) Vol. I1. Center for Lake Superior Env. Studies. Univ. Wis. Superior. • Leblanc, G.A. Acute Toxicity of Priority Pollutants to Water Flea (Daphnia manna). Bull. Environ. Contam. Toxicol., 24(5), pp 684-691, 1980. • Slooff, W., J.H. Canton and J.L.M. Hermens, Comparison of the Susceptibility of 22 Freshwater Species to 15 Chemical Compounds. 1. (sub) Acute Toxicity Tests. Aquatic Toxicology, 4 (1983) 113-128. o Van Leeuwen, C.J., F. Moberts and G. Niebeck. Aquatic Toxicological Aspects of Dithiocarbamates and Related Compounds. II. Effects on Survival, Reproduction, and Growth of Daphnia manna. Aquatic Toxicology, 7 (1985) 165-175. 1O Mattson, Vincent R., John W. Arthur, Charles T. Walbridge. Acute toxicity of selected organic compounds to fathead minnows. EPA-600/3-76-097, Oct. 1976. 11 Foster, L. Mayer and Mark R. Ellersicek. Manual of Acute Toxicity: Interpre- tation and Data Base for 410 Chemicals and 66 Species of Freshwater Animals. United States Depart. of the Interior, Fish and Wildlife Service, Washington D.C., 1986, pp 10-11. 12 Johnson, W.W. and M.T. Finley. 1980. Handbook of Acute Toxicity of Chemicals to Fish and Aquatic Invertebrates. U.S. Dept. Inter., Fish Wildl. Serv., Resource Publ. 137. 98 pp. APPENDIX _29_. 48 Hour Pauhnia pulex Screening Bioassay Appendix Aquatic Toxicology Group N. C. Division of Environmental Management The Aquatic Toxicology Group performs 48 hour static bioassays using the cladoceran Paphnia pulex to estimate the toxicity of waste discharge to aquatic life in receiving streams. All test and sampling glassware and equipment are washed with soap and hot water, then rinsed in nitric acid, acetone, and distilled/deionized water, to remove all toxins and contaminants. Effluent samples are collected by DEM Regional Office or Aquatic Toxicology personnel. All samples are collected chilled and above chlorination unless otherwise specified. Each sample is collected as a grab or 24 hour composite using an automatic sampler and is sent chilled to the Aquatic Toxicology Laboratory by state courier or bus. The sample must be received within 72 hours after collection. The effluent samples are prepared for testing by being thouroughly mixed, adjusted to standard test temperature, and aerated if dissolved oxygen is below 40X saturation. Hardness and alkalinity are measured. Chlorine is removed with sodium thiosulfate if applicable. The effluent is then diluted with D Dulex culture water, typically to seven concentrations (with replicates) from 0 to 90% effluent and initial pH and DO are recorded.. Each test chamber receives 100 mis total volume and ten D. pulex test organisms, 0-24 hours old. The test is conducted in a 20 degree centigrade incubator with a 16:8 hour light: dark cycle. Mortality of the D. oulex is recorded after 48 hours, along with final pH, dissolved oxygen, and temperature. A 48 hour LC50, or concentration of effluent lethal to 50% of the test organisms in 48 hours, is calculated from the mortality data. An instream waste concentration (IWC) for the effluent in the receiving stream is calculated using the treatment system permitted flow and receiving stream 7Q10 flow. The LC50 and IWC are then used to predict instream toxicity. If the effluent toxicity and/or the IWC are high, a persistance bioassay may be conducted. This involves a second 48 hour static D. pulex bioassay on the same effluent sample after it has been exposed to light and aeration for an additional 48 hours. If there is a 100% reduction in the LC50, the effluent is considered to be non-persistant. Guidance Document:1985. U. S. E. P. A. Methods for measuring the acute toxicity of effluents to freshwater and marine organisms. Third Ed. (EPA/600/4-85/013) 96 Hour On -site Flowthrough Bioassay Appendix Aquatic Toxicology Group N. C. Division of Environmental Management Candidacy for an on -site toxicity evaluation by the Aquatic Toxicology Group is determined on the basis of acute toxicity of the effluent in comparison with instream waste concentration. Acute toxicity is determined by a 48 hour screening static bioassay . For each on -site, flowthrough bioassay, a pre -test site inspection is performed in order to: 1) Determine appropriate areas for physical placement of the mobile laboratory. 2) Acquire proper equipment and installation needed for electrical service. 3) Determine appropriate areas for effluent sampling and equipment needed for such. Determine discharge schedule. Sampling is done above chlorination unless otherwise specified. 4) Determine possible areas for dilution sampling (actual receiving waters or other unstressed streams in the area) and equipment needed for such. 5) Collect additional samples of effluent and possible dilution waters for further static Daphnia pulex acute and static renewal Ceriodaphnia dubia reproduction bioassays to determine the range of concentrations of effluent for the flowthrough bioassay, to test for potential toxicity of possible dilution waters, and for fish acclimation to the chosen dilution water. 6) Determine route suitability to the facility for the mobile laboratory (eg. low clearances, poor road conditions) . 7) Discuss test procedures and requirements with appropriate facility personnel. 8) Determine appropriate sampling sites and techniques for benthic macroinvertebrate surveys. All test and sampling glassware and equipment are washed with soap and hot water, then rinsed in nitric acid, acetone, and distilled/deionized water to remove all toxins and contaminants. Upon actual arrival on -site with the mobile laboratory, dilution water is obtained and dilution and effluent pumping systems are set up and tested. Six to eight week old fathead minnows are wet transferred to the test chambers (containing approximately one liter of dilution water), ten fish to a chamber.This transfer is accomplished five fish at a time in a randomized order to each of the fourteen test chambers until two randomized sets of five have been transferred to each chamber. Seven concentrations (with replicates) including a control are used. The second day on -site the dilutor and the dilution and effluent pumping systems are turned on and the fathead minnow flowthrough bioassay is begun. A water bath is utilized to bring the effluent and dilution water to a constant 20 degrees centigrade. Test organisms are fed newly hatched brine shrimp twice daily throughout the test. A 7 day Ceriodaphnia dubia static renewal reproduction bioassay using newborn organisms is begun the first day on -site. The organisms are transfered to fresh dilution and effluent solutions daily and initial and final pH and dissolved oxygen are recorded. The number of young born per organism per day is recorded and mean cumulative reproduction is calculated for each concentration. The test is conducted in a 25 degree centigrade incubator with 16 light:8 dark hour photoperiod. Test organisms are fed 0.1 ml of a fermented trout chow mixture per organism per day. Individual chemical/physical parameter meters are calibrated daily according to DEM standards. At 15 minute intervals throughout the test, hydrolab systems measure and record dissolved oxygen, pH, temperature, and specific conductance in the test chambers with the highest and lowest concentration of effluent. These systems are calibrated at test initiation, the mid -point of the test, and test termination. Data from these systems is recovered daily and stored on floppy disc and hard copy. Samples of dilution water, effluent at the bioassay sampling point, final effluent, and the receiving stream upstream and downstream of the discharge point are analyzed for hardness as feasible. Where applicable, daily residual chlorine measurements will be made at the above sites. During the on -site evaluation, Biological Monitoring Group personnel collect benthic macroinvertebrate samples at the upstream, downstream, and dilution sites (see Benthic Macroinvertebrate Survey appendix). Where appropriate, electrofishing is undertaken upstream and downstream of the discharge to obtain resident fish population data. On a site -specific basis, various other efforts are undertaken, such as monitoring dissolved oxygen levels in the receiving stream. On a daily basis, test chamber screens are cleaned, effluent and dilution pumping systems are checked and adjusted as necessary, and pH, dissolved oxygen, and fish mortalities are recorded for each chamber. Dilution water is generally collected on an alternating day basis, depending on need. Two separate 24 hour composite samples of effluent are collected for chemical analysis by means of an automatic sampler. Receiving stream and dilution water samples are also taken for chemical testing. Static 48 hour Daphnia nulex bioassays are conducted on a 24 hour composite sample of the effluent and a grab sample of the influent. persistence test aging is begun on the effluent sample. Another static bioassay will be conducted after 96 hours of aging to determine persistance of toxicity. A tour of the facility is conducted. The actual treatment process is reviewed to ascertain the quality of the operation of the treatment system and to determine the treatment system's appropriateness to the type of waste being treated. An inventory of any industrial contributors to a municipal waste treatment facility is made. The manufacturing process at an industrial facility is reviewed to determine the nature and composition of the waste. An inventory of all chemicals used is made. A photographic record is made of the manufacturing and treatment facility, sampling points, receiving stream, and sampling procedures. At the end of the 96 hour test period, the dilutor is turned off and final mortality observations are made. Breakdown and packing routines are performed and the mobile laboratory is transported back to the Cary Aquatic Toxicology Laboratory. The Ceriodaphnia dubia reproduction bioassay is continued at the lab until the 7th test day. The persistance static bioassay is conducted. -32- Several special care operating procedures should be mentioned. At facilities that discharge for only a portion of the day, effluent samples are composited by the dilutor system into a large reservoir on board the mobile laboratory for use as the effluent while discharge is not in progress. If the effluent has a high oxygen demand, aeration systems for the test chambers are utilized and dissolved oxygen levels in the chambers are monitored closely in order to prevent levels from dropping below 40% saturation at test temperatures. In the event that actual receiving waters are deemed unfit for the test (i.e. potentially toxic), an alternate source of dilution water is sought in the vicinity. rariggAiilingt Alibi& Reproduction Bioassay Appendix Aquatic Toxicology Group N. C. Division of Environmental Management The cladoceran Ceriodauhnia dubia is used as test organism in a 7 day static renewal bioassay. This test estimates the effect of an effluent or other water sample on reproductivity. A control and 8 concentrations of effluent ranging from 0.01% to 100% are used. There are 10 animals per concentration, each animal in a one ounce polystyrene test chamber with 15 mis of solution. The test is conducted in a 25 degree centigrade incubator with a 16 light/ 8 dark hour photoperiod. All test and sampling glassware and equipment are washed with soap and hot water, then rinsed in nitric acid, acetone, and distilled/deionized water, to remove all toxins and contaminants. Effluent samples are collected by DEM Regional Office or Aquatic Toxicology personnel. All samples are collected chilled and above chlorination unless otherwise specified. Each sample is collected as a grab or 24 hour composite using an automatic sampler and is sent chilled to the Aquatic Toxicology Laboratory by state courier or bus. The sample must be received within 72 hours after collection. The effluent samples are prepared for testing by being thouroughly mixed, adjusted to standard test temperature, and aerated if dissolved oxygen is below 5 mg/1. Hardness and alkalinity are measured. Chlorine is removed with sodium thiosulfate if applicable. The test is initiated with 20-24 hours old animals, or neonates. Adults having brood sacs with eggs with visible eyespots (indicating eggs are about to be released) are isolated and checked periodically. Neonates are removed and grouped according to time of birth. Selected groups are then composited to make the youngest set of 90 or more neonates born within a 4 hour period. The test is begun when the neonates are introduced into the test chambers. Temperatures must be within 1 degrees centigrade for transfer. The animals are transferred daily to new test chambers containing freshly mixed solutions. Chemical/physical' parameters are measured twice for each batch of test solutions. The initial value is taken before the animal is introduced and the final value after the animal has been transferred out the next day. The animals are fed daily. Each organism receives 0.1 ml of fermented trout chow -yeast -alfalfa food. As reproduction begins, only the original test organism, now an adult, is transferred to the new chamber. A drop of concentrated nitric acid is added to the old chamber. This kills the young so they can be easily counted under a dissecting microscope. A mean number of young produced per adult is calculated for each concentration. Mortality of greater than 209E in control test organisms invalidates a test. Guidance Document: 1985. U. 3. E. P. A. Methods for estimaing the chronic toxicity of effluents and receiving waters to freshwater organisms. (EPA-600/4-85-014) -34- Benthic Macroinvertebrate Sampling Procedure Appendix Biological Monitoring Group N.C. Division of Environmental Management The sampling methodology requires that a stream or river be wadable. High water conditions may severely impair sampling efficiency by making critical habitats inaccessable. A fixed number of samples are collected for each station. These include: 2 kick samples of riffle and snag areas; 3 sweep net samples of bank areas and macrophyte beds; and 2 fine -mesh washdown samples from rocks, logs, leaves and substrate. The benthic macroinvertebrates are picked out with forceps and preserved in alcohol. A collection card is filled out with such data as canopy cover, substrate composition, stream morphology, dissolved oxygen, pH, and stream temperature. Organisms are identified to the lowest possible taxonomic level, generally to species. Density of each taxon is rated as Rare (1 or 2 individuals), Common (3 to 9), or Abundant (10 or more). Most organisms may be identified using only a dissecting microscope, but Oligochaeta and Chironomidae must be mounted and identified with a compound microscope. Reference collections are maintained and all samples are retained and stored by study area. The first level of data analysis summarizes the data by total number of taxa or "taxa richness" (S) and density (N) for each station. The second level of data analysis summarizes the data by taxonomic groups (mostly orders of insects) . The EPT or intolerant (to pollution) taxa richness value is the sum of the taxa richness values for the intolerant insect orders Ephemeroptera, Plectoptera, and Trichoptera. The final step in data analysis is to summarize the data separately for each taxa. The presence or absence of individuals of any tolerant species is, in itself, insufficient for characterizing water quality. In a stream of good water quality, both intolerant and tolerant species will be present. Tolerant species will become dominant only in polluted systems when the intolerant species have been eliminated. A rating or "bioclassification" of overall water quality is assigned to a particular site based on total and intolerant taxa richness values. Information on pollution tolerance of any given taxon, combined with quantitative data on its distribution, can often be related to specific chemical or physical changes in the environment. 0 List of Definitions Aquatic Toxicology Group N. C. Division of Environmental Management Acclimation - refers to the process of gradually adjusting organisms from water of one type to another so that the organisms are not stressed from radical changes in temperature, hardness, pH, ionic strength, etc. Acute toxicity - the effect a short term exposure to a chemical or substance has on an organism; usually defined as death of that organism. Application factor - a value which estimates an instream toxicant level that will be safe at a chronic level for resident organisms from acute toxicity data, usually defined by a fraction of the LC50. Aquatic - having to do with water. Aquatic Toxicology Group - the group within the Biological Services Unit (Water Quality Section) which performs aquatic bioassays for the Division of Environmental Management. The Group is located at the Cary laboratory facilities. All test organisms (including Ratan& Dulex, Ceriodaphnia SD., and fathead minnows) are cultured at these facilities by Aquatic Toxicology personnel. Benthos/Benthic macroinvertebrates - a wide assemblage of invertebrate animals (insects, crustaceans, molluscs, etc.) which live in streams, are an important food source for fish populations, and are used as long term water quality indicators. Bioassay - a test used to determine the effects of a chemical or substance on an organism. Cadmium - one of the toxicants recommended by EPA for quality assurance testing of the health of aquatic organisms. Calibration - the adjustment of meters or systems with standards of known values in order to assure the quality of data obtained from these meters or systems. Ceriodaphnia zo, - a small cladoceran crustacean. It is found throughout most of North America and obtains a maximum size of approximately 1 mm. This organism has been adopted for aquatic bioassay testing because of its small size, ease of culture under laboratory conditions, stability of genetic strains, and sensitivity to toxic substances. It is generally used in a 7 day static renewal "mini -chronic" bioassay testing for mortality, time to sexual maturity and reproductive rate. Ceriodaphnia is accepted in the field of aquatic toxicology for testing in moderately soft waters. Chronic toxicity - the effect of a chemical or substance on an organism, usually during a longer period of time than that measured for acute toxicity. This effect is usually measured as a non -fatal response (eg. reduction in growth, egg production, predator avoidance, feeding rate, etc.). Tests for chronic toxicity are frequently performed during the entire life cycle of the organism. Chronic value(ChV): A numeric value representing the geometric mean of the numeric values of concentrations analyzed as the No Oberserved Effect Concentration (N. O. E. C.) and the lowest Oberserved Effect Concentration (L. O. E. C.) by chroic toxicity testing. The chronic value is an estimate of the toxicant concentration that will be the actual no effect concentration based on the chronic effect tested. ChV=Antilog((Logi0L. O. E. C.+ Log10N. O. E. C. )/2) Composite - a sample or method of sampling used to obtain data on a substance which may vary over time or space. For example, a time or temporal composite of a stream would be one collected at intervals of time at the same location. This is frequently accomplished with automatic sampling devices. Daphnia Dulex (water flea) - a small cladoceran crustacean. It is found throughout most of North America and obtains a maximum size of approximately 3.5 mm. This organism has been adopted for aquatic bioassay testing because of its small size, ease of culture under laboratory conditions, stability of genetic strains, and sensitivity to toxic substances. It is generally used in a 48 hour static bioassay testing for mortality. k. pulex is widely accepted in the field of aquatic toxicology for testing in moderately soft waters. Design flow - the volume of water and waste that is initially planned to pass through a facility or waste treatment plant and still allow maximum operating efficiency. Design flow is usually expressed in millions of gallons per day (mgd). Dilution (water) - the water used in bioassay tests to dilute the waste water to various concentrations (expressed as percent) . Wherever possible, this water is from the actual stream that receives the waste, upstream from that waste. When this is not possible, other suitable water is obtained. Dilutor - refers to a modified Mount and Brungs design serial dilution apparatus which receives dilution water and effluent/waste and, through a series of chambers and electrical solenoid valves, mixes the effluent and dilution into a series of concentrations for the test (expressed as percentages of 100%6 effluent). Electrofishing - method for collecting fish using electrical shock to momentarily stun the fish so they float to the surface and are easily netted. Effluent - the waste water exiting a facility which is discharged as treated waste to a stream or as untreated waste to some other facility. Fathead minnow (Pime helas - a small fish which occurs throughout much of North America. It obtains a maximum size of approximately 100 mm and is raised commercially as bait fish. The fathead minnow has been raised for numerous generations in a number of laboratory cultures for use in toxicity testing. The fish can produce eggs year round in the laboratory environment under correct conditions, which provide test organisms as needed. Flow -through - the flow -through bioassay utilizes a mechanical dilutor which either continuously or occasionally replaces the effluent/toxicant concentrations throughout the test in an attempt to simulate stream conditions where new effluent and dilution water are continually flowing through an organism's habitat. Hydrolab* - a multiparameter instrument which measures and records temperature, pH, dissolved oxygen, and specific conductance of water. Instream waste concentration (IWC) - the percent concentration of an effluent/toxicant which is present in a stream under assumed worst case conditions. The IWC is derived from the formula: [DF / (7Q10 + DF)] x 100 = IWC (X), where DF is the design flow (in cfs) of the facility in question and 7Q10 is the 10 year, 7 day, low flow (in cfs) of the receiving stream. LC50 - that concentration or percentage of a waste/chemical/substance which is lethal to 50% of test organisms over a stated period of time Lowest Observed Effect Concentration(L. O. E. C. )- The lowest concentration of toxicant to which organisms are exposed in a life -cycle or partial life -cycle test, which causes a statistically significant adverse effect on the observed parameters(usually hatchability, survival, growth, and/or reproduction). NPDES - National Pollutant Discharge Elimination System. A system devised by the Federal Government and adopted by North Carolina for the permitting, monitoring, and pollution abatement of dischargers to surface waters. Neonate - roughly translated to newly born. In reference to Paphnia pulex, the neonate refers to the life stage in the first and early second instar, generally the first 24 hours of its life. No Observed Effect Concentration(N. 0. E. C. )- The highest concentration of toxicant to which organisms are exposed in a life -cycle or partial life -cycle test, which causes no statistically significant adverse effect on the observed parameters(usually hatchability, survival, growth, and/or reproduction.) Screening bioassay - a testing system established to determine general levels of acute. toxicity of compounds/discharges using 48 hour DauhniQ man tests. 7Q10 - the measurement of a stream's lowest average daily flow over a 7 day period during a 10 year span, generally stated as flow in cubic feet per second (cfs). Sodium pentachlorophenate - a chemical accepted by EPA as a toxicant for quality assurance testing of the health of aquatic organisms. This chemical is an organic pesticide. Static - refers to an aquatic ioassay in which toxicant/effluent concentrations are set up at the beginning of t e test and not changed for the rest of the test. This test is generally short term as compared to a flow -through or replacement test because of potential degradation of the toxicant/effluent. Taxa - refers to a group of genetically related organisms, (i. e. genus, order, species). Taxa richness - number of taxa. 30Q2 - the measurement of a stream's lowest average daily flow over a 30 day period during a 2 year span, generally stated as flow in cubic feet per second (cfs). Toxicity - the adverse effect of a chemical/substance on an organism. Toxicity is usually defined as a fatal or non -fatal response over a given period of time. UT - Unnamed tributary - a term given to streams which have no accepted name. *Use of this term .or system does not constitute an endorsement