Document 1g3Mmjvb6V9KmLJLdw0nD92KE

CHRONIC TOXICITY TO FISH TEST SUBSTANCE Identity:Perfluorooctanoiaccid,ammonium salt;may alsobe referredtoas 78.03,PFOA ammonium saltA,mmonium perfluorooctanoateP,FO, FC-1 16,FC-126, FC-169, or FC-143. (Octanoicacid, pentadecafluoro-a,mmonium salt,CAS # 3825-26-1) Remarks: The 3M productionlotnumber was 83. The testsample isFC-143. The testinglaboratorryefersto itas "78.03".It's puritywas notsufficientclhyaracterizedt,hough currentinformatioinndicates itisa mixtureof96.5 -100% testsubstance and 0 - 3.5% C6, C7, and Cg perfluoroanalogue compounds. METHOD: Method Followed: The methodology forthe egg and fryexposure closely followedthatpresented in"Proposed recommended bioassay procedureforegg and frystagesoffreshwater fish"(U.S.EPA, 1972). Test Type: Flow-through GLP: No Year study performed: 1978 Species: Fathead minnow (Pimephalespromelas) Supplier: U.S. EnvironmentalProtectioAngency's EnvironmentalResearch LaboratoryinDuluth, Minnesota. Test fishage: Eggs within48-hoursafterfertilization Analyticalmonitoring: Temperature, dissolvedoxygen concentrationa,nd pH were monitoreddaily.Weekly samples were taken from each aquarium fordeterminationof ammonium pertluorooctanoatceoncentrationA.ll samples taken duringthetestwere storedin polyethylenebottlesand shipped on May 31, 1978 tothe 3M Company. Exposure period: 30 days post-hatch StatisticaMlethods: Means ofmeasured biologicaplarametersfrom duplicateaquariawere subjectedtoanalysisofvariance(Steeland Torhe, 1960, completelyrandomized blockdesign,P=0.05). Data forpercentage survivaland percentage hatch were transformedto arcsinsquare rootof percentage priortoanalysis. Test conditions: Dilutiownaterw:ellwaterpumpedtoa concretreservowihrereit was aeratedbeforeflowingtotheexposure system throughaged PVC pipe. Dilutionwater chemistry (0-30days): Total hardness: 31 - 38 mg/L (asCaC03) Alkalinity: 26 - 32 mg/L (asCaC03) pH: 7.0-7.4 Speciflcconductance: 149-170pmhos/cm Stockand testsolutiopnreparationA:modifiedp,roportiondaillutweirtha 0.50 dilutiofnactorwas used. The dilutedreliveredfivenominal concentrationosfammonium perfluorooctanoatreangingfrom 100 to 6.2 mg/L and controlwater toduplicatetestaquaria.A 4 litegrlassmadotte bottletoxicandteliverysystem was used todeliver6.6mL ofa nominal ammonium perfluorooctanoatsetockconcentratioonf29.4 mg/mL indistilled water tothemixingchamber ofthediluter. Pretreatment: Eggs were placedina 60 mg/L malachitegreen solutiofnor 15 seconds toeliminatepossiblefungusgrowth. Egg cups:acrylitcubes(3cm O.D.,7 cm long)with40 mesh NitXeR screen on one end. An egg cup rockerarm apparatus,as describedby Mount (1968),was used togentlyoscillateheegg cups inthetestwater. Fry exposure vessels:Glasstestaquarium measuring30.5X 30.5X 30.5 cm witha 17.5cm highstandpipedrain,watervolume of 16 liters. Diluter:Delivered0.50literosftestwatertoeach aquarium 195 timesper day,yieldinag 90% testwater replacementtimeof approximately10 hours. Feeding: frywere fedlivebrineshrimpnauplitihreetimesdailyon weekdays and twicedailyon weekends throughouttheexposure period.The aquaria were brushedand siphoned toremove excessfood and fecalmateriatlwice each week. Temperature control:Water bathcontainingcirculatinwgaterheated by immersioncoilheaterand regulatedby a mercury column thermoregulator Number of replicatest:wo Number of eggs for hatchabilittyest: 60 Number of fryforfryexposure test:40 from each egg cup Number of concentrations:fiveplusa blankcontrol Water chemistry during the study: Dissolved oxygen: > 95% saturation pH: 7.0- 7.3 Temperature: 25 + IOC At theten-ninatiofnthetest,thefryfrom thecontroland the high concentratio(n100 mg/L)were preservedin10% bufferedfonnalinwhilethe fryfrom theothertestaquariawere frozen.Ten formalin-preservferdy(5 from each replicatef)rom the controland the high concentrationunderwent histopathologicaelxaminationofa transversesectionofthe nares and cephalicextensionofthe lateraline(See Table 2). The remainingpreserved fryand frozenfrywere analyzedat a laterdate (by3M Company) for ammonium perfluorooctanoatceoncentrations. RESULTS Nominal concentrations: Bk control6,.2,12.5,25, 50, 100 mg/L Element value: Not stated TABLE I PERCENTAGE HATCH OF EGGS, PERCENTAGE SURVIVAL, MEAN AND STANDARD DEVIATION TOTAL LENGTH, AND MEAN WET WEIGHT OF FATHEAD MINNOW (Pimephalespromelas)FRY EXPOSED TO AMMONIUM PERFLUOROOCTANOATE FOR 30 DAYS POST- HATCH. Nominal concentration (mgA) Control 6.2 12.5 25 50 100 Replicate A B A B A B A B A B A B Hatch % 98 95 95 94 93 88 98 100 90 1 95 30 Da atch Survival% Mean Mean lengthin weightin mm mg (+SD) 92 20(2) 62 95 21(3) 75 98 20(2) 59 88 22(2) 79 95 21(3) 70 100 21(2) 72 90 21(2) 74 95 21(2) 70 90 20(2). do 98 20(3) 65 88 19(2) 59 82 ____@0(2) 60 TABLE 2 RESULTS OF HISTOPATHOLOGICAL EXAMINATION OF PIMEPHALES PROMELAS FRY EXPOSED 30 DAYS TO 100 mg/l OF AMMONIUM PERFLUOROOCTANOATE3 TestMaterial Contrx)l Number of Observations 10 Ammonium 10 Perfluorooctanoate a. Work pertbrmedundercontracttoEG Histopathological Findings 3/10Normal 6tlOLiverFattyChange 3/10GillHyperplasia (EpitheliuIm) 5/10Normal 5/10LiverFattyChange 2/10GillHyperplasia (EpftheliuIm) & G BionomicsLaboratory NOTE: "Only thosetissueswhich were missingorcontained demonstrablechange are listedT.he onlytissuechanges observed were hyperplasiaofgillamellarepitheliumand fatty change ofthe liver.These changes were judged to be minimal and consistentwithchanges seen routineliynhealthyfish. Autolipiosf giltlissuewas observed inseveralfish.This change was probablydue tothe poor penetrationofthe buffered formalintothe posteriordorsalportionofthe gilslpace." CONCLUSIONS Biologicadlata generated inthisstudyindicatethatthe nominal concentration of 100 mg/L had no adverse effectupon thehatchabilitoyr eggs or upon the survivaland growth offatheadminnow frythrough30 days post-hatch. Submitter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St. Paul,Minnesota,55133 DATA QUALITY Reliab!14: Klimischranking= 2. This studymeets allthe criterifaorquality testingatthe timeitwas conducted,buthas severaldeficienciesI.tlacks informationon purityofthe testsubstance,and the productionlotnumber from which the testsample was taken.There isno informationavailableon the analysisofthe testsolutionconcentrationsor on the preservedfryand frozenfrysamples. REFERENCES This study was conducted by E G & G, Bionomics,Wareham, Massachusetts, 1978 on the requestof3M Company. Research Report"THE EFFECTS OF CONTINUOUS AQUEOUS EXPOSURE TO 78.03 (AMMONIUM PERFLUOROOCTANOATE) ON HATCHABILITY OF EGGS AND GROWTH AND SURVIVAL OF FRY OF FATHEAD MINNOW (Pimephalespromelas)."Report # BW-78-6-175, E G & G, Bionomics,AquaticToxicologyLaboratory,790 Main Street,Wareham, Massachusetts,June 1978. Research Report"SUMMARY OF HISTOPATHOLOGICAL EXAMINATION OF FATHEAD MINNOW (Pimephalespromelas) EXPOSED TO 78.03 (AMMONIUM PERFLUOROOCTANOATE) FOR 30 DAYS." Report# Bw- 78-9-301,E G & G, Bionomics,AquaticToxicologyLaboratory,790 Main Street,Wareham, Massachusetts,September 1978. OTHER Last changed: 5/25/00 THE EFFECTS OF CONTINUOUS AQUEOUS EXPOSURE TO 78.03 ON HATCHABILITY OF EGGS AND GROI-;TH A@NID SURVIVAL OF PRY OF FATHEAD MINNOW (Pimephales promelas). RESEARCH REPORT SUBMITTED TO 3M COMPANY ST. PAUL, MINNESOTA REPORT #BW-78-6-175 E G & G, Bionomics Aqua@L-ic Toxicology Laboratory 790 Main Street Wareham, ilassacl-iusetts Junc, 1978 -ABSTRACT Fathead minnow (PimephAles promela-S) eggs and fry were continuously exposed to nominal 78.03 concentrations ranging from 100 to 6.2 mg/Z through 30 days post-hatch. Observations were made on percentage hatch of eggs and on survival, mean total length and mean wet weight of fry. Results indicated that none of the above parameters were affected by continuous exposure to any of the 78.03 concentrations tested. SECTION I ii III IV v TABLE OF CONTENTS PAGE INTRODUCM@ION ..................................... 1 MATERIALS AND METHODS ............................ 3 A. Exposure System ............................... 3 B. Egg and Fry Exposure ......................... 4 C. Test llater Analysis .......................... 6 D. Statistics ..................? ................ 7 RESULTS .......................................... 8 REFERENCES ....................................... 9 TABLES ...........................................11 APPENDIX I ...................................... 12 SECTION I INTRODUCTION The objective of this study was to determine the effects of 78.03 on fathead minnow (Pimephales promelas) eggs and fry during continuous aqueous exposure. Exposures were initiated within 4.8-hours after egg fertilization and continued through 30 days post-hatch. The effects on egg hatchability and on survival and growth of fry were measured and would be used to make an estimate of the MTC (minimum threshold concentration). The @ir."C is virtually synonomous with the term MATC (maximum acceptable toxicant concentration) developed by Mount and Stephen (1967). Mount and Stephan's term, however, was estimated after the performance of a full, life-cycle, chronic-test where effects on reproduction and second generation fry were also measured. Macek and Sleight (1977) and McKim (1977) described egg and fry investigations as being reasonably accurate short-term estimations of potential long-term chemical hazards to fish, and as being similar to those estimations derived from definitive chronic toxicity studies. In the majority o'L the studies reported by the authors and of those performed at this laboratory, the embryos and fry during early stages of development were generally the most sensitive stages to chemical exposure. Rarely was reproduction or survival and growth of second generation frlr reduced at exposure levels lo-v;erthan those that reduced survival or growth of the first generation fry. 1 The authors demonstrated that for the great majority of toxicants, the quicker and more economical egg and fry tests yielded estimates of safe concentrations very similar to those derived from chronic toxicity studies. 2 SECTION II MATERIALS AND METIIODS The egg and fry study was performed according to methods developed at E G & G, Bionomics (Appendix I), which closely follow those presented in "Proposed recommended bioassay pro- cedures 1972). for egg and fry stages of freshwater fish" (U.S. EPA, The test material, labelled 78.03, a fine white powder, was obtained from the 3M Company, St. Paul, Minnesota in 2 shipments on March 17 and April 11, 1978. A. Exposure System A modified, proportional diluter similar to that described by Mount and Brungs (1967) with a 0.50 dilution factor was used in this study. The diluent water was well water which was pumped to a concrete reservoir where it was aerated before flowing to the exposure system through PVC pipe. This water was characterized as having a total hardness and alkalinity as calcium,carbonate (CaC03) of 31-38 mg/A",and 26-32 rig/Z, respectively (APHA, et al., 1975), a pH of 7.0-7.4 and a specific conductance of 149-170 micromhos per centimeter (Ijpihos/cm). The diluter delivered five nominal concentrations of 7E@.03 ranging from 100 to 6.2 mg/k and control water (well %%7ater)to duplicate test aquaria. Each test aquarium measured 30.5 x 30.5 x 30.5 3 centimeters (cm) and had a standpipe drain 17.5 cm in height to maintain a constant test water volume of 16 Z in each aquarium. The diluter delivered 0.50 of test water to each aquarium 195 times per day yielding a 90% test water replacement time of 10 hours (Sprague, 1969). To minimize the adsorption of 78-03 on surfaces, all exposure system components having contact with 78.03 were constructed of acrylic material rather than glass. Ethylene dichloride together. was used to cement acrylic components The aquaria rested in a water bath containing circulating water heated by immersion coil heaters and regulated by a mercury column thermoregulator designed to maintain the test water temperature at 25 + loc.; A 4 t glass Mariotte bottle toxicant delivery system was used to deliver 6.6 mZ of a nominal 78.03 stock concentration of 29.4 mg/mt in distilled water to the mixing chamber of the diluter. B. Egg and Fry Exposure On March 31, 1978, the exposure of fathead minnow eggs to 78.03 was initiated with eggs obtained within 48-hours after fertilization from the U.S. Environmental Research Laboratory, Duluth, Minnesota. Upon arrival at E G & G, Bi-onomics, the eggs were allowed to acclimate from 17.50C to the test teriperature 4 .of 250C over a two hour period. Sixty eggs were then randomly distributed to.each of 12 egg cups which were then placed in a 60 mg/t malachite green solution for 15 seconds to eliminate possible fungus growth. Ono egg cup was then suspended in each of the 12 test aquaria. Egg incubation cups were acrylic tubes (7 cm long, 3 cm O.D.) covered at one end with 40 mesh Nitex R screen. An egg cup rocker arm apparatus, as described by Mount (1968) was used to gently oscillate the egg cups in the test waters. Dead eggs were counted and removed daily,until hatching was complete. Percentage hatch calculations were based on the number of live fry per aquarium after hatching was completed compared to the number of eggs (60) per aquarium at the initiation of -,he exposure. To initiate the 30 day fry exposure, 40 fry were randomly selected from each egg cup and transferred to the respective aquaria. Upon completion of hatch, fry were fed live brine shrimp nauplii three times daily on weekdays and twice daily on weekends. Aquaria were brushed and siphoned twice each week to remove excess food and fecal matter. Observations on behavior and appearance of fry %%,eremade daily and fry counts were made weekly. At 30 days post-hatch the fry from each aquarium were anesthetized with Mc--222 (tricaine methanesulfonate) and percentage survival, mean total length, and mean wet weight were determined. The fry were measured individually to calculate mean and standard deviation total length while each fry group (fry from one aquarium) was wet weighed to cal- 5 culate mean wet weight. At the termination of the test, the fry from the control and the high concentration (100 mg/Z) aquaria were preserved in 10% buffered.formalin while the fry from the other test aquaria were frozen. Ten preserved fry (5 from each replicate) from the control and the high concentration were sent to the Environmental Pathology Laboratories, Inc., Carolina, Rhode Island.for complete histopathological examination with a transverse section of the nares and cephalic extension of the lateral line. The remaining preserved fry and frozen fry were sent to the 3M Company, St. Paul, Minnesota, May 31, 1978. C. Test Water Analysis Dissolved oxygen concentrations were measured in test aquaria using a YSI Model t54 dissolved oxygen meter with a combination electrode polarographic probe while pH was measured with an Instrumentation Laboratory Model '7175pH meter. Temperature was measured with a laboratory thermometer. Measurements were made dail.@-a.n,d alternated between aquaria such that each aquarium was measured once each week. One-hundred mt water samples were taken weekly from each test aquarium and stored in polyethylene bottles. Samples were shipped May 31, 1978 to the 3M Company, St. Paul, Minnesota for determination of 78.03 concentration. 6 D. Statistics Means of measured biological parameters from duplicate aquaria were subjected to analysis of variance (Steel and Torrie, 1960, completely randomized block design, P=0.05). Data for per- centage hatch and percentage survival were transformed to arc sin Vpercent prior to analysis. If treatment effects were indicated, the means of these parameters were compared to those from the controls using Dunnett's procedure (Steel and Torrie, 1960). When a treatment mean was significantly different from the control mean (P=0.05), that treatment was considered to be an effect level. 7 SECTION III RESULTS The daily measurements of water quality parameters demonstrated that the temperature remained at 25 + 10C and the dissolved oxygen concentrations above 95% of saturation throughout the entire exposure period. The pH norma lly ranged from 7 .0-7.3 and did not differ significantly between exposure aquaria. The biological data generated in this study indicate that nominal 73.03 concentrations as high as 100*mg/)' had no adverse effects upon the hatchability of eggs or upon the survival and arowth of fathead minnow fry (Table 1) through 30 days posthatch exposure. 8 SECTION IV REFERENCES APHA, AWWA, WPCF. 1975. Standard Methods for the Examination of Water and Wastewater. 14th Edition, New York, Hardness EDTA Titrimetric Method. 309B, pp. 203-206. Macek, K.J. and B.H. Sleight, II 1. 1977. Utility of toxicity tests with embryo and fry of fish in evaluating hazards associated with chronic toxicity of chemicals to fishes. Symposium Proceedings, ASTM, Memphis, Tennessee, October, 1976: 1 37-146. McKim, J.M. 1977. Eval uation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Bd. Can. 34: 1143-1154. Mount, D.I. 1968. Chronic toxicity of copper to fathead minnow (Pimephales promelas, Rafinesque). Water Res. 2: 215-223. Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Res. 1: 20-29. Mount, D.I. and C.E. Stephen. 1967. A method for establishing acceptable toxicant limits for fish, malatliion and the butoxyethanol ester of 2,4-D. Trans. Amer. Fish. Soc. 96: 185-193. 9 Sprague, J.B. 1969. Measurements of pollutant toxicity to fish. I. Bioassay methods for acute toxicity. Water Res. 3: 793-831. Steel, R.G.D. and J.H. Torrie. 1960. Principles and procedures of statistics. l@IcGraw-Hill,New York: 481 pp. U.S. EPA. 1972. Proposed recommended bioassay procedure for egg'aiidfry stages of freshwater fish: pp. 7. 10 Table 1 Percentage hatch of eggs, percentage survival, mean and standard dE total length, and mean wet weight of fa-L-headminnow fry (Pimephales continuously exposed to 78.03. Nominal concentration (mg/k) 100 50 25 12.5 6.2 control Replicate A B A B A B A B A B A B Hatch (%) 95 97 90 95 98 100 93 88 95 94 9@8 95 30 days Survival (%) post-hatch TQ-tal length (rLm)and (S.D.) 88 19(2.) 82 20(2) 90 20(2.) 98 '20(3) 90 21(2) 95 21(2) 95 21(3) 100 21(2) 98 20(2) 88 22(2) 92 20(2) 95 21(3) APPENDIX I PROCEDURES FOR CRITICAL LIFE STAGE TOXICITY TES-LS @9ITH FRESH'07ATP-R FISHES This describes standard toxicity testing procedures for egg and fry stages of freshwater fishes followed at the Aquatic Toxicology Laboratory of E G & G, Bionomics, !,.Iareham,Massachusetts. This procedure closely adheres to the Proposed RecoTimended Bioassay Procedure for Egg and Fry Stages of Freshwater Fish (EPA, 1972). A. Physical System 1. Diluter: A proportional diluter (Mount and Brungst 1967) with a dilution factor of 0.5 is employed for egg and fry exposures. A check is made of diluter function by daily observations. Five toxicant con- centrations, a control, and if necessary, a solvent control, are utilized in each test. 2. Toxicant mixing: A container to promote mixing of toxicant bearing solution and diluent water is used between diliiter and aquaria for each concentration. Separate delivery to each duplicate tubes are run from this container tank. Calibrations are performed before every test to insure that the correct pro- portion of toxicant solution and diluent i,..aL-eirs delivered to each duplicate tank. Toxicant concen- trations are monitored in each duplicate aquarium. 3. Tank: Each duplicate aquarium is constructed of glass and silicone adhesive and measures 39 x 20 x 25 cm. Water depth is maintained by a constant level glass drain tube 19.5 cm from the bottom of each test aquarium. The total test solution volume in each aquarium is thus maintained at 15 1. 4. Flow rate: Five-hundred-ml of test solution are delivered to each duplicate aquarium at a rate of 6-10 tank volumes per 24 hours. This is sufficient to maintain a dissolved of saturation. oyygen concentration >60% S. Cleaning: All aquaria are brus'@,ledand siplioned at least twice @.ieekly. G. Egg Cup: Egg incubation cups are maae from :'>cn U.-LT.-round glass and replaced jars with the boL--to-Iscut off stainless steel or NitexR screen (40 @-iiespher inch). Cups are oscillated in the test water by means of a rocl@er arm apprirci-tiis driven by a 2 RPM electric motor 'lount, 3.968) 7. Light. I-Ihennecessary for egg and 'ry survival Teg., salmonids),.the aquaria are shielded from all sources of light. 8. Temperature: Temperatures are controlled so as not to deviate from the specified test temoerature by more than 10C throughout the entire test period. 9. Construction materials: Construction ,qaterials which contact the test water are chosen @.7hichdo not either leach of sorb significant ar,.ountsof substances from the water. Glass, silicone adhesive, Nitex R , TygonR, silicone stoppers and unplasticized polyethylene are the construction materials used. 10. Water: A 125 meter deep bedrock well is the source of the diluent water. This water is pumped to a concrete holding tank where it receives ex-%L--ensiN,e aeration and is delivered through aged PVC pipe to the exposure system. B. Biological System i.. Beginning test: The exposures are initiated as soon as possible after the eggs are fertilized, and the stage ..of embryo deve.lopmen@L-is recorded. Depending upon availability of eggs, 35 to 50 eggs are rando,-nlydistri,buted to each of two egg cups or 60 eggs are placed in one egg cup per duplicate aquarium. Eggs are exposed for a minimum of 1/2 the expected egg incubation period. Egg mortality in each egg cup is recorded daily. If deemed necessary, eggs will be treated with an appropriate fungicide during incubation. 2. Pry exposure: If handling of eggs permits, a daily record is l@e-ptwhen hatching com,-Tiienceosf the number of eggs hatched, the nuriber of dead fry, and the number of de'Lormed fry in each egg cup. After complete hatch, 40 fry are randcmly selected ..from the egg cup or cups.and transferred to each aquarium. The fry are e>,posed to the '@-es-sLolution for a nini-,iumof 30 days post-hatch. This period Tnay be e.-tended if the da-L-ax...arranka'-slonger invest igatioi-i. The number of surviving -Lcrvis recorded twice weekly. At the end of the fry exposure period, percentage survival, individual mean total lenc.,L@lmie,an wet weight and deformities are recorded for each fry group. 3. Necessary data: Data that will be reported for 13 each duplicate in the egg and fry exposure are: a) percentage hatch (number of fry surviving after hatching is complete/number of eggs incubated), b) percentage fry survival at 30 days post-hatch, c) growth (mean total length and weight at 30 days), and d) deformities. 4. Food: unless otherwise deemed necessary, fish are Yed-live brine shrimp nauplii twice per day ad'libitum supplemented with dry pelleted food @q--hen the fish have reached a sufficient size. 5. Disease: Disease outbreaks are handled according to i-e-irn-arture. I-Ihentreatment is deemed necessary, all aquaria will.receive the same treatment. 6. Special_examinations: If required, extra fish and eggs are preserved for possible future physiological, biochemical, and histological investigations which may indicate certain toxican'L-related effects. C. Chemical System 1. Preparing a stock solution: Stock solutions are prepared by dissolving tti-etoxicant in @-iateror in an organic solvent if insoluble in %.7ater. The amount of solvent (reagent-grade or better) is kept at a minimuri. If solvent is used, a solvent control is also established. The concentration of solvent in the solvent control is equal to the highest solvent concentration found in any exposure aquarium. 2. Measurement of toxicant concentrations: The concentration of toxicant is measured Tne-ach duplicate aquarium at each toxicant concentration at least once per week. 1-4atersatrples are taken at a point approximately riiidwaybetween the water surface, bottom and sides of each aquarium. 1-7atersamples are either extracted irm-nediatelyafter sampling or anoropriately preserved until exL--rac-Lioio-ris analyses can be performed. 3. Measurement of other variables: Temperature and dissolved oxygen are rieasured in aquaria daily on an alternating basis, such that each aquarium is analyzed once each week. The pj:iis measureci @..,eeK-liyn the high ai-iol,ow test cc)nceiitrationaiideach coi-itrol, alternating between replica-L-eta,-iksfrom x-jeelt:o weel@. Total hardness is nacisured in 'the high anei'I.o@qconcentration and control weekly. If any of these parameters are affected by the toxicant, additional 14 analyses are performed to more closely monitor that parameter. 4. Residue analysis: When deemed necessary, exposed' fish and eggs are analyzed for toxicant residues. 5. Methods: Methods described in ilethods for Chemical T@,al@ys@isof Water and 1,7astes (EPA, 1971) are used unless other more efficient methods can provide more accurate information. Reference samples are analyzed periodically for each analytical Trethod. D. Statistics 1. Duplicates: True duplicates are used for each level of the toxicant being tested (i.e., no water connections between duplicat--eaquaria). 2. Distribution of test concentrations: The toxicant concentrations are assigned to aquaria by stratified random assignment. 3. Analysis of variance/Dunnett's E. Miscellaneous 1. Additional information:' All routine bioassay flowthrough methods not covered in this procedure (e.g., physical and chemical determinations, handling of fish) closely followed those described in Standard kiethods for the Examination of 1-7aterand Wastewater (American Public Health Association, 1975). 2. References: For additional information concerning flow-through bioassay tests iiith fish eggs and fry, the following references are listed: American Public Health Association. 1975. Standard methods for the examination of N,,aterand wastewater. 14 Ed. APHA, New Yor'r-1. Environmental Protection 7@gency. 1971. 1.'!eL-1-lofdosr Chemical Analysis of I-,latearnd 1-glas'L-esA.nalytical Quality Control Laboratory, Cincinnati, ol-lio. L@IcKim,J.M. and D.A. Benoit. 1971. Effec- of i.orloterm exposures to copper on survival, reproduction, and gro%qth of brook trou-L-(-Salvelinus fontinalis) (Mitchell). J. ri.sli.Res. Bd. Ca@iada, 28.-65-5--662. 15 Mount, Donald 1. 1968. Chronic toxicity of copper to fathead minnows (Pimephales p@@omelas, Rafinesque). Water Research, 2: 215 T23.Mount, Donald I. and I'lilliamBrungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Research, 1: 20-29. Sauter, Scott et al. '1976. Ef-Acectsof exposure to heavy.metal'g-on-selec'Led freshwater fish. iecological Research Series, EPA-660/3-76-105. Steel, R.G'.D.and J.II.Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York: 481 pp. U.S. Environmental Protection Agency. 1972.. Proposed RecorLrnended Procedure for Egg and Pry Stages of Freshwater Fish. 16 SUBMITTED BY: PREPARED BY: APPROVED BY: E G & G, Bionomics Aquatic Toxicology Laboratory 790 Diain Street Wareham, Massachusetts June, 1978 Brenda E. Wilson Aquatic Biolog'ist Stephen J. Ells AAaquu tetiiccTO@T'@xxZiicc'ologist George A. Cary Director, Aquatic Biology SUI-LXARY OF HISTOPATHOLOGICAL EXAMINATIONS OF FATHEAD MINNOW (Pimephales promelas) EXPOSED TO 78.03' FOR 30 DAYS. RESEARCH REPORT SUBbi@iTTE:D To 3M COMPANY ST. PAUL, MINNESOTA REPORT #BW-78-9-301 SUBMITTED BY E G & G, Bionomics Aquatic Toxicology Laboratory 790 Main Street Wareham, Massachusetts September, 1978 INTRODUCTION An "egg and fry test" was conducted between March 31 and May 4, 1978 (Bionomics Report #Bli-78-6-175, June, 1978) to assess the sub-lethal effects of 78.03 on fathead minnows (Pimephales promelas) . Effects on egg hatchability and on survival and growth of fry were measured. To further investigate possible effects due to exposure to 78.03, histopathological examinations of exposed fish were also performed. The results of these examinations are reported here. MATERIALS ALND METHODS Upon termination of the egg and fry study, five fish from each replicate of the high 78.03 concentration (100 mg/t, nominal) and control were prese'rved in 10% buffered formalin and sent to the Environmental Carolina, Rhode Island. Pathology. Laboratories, Inc. The fish were examined for gross lesions, and.sagital sections were prepared by a pathologist. Fish were placed with indentifying numbers into processing cassettes, dehydrated, cleared and infiltrated on an Auto-technicon tissue processor using the method of the Armed Forces Institute of Pathology. The fish were then placed in a vacuum oven and subsequently embedded in paraffin. All blocks were sectioned at 6 microns: one 2 slide was prepared from each block and stained with hematoxylin and eosin. Special staining procedures such as PAS, Trichrome and Acid Fast were used occasionally when requested by the p athologist. All blocks were sealed with paraffin and stored. Slides were labeled, boxed and delivered.w'ith all work sheets to the pathologist for examination., Two slides were prepared from each fish. Tissues processed and examined included but were not limited to; gill, thymus, liver, spleen, heart, gonad, kidney foregut,,hindgut,, olfactory mucosa (nares), brain, ear, skin, musclei pancreas and pharyngeal mucosa. RESULTS A summary of the examination of each fish is reported in Table 1. Only those tissues which were missing or contained demonstrable change are listed. The only tissue changes observed were hyperplasia of gill lamellar epithilium and fatty change of the liver. These changes were judged to be minimal and consistent with changes seen routinely in healthy fish. Autolipis of gill tissue was observed in several fish. This change was probably due .to the poor penetration of the buffered formalin to the posterior dorsal portion of the gill space'. 3 Based on these data, it is concluded. that 30 days exposure to a nominal 78..Ol concentration of 20 ug/i did not cause any significant, demonstrable, tissue changes in 30 day old fathead minnow fry. Table 1 examination of fathead minnow (Pimephales promelas) exposed 30 days to 100 mg/t nominal 78.03 and control water. -00 mg/.t 78.03 Fish 1 2 3 4 5 6 7 8 9 10 Sex Ukn a Uk-n Ukn Ukn F- p F Ukn F Organ Condition Missing Tissue Normal Normal Normal Normal Normal Liver-Fatty change Ic Gill-Hyperplasia (epithelium) I Liver-Fatty change I Gill-Hyperplasia (epithelium) I Liver-Fatty change I Liver-Fatty change I Liver-Fatty change I Spleen, gonad, nares, ear :Spleen, gonad, thymus Heart, gonad, nares Spleen, gonad, thymus Spleen, nares Na3@es, thymus Nares, ear Spleen, ear Spleen, gonad, nares, ear Spleen, nares Table 1 (cont.) Fish Sex Control Ukn .2 Ukn 3 F 4 5 Md 6 7 Ukn 8 Ukn 9 F 10 Ukn a Female. b Unknown. Lesions are minimal. d Male Organ Condition Missing Tissue Liver-Patty change I Gill-hyperplasia (epithelium) I Gonad, nares, thymus Liver-Fatty change I Spleen, gonad, nares Gill-hyperplasia (epithelium) I Thymus Normal Spleen, nares Normal Spleen, thymus, heart Liver-Fatty change I Spleen, heart Liver-Fatty change I Spleen, heart, gonad, thymus, nares, -Livet-Fatty change I Spleen, gonad, nares, thymus Liver-Fatty change I Gill hyperplasia (epithelium) I Spleen, nares Normal Gonad,, thymus SUBMITTED BY: PREPA-TMD BY: APPROVED BY: E G & G, Bionomics Aquatic Toxicology Laboratory 790 Main Street Wareham, Massachusetts September, 1978 Stephen J. Ells A4arti c r@n3ei col ogist George A. Cary JF D'irector, Aquatic-Biology