Document 4JyO7oX7EBRMxGVJVpxp2Z1VQ

The Effectsof Co 78.02 on Hatchab Fry of Fathead Mi Summary of histop Minnow (Pimepha Days CHRONIC TOXICITY TO EARLY LIFE STAGE OF FISH TEST SUBSTANCE Identity:Perfluorooctanesulfonatmea;y alsobe referredtoas 14C78.02,PFOS orFC-95. (1-Octanesulfoniaccid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafploutoarsos-i,um salt,CAS # 2795-39-3) Remarks field: The testsubstance isa white powder. Sample was radiolabeled.Sample puritywas notcharacterized. The followingsummary isabbreviateddue to thefactthatthisstudy has been superceded by a more recenttest. METHOD Method: Method was developed by E G & G, Bionomic and closely followedthose presented inthe "Proposed recommended bioassay procedureforegg and frystages offreshwaterfish",U.S. EPA, 1972. Type: Flow-throughchronic GLP: No Year completed: 1978 Species: Pimephales promelas RESULTS 30-Day NOEC: 30-Day LOEC: 30-Day MATC: 1 mg/L 1.9 mg/L >1 mg/L and <1.9 mg/L DATA QUALITY ReliabilityK:limischranking= 2. This study satisfiecdriterifaorquality testingatthe time performed,butthe analyticamlethodology was questionable. REFERENCES This study was conducted at E G & G, Bionomics,AquaticToxicology LaboratoryinWareham, Massachusetts at the requestofthe 3M Company. OTHER - Submifter: 3M Company, EnvironmentalLaboratory,P.O. Box 33331, St.Paul,Minnesota,55133 Last changed: 5/3/00 THE EFFECTS OF CONTINUOUS AQUEOUS EXPOSURE TO 14C-78.02 ON HATCHABILITY OF EGGS AND GROWTH AND SURVIVAL OF PRY OF PATHEAD MINNOW (Pimephales promelas). RESEARCH REPORT SUBMITTED TO 3M COMPANY tT. PAUL, MINUESOTA REPORT #BW-78-S-263 E G & G, Bionomics Aquatic Toxicology Laboratory 790 Main Street Wareham, Massachusetts August, 1978 ABSTRACT rathead minnow (Pimephales promelas) eggs and fry were continuously exposed to measured 14C-labelled 78.02 concentrations ranging from 1.9 mg/L to 0.12 mg/l. Data-were collected on percentage hatch of eggs and survival, total 1ength and wet weight of fry after 30 days exposure. Survival was the most sensitive indicator of 24 C-78.02.toxicity, while hatchability-and growth were not affe.cted. Based on the reduced survival of fish at 30 days post-hatch, the MTC value is estimated to be >1.0 mg/L and <1.9 mg/L 14C-78.02. SECTION TABLE OF CONTENTS PAGE I ii III IV v INTRODUCTION ....................................1 MATERIALS AND METHODS ........................... 3 A. Exposure System ...............................3 B. Egg and Fry Exposure ....................... 5 C. Stock Preparation ..........................7 D. Sampling Schedule and Techniques ..............a E. Method Sensitivity and Precision ............. a P. Statistics...............................11.... RESULTS ....................................12...... REFERENCES .....................................1.4 TABLES ......................... 16 TABLES NO. 1. Mean and standard deviation (S.D.), and range of measured concentrations of 14C-78.02 during exposure of eggs and fry of fathead minnow (Pimepha-l-es Bromelas) 2. Percentage hatch, percentage survival, mean and standard deviation (S.D.), total.length, and average wet weight of fathead minnow (Pimephales promelas) during exposure to 14C-78.02 Page 16 17 iv SECTION I INTRODUCTION The objective of this study was to determine the effects of 14C-78.02 on fa-thead minnow (Pimephales promelas) eggs and fry during continuous aqueous exposure. Exposures.were initiated within 48-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 used to make an estimate of the MTC (minimum threshold concentration). The MTC is virtually synonomous with the term MATC (maximum acceptable toxicant concentration) developed by Mount and Stephan (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 0.977) 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 of 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 fry reduced at exposure levels lower than those that reduced survival or -growthof the first generation fry. 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 METHODS A vial containing 3t@.96. grams of "C-labelled 78.02, a White crystalline material, was received from the 3M Company on February 20, 1978. Based on communications with Mr. Dale Bacon of the 3M Company, it was learned that the activity of the 14C-Jabelled 78.02 was 4.3 millicuries (mCi). The unlabelled 78.02, also a white crystalline material, used in this study was 'received from the-3M Company on January 23, 1978. This material appeared to be somewhat finer in texture than the labelled 78.02 compound. The egg and fry study was performed according to methods developed at B G & G, Bionomics (Appendix I), which closely follow those presented in the "Proposed recommended bioassay procedure for egg and fry stages of freshwater fish" (U.S. EPA, 1972). A. Exposure System A modified, proportional diluter similar to that described by Mount and Brungs (1967) with a dilution factor of 0.5 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 3 range as calcium carbonate (CaCO,) of 32-35 mg/L and 28-30 mg/t, respectively (APHA, et al., 1975), a pH of 7.0-7.3 and a specific conductance of 118-140 micromhos per centimeter (Umhos/cm). The diluter delivered five nominal concentrations of 14C-7$.02 ranging from 2.0 to 0.13 mg/i and control water (well water), and control water containing solvent (acetone) to duplicate test aquaria. Each test aquarium measured 30.5 x 30.5 x 30.5 cm and had a standpipe drain 16 cm in height to maintain a constant test water volume of 15 1 in each aquarium. The diluter delivered 0.5.0L of test water to each aquarium approximately 180 times per day yielding a 90% test water replacement time of 9 hours (Sprague, 1969). To minimize the absorption of 78.02 on surfaces, all exposure system components having contact with 78.02 were constructed of acrylic material rather than glass. Ethylene dichloride was used to cement acrylic components together. 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 + 10C. Due to the relatively low solubility of 14 C-78.02 in water, conventional toxicant delivery systems could not be utilized for this study. A metering pump (FMI Lab pump #RP-D) delivered a stock solution (19.4 mg/Z, nominal 14 C-78.02 concentration) from a 4 200 L stainless steel tank-to a premixing chamber on the diluter, which in turn delivered the appropriate volume of 14C-78.02 solution to the mixing chamber during each'diluter cycle. A McAllister (1972) delivery device was used to deliver acetone into the solvent control aquaria at a concentration equal to the highest concentration of acetone in the 78.02 exposure aquaria (43 pl/i acetone). B. Egg and Fry Exposure On June 3, 1978, the exposure of fathead minnow eggs to '4c.78..02was initiated. Eggs used were from the brood stock at the Aquatic Toxicology Laboratory of E G & G, Bionomics, Ilareham, Massachusetts. Sixty eggs were randomly distributed to each of 14 egg cups and one egg cup was then suspended in each of the 14 test aquaria. Egg incubation cups were acrylee tubes 7 cm long and 5 cm in diameter with 40 mesh NitexR screen bottoms. An egg cup rocker arm apparatus, as described by Mount (1968), was used to gently oscillate the'e'ggcups in the test water. Dead eggs were removed and counted daily until hatching was completed. Percentage hatch was calculated based on the number of live fry per aquarium after hatching was completed compared to the number of eggs per aquarium (60) at the initiation of the exposure. To initiate'the 30-day fry exposure, forty fry were randomly selected from each egg cup and transferred to the respective aquaria. Upon transfer of fry to the aquaria, the fry were fed newly hatched San Francisco Bay variety brine shrimp nauplii, ad libitum, three times daily throughout the exposure period. The aquaria were brushed and siphoned to remove excess food and fecal material three times per week. observations on behavior and appearance of surviving.fry were made daily and -.fry counts were made weekly. At 30 days post-hatch, the fry from each aquarium were anesthetized with MS-222 (tricaine .methane-sulfonate) and percentage survival,,mean total length, and average wet weight were determined. The fry were measured individually to calculate a mean and standard deviation total length while each fry group (fry from one aquarium) was wet weighed to calculate an average wet weight. At the termination of the test, the fry from the control and the high concentration (2.0 ng/t) were preserved in Bouin's solution while the fry from the other test aquaria were frozen. Ten Bouin's solution preserved fry (5 from each replicate) from the control and the high concentration were sent to the Environmental Pathology Laboratories, Inc., Carolina, Rhode Island, July 31, 1978, for histopathological examination of a mid-line saggital section. The remaining preserved fry and frozen fry 6 were sent to 3M Company,'St. Paiil,Minnesota, July 31, 1978. Temperature, dissolved oxygen concentrations, and pH were monitored daily, alternating between aquaria such that each aquarium was measured once each week. Temperature was measured with a mercury thermometer, dissolved oxygen with a YSI Model #54 dissolved oxygen meter and probe, while pH was measured with an instrumentation Laboratory Model #175 portable pH meter and probe. C. Stbck Preparation In.order to make-up the stock in the stainless steel tank, it was necessary to prepare a 14C-78.02 super-stock solution first. The super-stock was formulated by quantitatively transferring the contents of the vial containing 24C-78.02 and 56.16 g o f non-labelled 78.02 to a 2-1 volumetric flask and diluting to volume with pesticide quality acetone. The resulting super-stock bad a 14 C-78.02 concentration of 46.6 mg/mit and a theoretical specific activity of 102 disintegrations per minute per microgram (dpn,/Ug). To determine the measured specific activity of the super-stock, three 0.050 mt aliquots were pipetted directly into glass scintillation vials containing 15 mL of Monophase R (a xylene base counting solution with non-ionic surfactants and PPO + bis/MSB scintillators, 7 Packard Instrument Company), and then placed in a Model 12112 Packard Tri-Carb Liquid Scintillation Spectrometer for radiometric quantitation. The mean measuzed specific activity was determined to be 121 dpm/pl, 119% of theoretical. The stock in the stainless steel tank was prepared every 5 days by addding 83.3 mt of the super-stock solution to 200 t of well water and mixed thoroughly by stixxing. D. Sampling Schedule 'and Techniques One 5.0 mt water sample was taken from each aquaria (2 per .concentration) at the initiation of the test (day 0), when hatching was complete (day 3) and weekly thereafter (days 10, 17, 24 and 32) for radi6metric determination of 78.02 concentrations. Samples were taken with a 5.0 intvolumetric pipet from a point approximately midway between the surface, bottom and sides of each aquarium and added to glass scintillation vials containing 15 mi of Monophase. In addition, duplicate 5.0 mt samples were taken from each 200 L stock solution immediately after preparation. The radioactivity of each'sample was then quantified using liquid scintillation spectrometry. E. Method Sensi*tivity and Precision Recovery rates of the liquid scintillation spectrometer were determined prior to analyzing each set of samples by counting the activity of a standard reference material (New England 8 Nuclear Corporation) and comparing the measured value to the known theoretical'value of the standard. Recovery rates were determined to be 99-101% and experimental data were not adjusted for percentage recovery. Counting efficiencies of all experimental samples were determined according to the channel ratio method described by ]Kobayashi and Maudsley (1974). The counting efficiency for each water sample was determined by comparing the sample channel ratio to a series of quenched standards prepared monthly at E G a G, Bionomics. The standards were made by adding 15 mi of Monophase, increasing volumes (0.25 mt) of nitromethane (chemical quenching agent) and 25 Ut of a 24Ctoluene standard (New England Nuclear Corporation) to a series of scintillation vials containing 5 mt of well water. The measured activities of these samples reflect the change in the ratioof the sample count rate as it is altered by quenching. Days on which experimental samples were analyzed, the quenched standards were also quantitated and the data generated from the standards used to construct a channel ratio versus counting efficiency curve. From this curve, the counting efficiencies of all experimental samples were determined by calculating the sample channel ratio and interpolating the corresponding counting efficiency. Background levels of radiation for water (35 dpm/sample) were 9 determined by analyzing control water samples during the study. All samples were counted fora minimum of 100 minutes or until 5,000 counts were gen erated. Using these criteria and the calculations described in "Standard Methods for the Examination of Water and Wastewater" (1975), it was determined at the 95% confidence level that a minimum detectable activity (minimum net cpm) above the mean background level for all samples of 20 cpm had a 7.9% counting error associated with this measurement. This percentage was the maximum accepted counting error associated with the minimum detectable limit. The percentage counting error for each sample was dependent on the net cpm of that sample and decreased as the sample activity .increased. The calculations used in determining the concentration of 14 C-78.02 in each water sample were as follows: i. gross cpm = gross counts/unit time (minute) ii. counting efficiency (E) = from channel ratio method il'.i.total disintegrations per minute (dpm) = gross cpm/E iv. net disintegrations per minute (dpm) (total dpm)-(background dpm) v. total '4C-78.02 net dpm in sample specific activity of the 24C-78.02 x sample size (dpm/lig) (ML) 10 P. 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 percentage survival and percentage hatch were transformed to arc sin VpercentaTe prior to analysis. When treatment effects were indicated, the means of these parameters were compared to the control means using Dunnett's procedure (Steel and Torrie, 1960). When the treatment mean was.significantly -,different from the control mean (P--0.05),that treatment was considered to be an effect level. SECTION III RESULTS Water quality parameters measured during the egg and fry exposure exhibited little variation between test chambers with some variation between test days. Mean and standard deviation measured dissolved oxygen concentrations for all test aquaria was 8.6 + 0.4 mg/L and ranged from 8.0-9.9 mg/.t. Mean and standard deviation temperature was 25 + 10C and ranged 0 22-26 C. The pH ranged from 6.6-7.3, but was normally from above 7.0. The results-.of the radiometric analyses of weekly water samples are presented in Table 1. The mean measured 14C-1-78.02concen trations ranged from 90-to 112% of the nominal concentrations. The biological data generated during this study (Table 2) indicate no adverse effects due to 14C-78.02 exposure on percentage hatch of eggs or on mean length and average wet weight of fry at 30 days post-hatch. Percentage survival of 30 day old fry exposed to 1.9 mg/t measured 14 C-78.02--was 42%., significantly less than control fry. Mortality at this concentration was first observed after 23 days post-hatch, and continued through the remainder of the test. A majority of the fish in the 1.9 mg/t concentration were observed to be exhibiting stress behavior, erratic swimming and darkened coloration during the last 2 day% of the test. A similar syndrome was observed among a few fish in the A replicate of the 12 1.0 mg/L concentration the last two days of the test. This occurance suggests that the toxic action of 78.02 is cumulative and would increase with a longer exposure period. A longer fry exposure period would probably be more accurate in estimating the potential long-term hazard of 14 C-78.02 to fathead minnows. Based on the reduced survival of fry.at 30 days, the MTC for fathead minnow and 14C-78.02 is estimated to be >1.0 mglt and <1.9 mg/L. 13 SECTION IV REFERENCES APRA, AWNA, WPCF. 1975. Standard methods for the examination of water and wastewater. 14th Edition, Washington, D.C. 1193 pp. Kobayashi, Y. and D.V. Maudsley. 1974. Biological Applications of Liquid Scintillation Counting. New York Academic Press, pg. 28. Macek, K.J. and B.H. Sleight, 111. 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: 137-146. McAllister, W.A., W.L. Mauck, and F.L. Mayer. 19,72. A simplified device for metering chemicals in intermittent flow bioassay. Trans. Am. Fish. Soc., 161(3): 555-557. McKim, J.M. 1977. Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Bd. Can. 34: 1148-1154. Mount, D.I. 1968. Chronic toxicity of copper to fathead minnow (Pimephal*es promelas). Water Res. 2; 215-223. 14 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. Stephan. 1967. A method for establishing acceptable toxicant limits for fish, malathion and the butoxyethanol ester of 2,4-D. Trans. Amer. Fish. Soc. 96: 185-193. Sprague, J.B. 1969. Measurements of pollutant toxicity to fish. I.. Bioassay methods for acute toxicity. ilater Res. 3: 793-831. Steel, R.G.D. and J.H. Torrie. 1960. Principles and procedures of statistics. McGraw-Hi 11, New York: 481 pp. U.S.-EPA. 1972. Proposed recommended bioassay procedure for egg and fry stages of freshwater fish: 7 pp. 15 Table 1 Mean and standard deviation (S.D.), and range of measured concentrations of 14C-78.02 during exposure of eggs and fry of fathead minnow (Pimephales .prpmel-as Nominal concentration (mg/1) Measured 'concentration Mean and standard deviation ............. (mg/t) Range 19.4 (stock) 2.0 1.0 0.50 0.25 0.13 control solvent control 19.7 (.0.50) 1.9 (.0.3) 1.0 (0.2) 0.45 (0.08) 0.28 (0.12) 0.12 (0.02) <0.006 <0.006 18.7-20.5 1.5-2.3 0.7-1.2 0.34-0.57 0.17-0.49 0.09-0-17 16 Table 2 Percentage hatch, percentage survival, mean and standard deviation (S.D.) total length, and average wet weight of fatbead minnow fry (Pimephales promelas euring exposure to 14 C-78.02. mean measured concentration Replicate Hatch Survival 30 Days Post-Hatch Total length wet weight (mm) (mg) 1.9 A 93 a 42 21(2)b 75 a B 97 42 20(2) 69 1.0 A 95 62 20(3) 63 B 98 90 20(2) 62 0.4s A 9s 92 21(2) 69 B 98 as 20(2) 63 0.28 A 93 98 21(3) 66 B 98 90 21(2) 62 0-12 A 98 95 20(2) 67 B 97 95 20(3) 59 control A 98 98 19(3) S4 B 98 as 21(3) 69 solvent control A 90 100 21(l) 67 as 100 21(2) 62 a Significantly reduced at P-0-05. b Mean and (standard deviation). 17 PROCEDURES FOR CRITICAL LIFE STAGE TOXICITY TESTS IIITH FRESIIWATER FISIIES 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, T.Vareham, Massachusetts. This procedure closely'adheres to the Proposed Recommer@ded Bioassay Procedure for Egg and Fry Stages of Freshwater Fish (EPA, 1972). A. Physical System 10 Diluter: A proportional diluter (Mount and Brungs, 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 concentrations, 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 diluter and aquaria for each concentration. Separate delivery tubes are run from this container to each duplicate tank. Calibrations are performed before every test to insure that the correct proportion of toxicant solution and diluent water is delivered to each duplicate tank. Toxicant concentrations 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 am from the bottom of each test aquarium. The- total test solution volume in each aquarium is'thus maintained at 15 lo 4. Flow rate: Five-hundred-ml of test solution are deliv t-'oeach duplicate aquarium at a rate of 6-10 tank volumes per 24 hours. This is sufficient to maintain a dissolved oxygen concentration >60% of saturation. S. Cleaning: All aquaria are brushed and siphoned at least twice weekly. 6. Egg Cup: Egg incubation cups are made from 5 cm u.0. Found glass jars with the bottoms cut off and replaced with stainless steel or WitexR screen (40 mesh per inch). Cups are oscillated in the test water by means of a rocker arm apparatus driven by a 2 RP14 electric motor (Mount, 1968). 7. Li ht: I-Ihenecessary for egg and fry survival salmonids),, the aquaria are shielded from all sources of light. 8. Teamperature: Temperatures are controlled so as not to deviate from the specified test temperature by more than 10C throughout the entire test period. 9. Construction materials: Construction materials which contact the te-s@tw-ater are chosen which do not either leach of sorb significant amounts of substances from the water. Glass, silicone adhesive# NitexRg TygonR, silicone stoppers and unplasticized polyethylene are the construction materials used. 10. Water: A 125 meter deep bedrock well is the source oft-he diluent water. This water is pumped to a concrete holding tank where it receives extensive aeration and is delivered through aged PVC pipe to the exposure system. B. Biologiaal System 1. Beginning test-. The exposures are initiated as soon as possible alter the eggs are fer' tilized, and the stage of e?-nbryodevelopment is recorded. Depending upon availability of.i!ggs,35 to 50 eggs are randomly distributed 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. Fry exposure: If handling of eggs permits, a daily record-rs=ept when hatching commences of the number of eggs hatched, the number of dead fry, and the number of deformed fry in each-,-eggcup. After complete hatch, 40 fry are randomly selected -from the egg cup or cups,and transferred to each aquarium. The fry are exposed to the.test solution for a minimum.of 30 days post-hatch. This period may be extended if the data warrants a longer investigation. The number of surviving fry is recorded twice weekly. At the end of the fry exposure period, percentage survival, individual mean total length, mean wet weight and deformities are recorded for each fry group. 3. Necessary data% Data that will be reported for each duplicate in*the egg and fry exposure are: a) percentage hatch (number of fry surviving after hatching is cpmplete/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 Te-d-li-ve brine shrimp nauplii twice per day ad*. libitum supplemented with dry pelleted food w-gen the fish have reached a sufficient size. Disease: Disease outbreaks are handled according to their'rnature. When treatment is deemed necessary, all aquaria will receive the same treatment. 6. Special examinations: if required, extra fish and eggs are preserved for possible future-physiol'ogi-cal, biochemical, and histological investigations which may indicate certain toxicant relatiedeffects. C. Chemical System 1. Preparing a stock solution: Stock solutions are prepared by dissolving t'7e- toxicant in water or in an organic solvqnt if insoluble in water. The amount of solvent (reagent-grade or better) is kept at a minimum. 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 -x'n@eac@duplicate aquarium at each toxicant concentration at least once per week. Igater samples are taken at a point approximately midway between the water surface, bottom and sides of each aquarium. Water samples are either extracted immediately after sampling or admropriately preserved until extractions or analyses cl; be performed. 3. Measurement of other variables: Temperature and dissofv-e-coTx-ygen are measurecl in aquaria daily on an alternating basis, such that each aquarium is analyzed. once each week. The pH is measured weekly *in the high and low test concentration and each control, alternating between replicate tanks from week to week. Total hardness is measured in the high and low concentration and control weekly. If any of these parameters are affected by the toxicant, additional analyses are performed to more closely monitor that parameter. 4. Residue analysis: When"deemed necessary, exposed T2'-s-h-anedggs are analyzed for toxicant residues. 5 Methods: Methods described in Methods for Chemical Analysil@sof Water and llastes (EPAT 1971) are used unless other more efficient methods can provide more accurate information. Reference samples are analyzed periodically for each analytical method. D. Statistics 1. Duplicates: True duplicates are used for each level of the toxicant being tested (i.e., no water connections between duplicate aquaria). 2. Distribution of test concentrations: The toxicant concentrations are assigned to aquaria by stratified random assignment. 3. Analysis of variance/Dannett's E. Miscellaneous I.. Additional informa*tion: .All routine bioassay flow- through methods not covered in this procedure (e.g., physical and chemical determinations, handling of fish) closely followed those described in Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1975). 20- References: For additional information concerning 3EIow-EFro-ughbioassay tests with fish eggs and fry, the following references are listed: American Public Health Association. 19,75. Standard methods for the examination of water and wastewater. 14 Ed. APHA,.New York. Environmental Protection Agency. 1971. Methods for Chemical Analysis of Water and Wastes. Analytical Quality Control Laboratory, Cincinnati, Ohio. McKim, J.M. and D.A. Benoit. 1971. Effect of long- term exposures to copper on survival, reproduction, and growth of brook trout (Salvelinus fontinalis) (Mitchell). J. Fish. Res. Bd. Can 28: 65 62. Mount,, Donald 1.' 1968. Chronic toxicity of copper to fathead minnows (Pimei2hales promelas, Rafinesque). Water Research, 2: 2-15-223. Mount, Donald I. and William Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water Research, 1: 20-29. Sauter, Scott et al. '1976. Effects of exposure to heavy. metals-dn-selected freshwater fish. Ecological Research Series, EPA-660/3-76-105. . Steeljp*R.G*.D. and J.H. Torrie. 1960 . Principles and Procedures of Statistics. McGraw-Hill, New York: 481 pp. U.S. Environmental Protection Agency. 1972. Proposed Recommended Procedure for Egg and Fry Stages of Freshwater Fish. SUBMITTED BY: PREPARED BY: AP PROVED BY: E G G, Bionomics Aquatic Toxicology Laboratory 790 Main Street Wareham, Massachusetts August, 1978 Jerry Dean Aquatic Biologist Stephen J. Ells Aquatic Toxicologist George A. Cary Director, Aquati6'@@Biology or -13- ITEFERENCES -ikeemY,@D. L. and It.L. Bohon: "A Proposal to Study the Fate ()fFluorochemicals in the Environment," April 4, 1975. (2) M&anjdi6lA4. Technical Report - Analytical Methodology on FM 3422, November 15, 1977. A. Memo: Additions to Technical Report dated November 15, 1977. December 27, 1977. IA % -----kvtwEr., A. Technical Report - Biodegradation Fluorocarbons II, January 9. 1978. Studies of gw4&4 40i R-.A.10, Greg. Interoffice memo to Dale Bacon: Gas Chromat- ographic Analysis of FU 3422. March 26, 1976. md6sdog A. to A. N. Welter, Personal Communication. A. Technical Report in preparation. leimiptS. K. Interoffice Memo to Dale Bacon: FC Project Soil Adsorption, September 22, 1977. S. K. Technical Report in preparation: Adsorption of FU 3422 on Soil. ---harawy,M. T. Technical Report: Biocancentration of FM 3422 in Bluegill Sunfish and in Channel Catfish, May 17, 1977. echnical Report: Aquatic Fate of a Fluoro#0",-Cllemical,FM 3422. October 14, 1977. (12) WL-1-tc.-Ar., N. Technical Report: [Biocon cent ration and Clearance Studies of FM 342 August 16, 1978. 1:1) Hamaker, J. W. "Int rpretati!onof Soil Leaching Experiments" in Chemicals, Human ealth and the Environme&t, A Collection of Dow Scientific P pers, Vol. 1, Dow Chemical USA, Midland, Michigan 48640. A IJLJtkt" ANW/cen SUMMARY OF HISTOPATHOLOGICAL EXAMINATIONS OF FATHEAD MIN'--40W (Pimephales promelas) EXPOSED TO 78.02 FOR 30 DAYS. RESEARCH REPORT SUBMITTED TO 3M COMPANY ST. PAUL, MILINESOTA REPORT #BW-78-11-352 SUBMITTED BY B G & G, Bionomics Aquatic Toxicology Laboratory 790 Main Street Wareham, Massachusetts Decer&er, 197S INTRODUCTION An "egg and fry test" was conducted between June 3 and July 6, 1978 (Bionomics Report #BW-78-8-263, August, 1978) to assess the sub-lethal effects of 14C-78.02 on fathead mi=ows (Pimephales promelas). Effects on egg hatchability and on survival and growth of fry were measured. To further investigate possible effects due to exposure to 14C-78.02, histopathological examinations of exposed fish were also petformed. The results of these examinations are reported here. MATERIALS AND METHODS Upon termination of the egg And fry study, five fish from .-each replicate of the high 14C-78.02 concentration (1.9 mg/j, measured) were preserved in 10% buffered formalin and sent to the Environmental Pathology Laboratories, Inc., Carolina, Rhod e Island. Control fish were also examined. %The fish were examined for gross lesions, and sagital sections were prepared by a pathologist. Fish were placed with identifying numbers into processing cassetts, 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 parrafin. 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 pathologist. All blocks were s'ealedwith paraffin and stored. Slides were labeled, boxed and delivered with 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, muscle, 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 demonstrabl e change are liste@d'. %The only tissue changes observed in fish in the study were fatty change of the liver of fish from both groups and autolysis of several parts of the digestive tract of fish from the control. The condition of the livers was consistent with that seen in healthy fathead minnows. In several fish it was obvious that only the deep portion of the 3 affected organs were autolysed while the more superficial areas were well preserved. This autolysis appears to be the result of poor generation of fixative. Based on these data, it is concluded that 30 days exposure to a measured 14C-78.02 concentration of 1.9 mg/i did not cause any significant, demonstrable, tissue changes in 30 day old fathead minnow fry. SUB14ITTED BY: E G G, Bionomics Aquatic Toxicology Laboratory 790 Main Street ;Iareham, Massachusetts December, 1978 PREPARED BY:- Stephen J. Ells A atic Toxicologist APPROVED BY: George A. Cary Director, Aquatic Biology Table 1 Histopathological examination of fathead minnow (Pimephales promelas) exposed 30 days to 1.9 mg/L measured 78.02 and control water. Fish Sex 1.9 mg/L 78.02 1 male 2 male 3 female 4 Unknown 5 female 6 male 7' male 8 male 9 unknown 10 female Control 1 2 unknown unknown 3 unknown 4 female Organ Condition 14issing Tissue fatty change in liverail fatty change in liver 11 fatty change in liveiII fatty change in liver II normal fatty change in liver II fatty change in liverIi fatty change in liver Ii no-mal :Eatty.change in liver I none none ear, thymus gonad ear nares spleen none gonad thymus, spleen fatty change in liver II bacterial gill disease fatty change in liver autolysis in pharynx, stomach, intestine, and pancreas fatty change in livdr 11 autolysis in pharynx fatty change in liver Ii autolysis in intestine gonad gonad gonad, nares spleen Table I (continued) Fish Sex Organ Condition 14issing Tissue Control 6 7 8 9 10 unknown.male female unknown unknown unknown fatty change in liver II autolysis in pharynx, intestine, and pancreas fatty change in liver I autolysis in pharynx, stomach, and intestine fatty change in liver II autolysis in pharynx, stomach, and'intestine fatty change in liver I autolysis in pharynx, stomach, and intestine fatty change in liver II autolysis in pharynx, stomach, intestine, and pancreas autolysis in pharynx, stomach, and intestine nares, spleen gonad none nares spleen, nares and gonad nares, gonad spleen, gonad a Lesions graded as follows: I II III IV minimal moderate marked severe