Document baKL72jE4vxgJX3Ny9L6DZkMO

AR226-9 10% TTHEEOP0ES0FU0FR5EECATNTSDo OG1F4RCOC-W7OT8NH.T0IA2NNUDOOUNSSUHRAAVQTIUCVEHAOALUBSIOLFITY . FRY OF FATHEAD MINNOW (Pimephales pro-moemlealass)) . Fe-95 : RESEARCR REPORT - SUBMITTED TO am company BT. PAUL, MINNESOTA REPORT t30-76-8-263 AquatiEoGTo&xGi,coBlioognyomLiacbsoratory Vazeh7a9An0a,gMusMaeia,nssaS1ct9hr7eu8estetts | 001476 ABSTRACT Fathead minnow (Pimephales promelas) eggs and fry were continuously exposed to measured !C-labelled 78.02 concentrations ranging from 1.9 mg/% to 0.12 mg/f. Data were collected on percentage hatch of eggs and survival, total length and wet weight of fry after 30 days exposure. Survival was the most sensitive indicator of 1*C-78.02 toxicity, while hatchability and growth were not affected. Based on the : reduced survival of fish at 30 days post-hatch, the MIC value is estimated to be >1.0 mg/% and <1.9 mg/2 1C-78.02. i 001477 t TABLE OF CONTENTS SECTION PAGE 1 1 111 Cow v INTRODUCTION. .eevnnenneennnnnnnnsnnneneaneen 1 MATERIALSANDMETHODS. +eevuevnsrnrsnrnenneenens 3 AB..EEgXgPOaSnUdEFE rSyYSEEXEPMO.S.UeZ.ee.e.u.r.v.s.en.e.r.n.seeeneeirensrennneeenseeannss 35 DC.. SStaomcpklinPgrepScahreadtuilOef.a.n.d...T.e.c.h.n.i.g.u.e.s..............o.o.o.o.e.s. 78 EF.o MSeEtAhLoidStSieCnSs.i.t.i.veietvyunasnednnPerrencniaseinonna.r.i.n.i.o.n.n.i.o.n.e.y. 118 RESULTS. .eevuernnennsernnnnnnrnnesenennnanennes 12 REFERENCES..vunenrunnennennnrnnnnsuenenneenees 14 TABLES. .vvstrrrusinersessnsesnsnerireesnsenses 16 11 001478 TABLES Yo. rage 1. Mmeeaasnuraendd csotnacnedanrtdratdieovnisatoifon14(CS-.7D8..),02adnudrirnagngeexopfosure porfomeeglgass)and fry of fathead minnow (Pimephales 16 2. Psetracndeanrtdagedevhaitacthi,onpe(rsc.eDn.)t,agteotaslurlvievnagl,n,meaanndanadverage dwuertinwgeiegxhptosoufrefattohea14dCm-i7n8.n0o2w (Pimephales promelas) 17 : 001479 iv 0 secrIoN 1 wrRopuCTION he objective of this study was to determine the effects of 14c-78.02 on fathead minnow (Pimephales promelas) eggs and fry during continuous aqueous exposure. Exposures were initisted within 48-hours after egg fertilization and continuedthrough 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 MIC 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 ana 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 of the studies reported by the authors and of those performed at this labor- atory, 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 : 001480 ` that reduced survival or growth of 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 001481 SECTION II ! MATERIALS AND METHODS A vial containing 36.96 grams of `C-labelled 78.02, a white crystalline material, vas received from the 3M Company on February 20, 1978. Based on communications with Mr. Dale Bacon of the 34 Company, it vas learned that the activity of the 1%C-labelled 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 E 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 001482 range as calcium carbonate (Cac0,) of 32-35 mg/% and 28-30 ng/s, respectively (APHA,etal., 1975), a pi of 7.0-7.3 and a specific conductance of 118-140 micromhos per centimeter (umhos/cm). The diluter delivered five nominal concentrations Of 1-78.02 ranging from 2.0 to 0.13 mg/% and control water (well water), and control vater 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 0.50 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 vater 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 + 1. Due to the relatively low solubility of 1%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/y, nominal'*C-78.02 concentration) from a 4 001483 : 200 & 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 ut/2 acetone). B. Egg and Fry Exposure On June 3, 1978, the exposure of fathead minnow eggs to !C78.02 was initiated. Eggs used were from the brood stock at the Aquatic Toxicology Laboratory of E G & G, Bionomics, Wareham, 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 acrylic tubes 7 om long and 5 cm in diameter with 40 mesh Nitex screen bottoms. An egg cup rocker arm apparatus, as described by Mount (1968), was used to gently oscillate the `gg cups in the test vater. 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 5 001484 : 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 | iy counts vere 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 Rg/2) 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 vere sent to the Environ- mental 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 001485 ; were sent to 34 Company, St. Paul, 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 vas measured . with an Instrumentation Laboratory Model #175 portable pH meter and probe. . stock Preparation In order to make-up the stock in the stainless steel tank, it was necessary to prepare a 1%C=78.02 super-stock solution first. The super-stock was formulated by quantitatively transferring the contents of the vial containing }'C-78.02 and 56.16 g of non-labelled 78.02 to a 2-% volumetric flask and diluting to volume with pesticide quality acetone. The resulting super-stock had a 1'C-78.02 concentration of 46.6 mg/me and a theoretical specific activity of 102 disintegrations per minute per microgram (dpm/ug). To determine the measured specific activity of the super-stock, three 0.050 my aliquots were pipetted directly into glass scintillation vials containing 15 ms of MonophaseTM (a xylene base counting solution with non-ionic surfactants and PPO + bis/MSB scintillators, 7 001486 Packard Instrument Company), and then placed in a Model $2112 Packard Tri-Carb Liquid Scintillation Spectrometer for radiometric quantitation. The mean measured specific activity vas deternined to be 121 dpm/ug, 119% Of theoretical. The stock in the stainless steel tank was prepared every 5 days by addding 83.3 mi Of the super-stock solution to 200 of well water and mixed thoroughly by stirring. D. sampling Schedule and Techniques One 5.0 me water sample was taken from each aguaria (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 radiometric determination of 78.02 concentrations. Samples were taken with a 5.0 mi volumetric pipet from a point approximately midway between the surface, bottom and sides of each aquarium and added to glass scintillation vials containing 15 ms Of Monophase. In addition, duplicate 5.0 mg samples were taken from each 200 1 stock solution immediately after preparation. The radioactivity of each Sample was then quantified using liquid scintillation spectrometry. E. Method Sensitivity 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 001487 { 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 & G, Bionomics. The standards were made by adding 15 me of Monophase, increasing volumes (0.25 ms) of nitromethane (chemical guenching agent) and 25 ut of a }`c- toluene standard (New England Nuclear Corporation) to a series of scintillation vials containing 5 m2 of well water. The measured activities of these samples reflect the change in the ratio of 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 $ 001488 determined by analyzing control water samples during the study. All samples were counted fora minimum of 100 minutes or until 5,000 counts were generated. 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 met 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 13C-78.02 in each water sample were as follows: i. gross cpm = gross counts/unit time (minute) ii. counting efficiency (E) = from channel xatio method iii. total disintegrations = gross cpm/E per minute (dpm) iv. pneert mdiinsuitneteg(raapmt)ions = (total dpm)-(background dpm) v. total }'c-78.02 (ug/me) = ac __nettdSpepcifmc iiv n si amopltfe yThe 3%C(-ap7m8/.u0g2) x sample (ne) size 10 001489 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 /percentage 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 meanwas significantly different from the control mean (P=0.05), that treatment was considered to be an effect level. un 001490 SECTION IIT 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/% and ranged from 8.0-9.9 mg/%. Mean and standard deviation temperature was 25 + 1C and ranged from 22-26%. The ph ranged from 6.6-7.3, but was normally above 7.0. The results of the radiometric analyses of weekly water samples are presented in Table 1. The mean measured 1Cr78.02 concen- trations ranged from 90.to 1128 of the nominal concentrations. The biological data generated during this study (Table 2) indicate no adverse effects due to !'C-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/s measured 1'C-78.02-was 42%, significantly less than control fry. Mortality at this concen tration 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/3 concentration were observed to be exhibiting stress behavior, erratic swimming and darkened coloration during the last 2 days of the test. A similar syndrome was observed among a few fish in the A replicate of the 12 001491 1.0 mg/2 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 }C-78.02 to fathead minnows. Based on the reduced survival of fry at 30 days, the MIC for fathead minnow and 1'C-78.02 is estimated to be >1.0 mg/t and 1.9 mg/L. 5 001492 SECTION IV REFERENCES APHA, AWA, WPCF. 1975. Standard methods for the examination Of water and wastewater. 14th Edition, Washington, D.C. 1193 pp. Kobayashi, . and D.V. Maudsley. 1974. Biological Applications of Liquid Scintillation Counting. New York Academic Press, pg. 28. Macek, K.J.'and B.H. Sleight, III. 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. Mohllister, W.A., W.L. Mauck, and F.L. Mayer. 1972. A simplified device for metering chemicals in intermittent flow bioassay. Trans. Am. Fish. Soc., 101(3): 555-557. MoKim, 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. Chromic toxicity of copper to fathead minnow (Pimephales promelas). Water Res. 2: 215-223. 1 001493 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. Water Res, 3: 793-831. Steel, R.G.D. and J.H. Torrie. 1960. Principles and pro- cedures of statistics. McGraw-Hill, New York: 481 pp. U.S. EPA. 1972. Proposed recommended bioassay procedure for egg and fry stages of freshwater fish: 7 pp. 15 001494 Table 1 -- Mean and standard deviation (S.D.), and range of measured concentrations of }*C-78.02 during exposure of eggs and fry of fathead minnow (Pimephales promelas.) concNeonmtirnaatlion (mg/1) a Tann Measured g concentrae tion (mg/n t) . standard deviation 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 = - 1 001495 Table 2 -- Percentage hatch, percentage survival, mean and standard deviation . (5.0.) total length, and average wet weight of fathead minnow fry (Pimephales promelas) during exposure to ''C-78.02. Mean measured concentration (=9/2) replicate 30 Days Post-natcn match . Sorvival Total length Wet weight (V) 3) (om) (mg) 1.9 a 93 * 22 7s 5 97 o* 2002) 6 1.0 a 95 a 2003) 6 5 8 % 2002) 62 0.5 a 9 02 2@ 6 2 8 202) 6 0.28 a 03 8 2m) 6 % %0 2@ 6 02 a 98 os 0@ 6 Bs 9 9 2003) 59 control a % 58 100) 54 5 % 8 2) 6 solvent control a % 100 2a 67 s 88 100 2@ 6 bSignificantly reduced at Pe0.05. Mean and (standard deviation). v 001496 PROCEDURES FOR CRITICAL LIFE STAGE TOXICITY TESTS WITH FRESHWATER FISHES Tfhriyssdteasgcersiboefsfsrteasnhwdaatredrtofxiischietsyfotlelstoiwnegd patroctehdeurAeqsuatfiocr Teogxgicaonldogy pLarboocreadtuorreyclofoseElyG a&dhG,ereBsiontoonitchse, PWraorpeohsaemd,RMeacsosmamcehnudseedttBsi.oassThaiys Procedure for Egg and Fry Stages of Freshwater Fish (EPA, 1972). A. Physical system 1. D5i6l7u)tewri:thAaprdoiplourttiioonnaflacdtoirlutofer0.(5MoiusntemapnldoyBerdungfso,r efgugncatnidonfbryy deaxipolsyuroebss.ervaAticohnesc.k isFimvaedetooxficdainltutceorn ccoennttrroalt,ionasr,e uaticlonitzreodl,inaendachiftensetc.essary, a solvent 2. EToonxiiccaannttbmeiaxriinng:g sAolcutoinotnainaenrd dtiolupernotmotweatmeirxiinsguosfed bSeetpwaereanteddielluitveerryandtuabqeusarairae frourn efarcohm cthoinscenctornattaiionne.r bteofeoarcehedvueprlyictaetset ttaonki.nsurCealitbhraattitohnescaorrerecpterpfroor-med dpeolritvieornedofttooexaiccahntdupsloilcuattieontaannkd. diTlouxenitcawnattecroncisentrations are monitored in each duplicate aquarium. 3. Tgalnaks:s anEdachsidluipcloinceataedheasgiuvaeriaunmdismeacsounrsetsruc39tedx o20f x 2l5evceml. glWaassterdradienpthtubies m1a9i.5ntacimnefdrobmytahecboontsttoanmtof : eiancehacthestaqauqaurairuimumis.' thTuhse mtaoitnatlaitneesdt astol1u5tio1.n volume 4. Flow rate: Five-hundred-nl of test solution are d6e-l10ivtearnekd tvooluemaecsh dpueprli2c4athoeurasq.uarTihuimsatisasruaftfeicoifent otfo msaaitnutraatiinona.dissolved oxygen concentration >60% 5. Col3eiaTnintgi:ceAwlelekalgyu.aria are brushed and siphoned at 6. TE.gDg:CFuopu:ndEggglaisnscujbaartsiwonithcuptshearbeotmtaodmes cfurtomof5fcm a(n4d0 rmeesphlacpeedr wiincthh).staiCnulpsessarseteoeslcilolratNeidtexin sthcereen dtreisvtenwatbyeraby2 RmePaMnseleofctraicromcokteorrar(mMoaunptp,ara1t9u6s8). 001497 7. Light: When necessary for egg and fry survival wh salmonids), the aquaria are shielded from all sources of light. 8. Zemerature: Temperatures are controlled so as mot to deviate from the specified test temperature by more than 19C throughout the entire test period. : 9. Construction materials: Construction materials which contact the test water are chosen which do not either leach of sorb significant amounts of asduhbesstiavnec,esNiftreoxm,thTeygwoantRe,r. silGilcasosn,e ssitloipcpoenres and unplasticized polyethylene are the construction materials used. 10. oWfattehre: dAilu1e25ntmweatteerr.deeTphibsedwraotcekrweilslpuimspetdhetosoaurce aceornactrieotne haonlddiisngdetlainvkerwehderethirtougrheceaigveedsPVeCxtepnipseive to the exposure system. 2 B. Biological System 3 1. PoBesgsiinnbiln[eg sattfeetesertr: thTeheegegsxpoasruerefseratrieliziendi,tiaatneddthaes sstoaogneas .of embryo development is recorded. Depending upon bauvtaeidlatboilietaychoOffegtgwso,eg3g5 ctuops50oregg6s0 aegrgesraanrdeompllyaceddistirni- ofnoer egg cup per duplicate aquarium. Eggs are exposed 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 appro- priate fungicide during incubation. 2. FTryEcexpoTo5sTurdeKe:pt WIhfenSahsadtlcihnigongfceogmgmsencpeersmiotfs,thea daily number of eggs hatched, the number of dead fry, * and the number of deformed fry in each-egg cup. -fArfotemrtcheompelgegtecuphatocrh,Cup40s.afnrdy atrreanrsafnerdroemdlytoseelaeccthed afqouraraiummi.nim"uTmheoffry30adraeysexppoosste-dhattocht.he.Ttheisst pseorliuotidonmay * Tbheeenxutmebndeerdofifsutrhveidvaitnag wfarryrainstsreacorldoendgertwiicnevewseteikglayt.ion. At the end of the fry exposure period, percentage survival, individual mean total length, mean wet wgeriougph.t and deformities are recorded for each fry 3. Necessary data: Data that will be reported for 001498 ae)acpherdcuepnltiacgateehaitnchthe(neugmgberanodf ffrryy esxuprovsiuvriengarea:fter bh)atcpheirncgentiasgecomfprlyetseu/rnvuivnableratof3e0gdgsaysincpuobsatt-ehda)t,ch, ca)ndgdr)owdthefo(rmmeiatnietso.tal length and weight at 30 days), 4. FFeododl:iveUnblersisneotshherriwmipsneaudpeleimiedtwniecceesspaerry,dayfisahd are `ltihbeiftiusmhshuapvpelemreenatcehdedwiathsufdrfyicipeenltletseizde.food when 5. TtDoihseealser: naDtiusreea.se Vohuetnbretarkesatmaernethainsdldeedemeadccnoercdeisnsgary, all aquaria will receive the same treatment. 6. Spsp2e5rc5ieaalrseeexamrinvateiodns:for pIofssriebqulierefdu,tureextprhaysfiioslhogaincdal, mbiaoychienmdiiccaalt,e caenrdtahiinstotlooxgiciacnatl rienlvaetsetdigeaftfieocntss.which C. Chemical system 1. F Prepr arino g Bn ya Gs1a tSoscokr Ivsioe nlugttd ihoen: toSxtioccakntsoilnuwtiaotnesr oarrein aanmoourngtanoifcsoslovlevnetnt (irfeaignesnotl-ugbrlaedeinorwabteetrt.er) Thies kept caotntaromliniismma.lsoIefstsaobllviesnhtedi.s usTehde, caoncseonltvreanttion hoifghseosltvenstolvinenttheconsoclevnetnrtaticoonntrfoolundisienqaunayl etoxpotsheure aquarium. A 2. Measurtrateionmeofnttoofxitcoaxnitcanistmecaosnucreendtriantioeancsh: dTuhpeliccoantceen+ paeqruawreieukm. atWeaatcehr tsoaxmipclaenstarceontcaeknetnrataitona paotinlteaasptproonxciemsaitdeeslyomfidewaacyh bageutawreieunm.theWawtaetrersasmurpflaecse,arbeotetiothmerand perxetsrearcvteedd uinmtmieldieaxtterlayctaifotnesr osramapnlailnygseosr caapnprboeprpieart-ely formed. 3. MeasSoTvuedremOxeyngtenofaroethmeeravsaruirabeildnes:aquTaermipaedraaitluyreonandandis- - oanlcteerneaacthinwgeebka.sis,Thesucphi tihsatmeaesaucrhedaqwueaerkiluym iins atnhaelyzed - ahilgthernaantdinlgowbettewsetencornecpelnitcraatteiotnanaknsd fearcohm wceoentkrotlo,week. cTeonttarlathiaorndneasnsd cisontmreoalsuwreeedkliyn. theIfhiagnhy and low conof these para- meters are affected by the toxicant, additional 001499 analyses are performed to more closely monitor that parameter. 4. E ResidauneaaR enaaslsysairse: aWnhaelnyzdeedemfeodr tneocxeiscsaanrty,resiedxupeoss:ed 1 ' 5. MAentahloydssi:s oMfeWtahotdesr daensdcWraisbteedsin(EMPeA,tho19d7s1)forarCeheumsiedcal uanclceusrsateothinefrormmoarteioenf.ficRieefnetremnectehodssampclaenspraorveidaenamloyrzeed periodically for each analytical method. Dp. statistics 1. DuplicOfthetatoexsi:canTtrubeeidunpglitceastteesd a(rie.c.u;sednofwoarteerachconleevel nections between duplicate aquaria). 2. CDisotrnibuctieon noaftxteerstasasciogtnnceedintor0atnaiogsnusa:riaTbhye tstorxaitciafnited random assignment. 3. Analysis of variance/Dunnett's E. Miscellaneous 1. tAhdrdoiutghionmaelthiondfsormnoattiocno:vereAdllinrotuhtiisneprboicoeadsusraey f(leo.wg.-, pihsyhs)icacllosanedlychfeomlilcoawleddettheorsmeidnaetsicornisb,edhainndlSitnagndaorfd M(eatmheordiscafnoPrubtlheicEHxeaamlitnhatAiosnsocoifaWtaiotne,r a1n9d75)W.astewater 2. RFelfoewr-etnhcreosu:gh Fbioorasasdadyititoensatls wiintfhorfmiasthionegcgsoncaenrdnifnryg, the following references are listed: AmemtehroidcsanfoPrubltihce eHxeaamlitnhaAtsisooncioaftwioant.er 1a8n7d5.wastSetwaantdearr.d 14 Ed. APHA, New York. EChnevmiircoanlmeAnntaallysPirsoteocftiWoanteArgeanncdy.Wast1e9s7.1. Methods for Analytical : Quality Control Laboratory, Cincinnati, Ohio. MtceKrimm,expJo.Ms.ureasndtDo.Ac.opBpeenroiotn. sur1v9i71v.al,Efrfeepcrotduocftiloonn,ga(ntditgcrheolwlt)h.ofJ.broFioskh.trRoeust. B(Sda.lCvaenaldian,us ZfoBn:tin6a5l5i-s6)62. 001500 | i Mtoounfta,theDaodnalmdinnI.o' ws 19(6P8i.mepChhalreosnicprotmoexliacsi,ty Roaffinceospqpueer). Water Research, 3; FIEFE: Msoiumnptl,ifDioendadlodsiI.nganadppWairlaltuisamfoBrr.unfgiss.h to1x96i7c.ologAy studies. Water Research, 1: 20-29. Staoutheera,vySmceottatlsetoanl.sel`e1c9t7e6.d fErfefsehcwtasteorffeisxhp.osurEecological Research Series, EPA-660/3-76-105. aStnedelP,rocRe.Gd:uDr.esaonfd JS.tKa.tiTsotricrsi.e. Mo1G96r0a.w-RiPlrli,nciNpelwes . York: 481 pp. pUo.Ss.edEnRveicroomnmmeenndteadlPrPorcoetdeucrteionfoArgeEngcgy.and19F7r2y. StPagreos- of Freshwater Fish. : 001501 .i suBMITTED BY: ` PREPARED BY: APPROVED BY: AquatiEcG79T0oxMiG,acionlBolgoSyntormeLieatcbsoratory Wareha`Amu,guMsta,ssa1c97h8usetts Jerry Dean Yeo Deon' Aquatic Biologist Stephen J. Ells 2 etcotostar George A. Cary 7 Director, Aquaticsiolosy 001502 - -13- i EFERINCES 1 ldojibreorer=D. L. and R. L. Bohon: "A Proposal to Study the Fate of Fluorochemicals in the Environment," April 4, 1975. MolndFiad34e22, A.NovTeemcbhenric1a5l, R1e9p77o.rt - Analytical Methodology om detNtoveem,oerA.15, Me1m9o7:7. AdDdeicteimobnesr 2t7o, Te1c9h77n.ical Report dated 3 amide, E. A. Fluorocarbons Technical Report - Biodegradation II, January 9, 1978. Studies of i I bij, Greg. Interoffice memo to Dale Bacon: Gas Chromat- it ographic Analysis of FM 3422. March 26, 1976. i wibiddianded, A. to A. N. Welter, Personal Communication. } wiper], A. Technical Report in preparation. | 5) MSaolile,AdsS.orpKt.ionI,ntSeeropftfeimcbeerNe22m,o t1o977D.ale Bacon: FC Project 1i whSlla`odfusFmH, 34S.32K.on TSoeiclh.nical Report in preparation: Adsorption 1 { Godiiasbarawy, M. T. Technical Report: Bloconcentration of F1M9773.422 {n Bluegill Sunfish and in Channel Catfish, May 17, E STIISChComricoalm, mFmM m34s22e.chmniOgccaetlobeRrepoIr,t: 197A7q.uatic Fate of a Fluoro?- (12) Welter, A. N. Technical Report: [Bioconcentration and Clearance Studies of FM 3422)| August 16, 1978. i (13) HoiafnmaCDkhoeewrm,iSccaiJl.esn,tW.ifHiucm"aInPngtpdorrpJesra,eltthaVtoalin.odn t1,ohfeDoSEwonivliCrhoLenmemiaeccnahtli,ngUSAAE,xCpoelMrilidelmcaetnnidto,sn" Michigan 48640. re ---- AN Wcaolnbn Covet Ageooc Elian. of oe . Borocancastictin [ladof F-37122 w 001503 E ------E -------------------------------- ---- ---- --0 -------- Lo | 001504