Document VJG5GxkRjzY2N41Brwdv9q68w

|5 | TEEGHXGEPSOSEUAFRNFEDECTG1S0ROOW7TF5H.0C3AONNTDOINNSUHUOARUTVSICVHAAAQLBUIEOLOIFUTSYFROYF OF FATHEAD MINNOW (Pimephales promelas). | Fe143 | I RESEARCH REPORT 3 su company | ST. PAUL, MINNESOTA | REPORT $30-78-6-175 | AquatiEcGTo&riG,tolBoigoynomLiecpseratory : Wareh7a9m0, MaMianssaScthrueseettts J June, 1978 ` 03753 | 3 ABSTRACT Fathead minnow (Pimephales promelas) eggs and fry were continu- ously exposed to nominal 78.03 concentrations ranging from 100 to 6.2 mg/2 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 any oafbovtehepa7r8.a0m3etecrosncweenrtreataifofnesctetdestbeyd.continuous exposure to | | . 03754 ) TABLE OF CONTENTS . sEcrron PAGE xT pod 1x v ) v INTR+eOeeD etU vteC veeT neiIeaO eneN ae . LL 1 MATERIALSANDMETHODS.....vevornenisennnnnn...... 3 A. EXpOSUre SYStem.............eeiiieeeeniniii... 3 B. Egg and Fry EXPOSUXE...evurrnirnieunenni..l. 4 C. Test Water Analysis.....................,.... 6 Do SEALISEICS.......ieiiiiiiiiii iii... 7 RESULTS...ovitiiiitiiiii ieee. 8 REFERENCES... oeuiiniininniniiiiiniiasnins. 9 TABLES...uuniiiiiitit eee, 11 APPENDIX T..euiiiininiiininiiiiinininenana 12 03755 \ SECTION I . INTRODUCTION 1 | 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 48-hours after egg fertilization and continued through 30 days I post-hatch. The effects on egg hatchability and on survival 1 and growth of fry were measured and would be used to make an estimate of the MIC (minimum threshold concentration). The MTC | is virtually synonomous with the term MATC (maximum acceptable toxicant concentration) developed by Mount and Stephen (1967). | ) Mount and Stephan's term, however, performance of a full, life-cycle, was estimated after the chronic. test where effects on ! reproduction and second generation fry were also measured. | A Macek and Sleight (1977) and McKim (1977) described egg and I 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- | were generally the most sensitive stages to chemical exposure. Rarely was reproduction or survival and growth of second I" generation fry reduced at exposure levels lover than those 7 that reduced survival or growth of the first generation fry. | 03756 1 Y The authors demonstrated that for the great majority of toxi- cants, the quicker and more economical egg and fry tests yielded estimates of safe concentrations very similar to those derived from chronic toxicity studies. ) : J 03757 2 y SECTION IT MATERIALS AND METHODS ) 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 procedures for egg and fry stages of freshvater fish" (U.S. EPA, 1972). ! 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 (CaCO) of 31-38 mg/2 and 26-32 mg/%, respeci tively (APHA, et al., 1975), a pH of 7.0-7.4 and a specific | conductance of 149-170 micromhos per centimeter (umhos/cm). The diluter delivered five nominal concentrations of 78.03 ranging ! rom 100 to 6.2 mg/g and control water (well water) to duplicate J test aquaria. Each test aquarium measured 30.5 x 30.5 x 30.5 | C3758 | 3 y centimeters (cm) and had a standpipe drain 17.5 cm in height to maintain a constant test water volume of 16 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 was used to cement acrylic components i together. 3 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 + 1%. | A 4 2 glass Mariotte bottle toxicant delivery system was used to deliver 6.6 mi of a'nominal 78.03 stock concentration of I 29.4 mg/m? in distilled water to the mixing chamber of the 1 diluter. | 5. rag ana sry seposure | 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, I blush, Minnesota. upon arrival at E G&G, mionomics, the { eggs were allowed to acclimate from 17.5C to the test temperature i 4 C03759 of 25% over a two hour period. Sixty eggs were then randonly distributed to each of 12 egg cups which were then placed in a 60 mg/2 malachite green solution for 15 seconds to eliminate possible fungus growth. One egg cup was then suspended in each Of the 12 test aquaria. Egg incubation cups were acrylic tubes (7 om long, 3 em 0.D.) covered at one end with 40 mesh Nitex 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 J a the initiation of the exposure. To initiate the 30 day fry exposure, 40 fry were randomly ! | selected aquaria. fproomnecacohmpleegtgiocnupofanhdattcrha,nsfferyrrweedretofetaheLirveespeecitnieve shrinp nauplii three times daily on weekdbys 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 were made daily and fry | counts were made weekly. At 30 days post-hatch the fry from I ecuatczhonaaqtuearriuamnswpeerceceannteasgtehetsiuzrevidvewdi,thmeMaSn-22t2ota(1trtiucnagienne, meatnthanpeo-em | wet weight vere determined. The fry were measured individually J) to calculate mean and standard deviation total length while | each fry group (fry from one aquarium) was wet weighed to cal- | s 03760 ) culate mean wet weight. At the termination of the test, the fry from the control and the high concentration (100 mg/2) 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 extnsion of the lateral Line. The remaining preserved fry and frozen fry were sent to the 3M Company, St. Paul, Minnesota, May 31, 1978. Dc. mest water marysis Dissolved oxygen concentrations were measured in test aquaria using a SI Model #54 dissolved oxygen meter with a combination ' electrode polarographic probe while pi was measured with an Instrumentation Laboratory Model $175 pH meter. Temperature was measured with a laboratory thermometer. Measurements were made daily and alternated between aguaria such that each aquarium was measured once each week. One-hundred mf water samples were taken weekly from each test aquarium and stored in polyethylene bottles. Samples were ) chipped May 31, 1978 to the 34 Company, `St. Paul, Minnesota for determination of 78.03 concentration. 03761 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 /percentage 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. ) . J 7 03762 | section 111 : RESULTS The daily measurements of water quality parameters demonstrated that the temperature remained at 25 + 1C and the dissolved | oxygen concentrations above 95% of saturation throughout the entire exposure period. The pH normally ranged from 7.0-7.3 ] and did not differ significantly between exposure aquaria. | The biological data generated in this study indicate that ] nominal 78.03 concentrations as high as 100'mg/% had no adverse ' effects upon the hatchability of eggs or upon the survival and i growth of fathead minnow fry (Table 1) through 30 days post- ) hatch exposure. i | | ] ] J | i J | 03763 SECTION IV D REFERENCES APHA, AWWA, WECF. 1975. Standard Methods for the Examination | of Water and Wastewater. ldth Edition, New York, Hardness | EDTA Titrimetric Method. 3098, pp. 203-206. ] 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. y McKim, J.M. 1977. Evaluation of tests with early life stages | of fish for predicting long-term toxicity. J. Fish. | Res. BA. Can. 34: 1143-1154. ] Mount, D.I. 1968. Chronic toxicity of copper to fathead minnow (Pimephales promelas, Rafinesque). Water Res. 2: ] 215-223. 1 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 J acceptable toxicant limits for fish, malathion and the | butoxyethanol ester of 2,4-D. Trans. Amer. Fish. Soc. 96: 185-193. ] 9 03764 \ ! 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. McGraw-Hill, New York: 481 pp. | U.S. EePqAs! ana197f2a.y sPtargoepsoseofd rfreecsohmwmaetnedredfbisiho:assappy. pr7.ocedure for ` J 1 ie C03765 ---- ---- ~ Table 1 = PteortcalentLaegnegthh,atcahndoefaenggsv,etpweericgehnttaogfe sfuartvhievaadl,mimnenaonw afnrdy s(tiannedpahradledsevipraotsieolnss()5.0.) : continuovaly exposed to 78.03. concv[eonnti7znaation 10 = so x 12. 62 control Replicate nawch A s"s a 5%5 a s 5 200 a 2 a > s2s4 as s%s SEwER20 cays ROpoTseoannaTateennior--nveTgeRl)e o"y 2 w@ 5"0 . 5=0 2nIe os ugEFfd s0s naa "n < 0505 namm ""0 5 w2aa) ss 5o2s anea) @= 8g33 8 EP APPENDIX I PROCEDURES FOR CRITICAL LIFE STAGE TOXICITY TESTS WITH FRESHWATER FISHES in ! LTfharibysorsadtteaogsercsryibooeffsEfsrGteasnh& dwGaa,tredrBitofonixosimhcieicstsy,fotWlealsrotewihenadgm,aptrMoactsehsedaucrAheqususaetftiotcrs.TeogxgTihcaionsldogy | Pprroocceedduurree cflorosEeglgy aanddherFersy Sttoagtehse oPfropForseesdhwaRteecromFtiesnhded(EBPAi,oas1s97a3y). A. Physical System ] 1. e1Dg9i6lg7u)taenWrdi:tfhryAaepxdrpioolpsuoutrrtieioson.naflaAcdtiocrlhuectokefri0s.(5MmoaiudsneteomafpnldodyiBelrduutngefsro,r | ccfoeunnnttcrrtoailto,inonbasyr,edauaitilcloyintzorebodsle,rivnaateniadocnhisf.tensetcF.eisvseartyo,xiacaSntolvcoennet | 1 2. bTeotxwieEcoexaincnanttbdeimliaxuritinengr:gansdAolaucqcouinoatnraiiananedrfoGrtioelahpcerheomrocteoenecnemsnitxriianntgiuosonef.d i ) Stoepacraachtedudpelliicvaetrey before every test ttaunbke.s aCraelirburnatfiroonmsthairse cpoenrtfaoirnmeerd to insure that the correct pro- ! dpteorlraittviieoonrnsedoafrteotomxeoainccihatnotdruepsdloilcuianttieocnacthaannkdd.updliilTcuoaextnietcawnataqtuaecrroinucgmee.n- | 3. 2gT5alnaksc:ms. aEnWadacthseirdluidpcelopinetcahtaedisheamsgaiuivanertiaauinmndeidmseabcsyounrsaetscroucn39tsetdxanto20f x ] eilanecvheealcthegsltaasqsuaaqdurrairauiimunmi.stubteThhues19m.ta5oitnactlmaiftneresodtm sthoelubtoiotntovmoloufme at 15 1. ] 4. d6Fe-l1lo0iwvetraartneekd:vtoolFueimavecesh-hudpneurdprlei24dc-amthleouraosfq.uatreTishtuimssaotluatiornatearoef otfo smaatiunrtaatiinona. dissolved oxygen concentriastisounffi>c60i6ent | S- TCelaesatnintgw:iceAwlelekalgyu.aria are brushed and siphoned at || 6. 0Eg.gDTCuopu:ndEggglasisncujbaartsiownithcuptsheabreottmoamdse cfurtomof5fom iJ ta(en4sd0tnrweeaspthleacrpeedrbywiimntcehah)n.sstaofiCunplasesrsoacreksetreoeslcairlmolraatpNepidatreaixtnRusstchreeen driven by a 2 Rui electric motor (lount, 1968). | 12 03767 3 7. Light: When necessary for egg and fry survival Te.g., salmonids), the aquaria are shielded from all sources of light. # 8. Temperature: Temperatures are controlled so as not | mtooreSevtihaante19fCromthtrhoeughsopuetcifthieedentteisrte tteemspterapteruiroed.by 9. Construction materials: Construction materials | which contact the test water are chosen which do not either leach of sorb significant amountsof ] uasnduphbelssatisavnteci,ecsizNieftdreoxpmo,ltyheeTytgwhoayntlRee,rn.e sairlGeliacsotsnh,ee cssotinolspitpcerorunscetiaonnd materials used. | 0 the exposure system: 1 10. WOaftetrh:e diAlu1e25ntmewatteerr.deepThibsedrwoatcekrweilslpuimspetdhetosoaurce concrete aeration holding tank where it receives extensive and is delivered through aged PVC pipe 3 B. Biological System | ) 1. oBPefogsis`neinmbiblnregyaoardfteeteseavtres:lotphmeTehneetggesxipsoasrreuercoefrsedretadir.leizieDndei,pteinaadntidendgthaews pssetoaogne as v availability of eggs, 35 to 50 eggs are randomly distri- ] obfunoteredeagmgtionciuempaucmhperooffdut1p/w2loicetaghtege ceuxappqseucatroierudm6.e0ggegEggisgnscuabraearetipoelnxacpeopdesreidiond. ] dEegegmmeodrnteacleistsyariyn, eeagcghsegvigllcubpeistreraetceodrdweidthdaialny.apprIef- priate fungicide during incubation. ] 2. FFryEeCxp1oossuKrreepd:t wIhfenhahnadtlicnhginogfceogmgmsencpeesrmiotfs,Lhea daily ] ' number of eggs Aafntderthceomnpulmebteer hhoafattccdhhe,efdo,4r0metfdhreyfnrauyrmebienrraenoadfcohmdleeyaqdgsecflurepyc.,ted rom the egg cup or Cups.and transferred to each | abfqeouraerxaituemmni.dneidmu"Tmihfeoftfhrey30daardteaaysewxapproorssate-ndhtasttocaht.hleongTteherisstipnsevorelisuottdiigoanmtaiyon. | ATsthuervtinhveuamlbe,enrdionoffdivstihuderuvaiflvriymnegeaxnpforystuortieaslpreelrceinoogrdtd,he,dpmeterwacinceentwawegepeekly. gwreoiugph.t and deformities are recorded for each fry | J 3. Necessary data: Data that will be reported for 13 03768 D ea)acpherdcuepnltiacgaetehaitnchthe(neugmgberandof ffrryyexspuorsvuirveingarea:fter oe bh))atgpcrehroicwnetgnhtias(gmeceoanmfprlyteottsaeul/rnviulvmeabnlgetrhatoafn3d0egdgwaseyisgihntpcousbatat-the3ad0t)c,dha,ys), and a) deformities. 4. Foeodd:LiveUnlbersisneosthhreirmwpisenaudpeleimiedtnweicceesspaerry,dayfiashd are `lthiebfitiusmhshuapvpelermeenatcehdedwiathsudfrfyicipeenltletseidze.food When 5. attDoilhsleeaalsqeur:arinaaDtiuwsreieal.slerWoeuhcteebinvreetarketshaetmaesrenamtehaistnrdedlaeetedmmeeandctc.noercdeisnsgary, 6. bpSipoercchieeaShmlositsceaexla,mrinaeantdaiohntiss:ttoorlopIgofiscsraielbqlueiirnevfdeu,tsutrieegxattprihaoynsfsiioslwhohgiaicnchdal, may indicate certain toxicant related effects. C. Chemical system d 1. aP Pnrepor arrgiane ngibcyp asGos1lta Svoeceoknr Ttvsioie nflugtiid neosnneo:lutbolpSettooecinkntwsaoTthleurtv.iaotnesTrheoarrein caatomnotuarnmotilnoifimsusmao.llsvoenItfests(aorblelvaiegsnehtnetd-i.sgrausdTeehde,orcaobnecsteotnletvrer)nattiiosnkept | hOfighseosltvenstolvinenttheconscoelnvtenrtaticoonntrfoolundis inequaanly aquarium. etxepotsheure | 1 2. Mteraastuiornemoefntfoofxictaonxticainstmecaosnucreendtraitnioeancsh: The concen- duplicate aquarium at each toxicant ; mpeartevleyekm.idwaWyatebretwseaemnpletsheawrcaeotnectreanketsnruraftaaitcoena, paobtiontltteoasmatpapnrognocxeisides of each aguarium. i perxetsrearcvteedd uinmtmieldieaxtterlayctaifotnWesrateosrramapsnlaaimlnpyglseesosrarcaeapnpreboietphrpeierartwely formea. 3. SMa0elIatvseeurdrneaOmtxeiynngtgenobafasrioest,hmeersausvcuahrreitdahbailtnesea:aqcuhaTreaimqapueardraaiitulumyreisomaanndaanldyizse-d hoingche aenadchlwoewekt.estThceoncpieintirsatmieoansuraendd alternating between replicate tanks weeaeckhlyconitnrotlh,e from week to week. 0) mcTeeotntetalrrsathiaaorrnedneaasnfsdfeccitsoendtmreobalysurwteehedekltiyon.xitchaeInfth,iagnhyaddoafnidtitohlneoaswlecpoanra- 14 C3769 y 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 Methods forChemical Analysis of Water and Wastes (EPA, 1971) are used unless other more efficient methods can provide more paecrciuordaitcealilnyforfmoartieoanc.h aRneafleyrteinccael mseatmhpolde.s areanalyzed D. Statistics 1. Duplicates: True duplicates are used for each level of e toxicant being tested (i.e., no water connections between duplicate aquaria). 2. Distribution of test concentrations: The toxicant croanncdeonmtraastsiigonnmsenatr.e assigned to aquaria by stratified 3. Analysis of variance/Dunnett's E. Miscellaneous ) 1. tAdhdriotuigohnmaelthiondfosrmnaottionc:o'vereAdll inrouthtiisneprboicoeadsusraey flow- (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). 2. References: flow-through For additional bioassay tests information concerning with fish eggs and fry, the following references are listed: American Public Health Association. m14ethEao.ds AfPoHrA,theNeewxaYmorikn.ation of water 1975. Standard and wastewater. CEhnevmiircoanlmenAtnaallysPirsotoefctWiaotnerAgeanncdy.Wast1e9s7.1. AnMaeltyhtoidcsalfor Quality Control Laboratory, Cincinnati, Ohio. aMtcneKdrinmg,eroxwpJt.oMhs.uroefasndbrtoDo.oAkc.optpBreeornutoiotn(.Ssaulr1vv9ei7lv1i.anlu,s Erffefopenrtcoitndauolcfitsil)oonn,g- (Mitchell). J. Fish. Res. Bd. Canada, 287 655-662. J 15 93770 9 WMtaootuneftra,tRheeDsaoednaarmlcidhn,nI.ow2:s19(62P8i1.m5e-ph3Cah3lr3eos:nicprotmoexliacsi,tyRaoffinceospqpueer). o | Msoiumnptl,ifiDeodnadlodsiI.ngaanpdpaWrialtluisamfoBrrunfgiss.h to1x96i7c.ologAy studies. Water Research, 1: 20-29. | tlSaoougtiheceraa,lvyRSmceeostteatalrscehtonSale.rsieelse`,1c9t7eE6.dPA-f6rE6ef0sf/eh3cw-ta7st6e-ro1f0f5ei.sxhp.osurEeco- ] . SatnedelP,rocRe.Gd:uDr.esanofd York: 48lpp. JS.tHa.tisTtoircrsi.e. Mc1G9r60a.w-HiPlrli,ncNiepwles 1. | OUp.ofSs.eFdreEsnRhvewicarotomenmrmeenFnditesadhl.PrPorcoetdeucrteionforAgeEmgcgy.and 1F9r72y. StaPgreos- , : : | | 03771 | 16