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AR 226 _ O743 ACUTE TOXICITY TO FISH (MUMMICHOG) TEST SUBS" ANCE Identity: A mixturecontainingperfluorooctanesulfonate; may also be referred to as PFOS, FC-95, oras a component of FC-203C a1,n1d,2L,-25,434,03.,4,(41,-5O,c5t,a6n6e,s7u,l7f,o8,n8i,c8a-chiedp,tadecafluoro-, potassium salt, CAS # 2795-39-3) Remarks: sample is The 3M production lot number FC-203C and is referred to by the was not noted. The test testing laboratory as "81-1/8". Current information indicates it is a mixture of 1.47% test substance, 60% `hwyadterro,xy30fo%amdeiret,h1yl0e%nepgollyycooxlybeutthyylleetnheer`,mo3n%ooscotydlipuhmenoycltyeltshuelrf,at0e.,132%% sodium lauryl sulfate, and 0.05% benzotriazol. Ttehset fsoulblsotwainngcesiunmwmaaterry saoplpultiieosnt`owitthheitnecsotmspalmeptelleyacshaaramcitxetruirzeeodfthe concentrationof impurities. Data`may not accurately relatetoxicity of the test sample with that of the fluorochemical component. METHOD: Method: EG&G, Bionomics protocol entitled "Protocol for Flow-through Toxicity Tests with Fishes," January, 1980. Test type: Flow-through acute GLP: No `Year Completed: 1981 SSuppepcliieesr:: FCuonmdmuelrucshieatlersoucplpiliteurs Analytical monitoring: Temperature, pH, DO, and salinity Exposure period: 96-hours Statistical methods: LCso values calculated using the computer program (created by Stephan, 1978) based on the `moving average angle analysis (24-, 48-, and 72-hour LCsos), Of binomial `probability (96-hourLCso). Test fish age: Adult. Length and weight: Length = 31 = 37 mm with a mean of 35 mm Weight = 0.43 --0.809 with a meanof 0.63 9 Loading: 0.13 g/L Pretreatment: Not noted. Test conditions: Dilution water: Natural, filtered seawater from Big Lagoon, a Gulf of Mexico estuary, Florida. Dilution water chemistry: Salinity: 28 -- 29 parts per thousand 001022 Lighting: Fluorescent lights provided a daily photoperiod of 12 hours light and 12 hours dark with an intensity of 1,100 lux incidenttothe water surface. n preparation: A primary stock solution of Stock andtestsolutio 523,000 mg/L was prepared by adding 523 gramsof test sample to a 1-L. volumetric flask and bringing to volume with deionized water. All test solutions were prepared by a diluter delivering five nominal `concentrations oFfltohwe-ttehsrtosuagmhprlaetea:ndApcpornotrxoilmawtaetelry.3 aquarium volume replacements per 24 hour period. `Stability ofthe test chemical solution: Notnoted. Exposure vessels: 30 x 30 x 91 cm glass tanks. containing ~ 50 liters of solution volume. Number of replicates: two Number of fish per replicate: 10 - WNautmebrecrhoefmicsotnrcyendturraitnigontsh:e 5stpulduys:a blank control pH range (0-96 hours): 7.8 7.6 8.0 -8.0 ((c2o0n0tr0olm)g/L) Temperature range (0-36 hours): Dissol2v2ed-- o2x4ygCe(nforr aalnlgaequ(a0r-i9a6) hours): 6.3 -- 7.1 mg/L (control) 3.87.1 mg/L (2000 mg/L) Salinity range (0-96 hours): 28 -- 29 parts per thousand (for all aquaria) RESULTS Nominal concentrations: Bk control, 125, 250, 500, 1000, and 2000 [onJom PRwRr mg/L Percent mortality (20 fish per concentration) Nominal [Comm ms | |o 0 | 0 _ 1 | 0 0T o [0 o1 2s [so |o 0 | oT |o oo o T7T 0 0| | To2o 00 [ | 0o [| 5 | _ [ o es T | o o 0J Element value: 24-hour LCso = 1,500 (1,400 1,600) mg/L 4782--hhoouurr LLCCssoo == 11,,550000 ((11,,440000 11,,660000)) mmgg//LL. 96-hour LCso = 1,400 (1,000 --2,000) mg/L. 001023 Element values based on nominal concentrations. dReesmcarrikbsed: iTnetshteintgeswtassubcsotnadnucceterdemoanrkthsefimelidx.tuTrheeofvtahltueesrtepsourtbesdtaanpcpleiaess 1o that mixture and not the fluorochemical proportion alone. CONCLUSIONS Tbhee1,F4C0-02m0g3iCL9w6i-thhoaur9L5C%socofonrfiFduenndcueluinstehrevtaelroofci1t,u0s0w0atso 2d,e0t0e0rmmign/Le.d to `PSauublm,iMtitnern:es3oMtaC,o5m5p1a3n3y, Environmental Laboratory, P.O. Box 33331, St. DATA QUALITY _ tReesltiianbgi.litHyo:weKviemri,scshamrpalnkeipnugr2t.y Twhaes sntoutdpyrompeeertlsy cahlacrraicttereiraizfeordqaunaldittyhe psrtoupdoyrtliaocnksinantahleytsioclaulticono.nfirmationofthe amount of fluorochemical REFERENCES TLhaibsorsattuodryywoafsPecnosnadcuocltae,dFbLyaEt Gth&eGrBeioqnouromeficstshteMa3riMneCoRmepsaenayr,ch3M Lab Request number 7038, 1981 OTHER Last changed: 6126/00 001024 EAT# S120 LR No. 703% FC-203C Ascoultuetiotnoxi(c8i1t-y1/8o)f atosuthrefamcutmamnitchog Fundulus heteroclitus in a flow- through system Toxicity Test Report Submitted to Minnesota Mining & Manufacturing Company Environmental Engineering and Pollution Control Environmental Laboratory St. Paul, Minnesota 55133 : \ Project Number R36 Report Number BP-81-10-163 EG&G Bionomics. Marine Research Laboratory 10307 Gulf Beach Highway Pensacola, Florida 32507 October 1981 001025 MarineEGR&eGseBairocnhomLiacbsoratory 10307 Gulf Beach Highway Pensacola, Florida 32507 TOXICITY TEST SUMMARY SHEET Client: Minnesota Mining and Manufacturing Company Client Contact or Principal Investigator: Mr. Eric Reiner Report date & number: October 1981; BP-81-10-163 Bionomics' project number: R36 Study Director: Mr. G. Scott Pest material: Surfactant solution 81-1/8 . - Ward Description: Deep orange-yellow liquid Date material received: 12 August 1981 - Date of definitive test: 5-9 October 1981 Test condition: Flow-through, 96-hour duration, 23:l degreesCelcius Test procedure: BTMeRsLtstweistthprFoitsohceosl,," "dPartoetdoco2l9 for Flow-through January 1980. Toxicity Pest animal: Mummichog; Fundulus heteroclitus : Source and age: Purchased from a commercial supplier, adults Seawater: Nsaatluirnailt,y filtered, from Big Lagoon, 28-29 parts per thousand Solvent/carrier: none Nominal test 7 concentrations: ..p1e2r5, l2i5t0e,r 5(0m0g,/%)1,000 and 2,000 : milligrams Effect criterion: Mortality 96 -hour LC50: 1,400 mg/% 95% confidence limits: 1,000 - 2,000 mg/% 001026 1 A flow-through marine toxicity test was conducted at EG&G Bionomics Marine Research Laboratory (BMRL), Pensacola, Florida, to determine the acute effect of a surfactant solution (81-1/8) on the mummichog Fundufus heteroclitus. The criterion for effect was death. Results of the test are reported as 24-, 48-, 72-, and 96-hour 1050 values (the concentrations of test material estimated to be' lethal to 50% of the test animals at the specified times of exposure) . All data related to this study are stored at BMRL. MATERIALS AND METHODS Test material The test material was received at BMRL on 12 August 1981 in one 9.5 liter (%) cubetainer labeled "3M SAMPLE 81-1/8." a deep orange-yellow liquid, was tested as received. The test material, All test concentrations are reported here as milligrams (mg) of surfactant solution per of seawater or as parts per million (ppm). Test animals Mummichogs were purchased from a commercial supplier and maintained at BMRL in flowing, natural seawater for 5 days. During the 96-hour period immediately before the test, the salinity of the holding water was 28-29 parts per thousand (/oo) and the temperature was 19-21 degree Celsius (C). No mortality was observed during the entire holding period. The control fish were 31-37 millimeters (mm) standard length and 0.43-0.80 grams (g) wet weight; mean length and weight were 35 (s.D.2) mm and 0.63 (S.D.:0.10) g, respectively. The control fish appeared to be representative of the test population. 001027 2 Test water Water used to maintain and test the fish was natural seawater which was pumped from Big Lagoon, a Gulf of Mexico estuary adjacent to BURL. The pump intake was located about 80 meters (m) offshore at a depth of approximately 3 m. Water was pumped by a 316 stainless steel pump through hard polyvinylchloride (PVC) pipes, through sand-filled fiberglass filters, through charcoal-filled fiberglass filters, and through 10-micrometers (um) pore size polypropylene core filters into an elevated fiberglass reservoir. Water was chilled by Teflon heat exchangers and flowed by gravity into the laboratory. Test conditions Methods for the 96-hour flow-through test were those described in BMRL's "Protocol for flow-through toxicity tests with fishes" dated 2 January 1980. The test was conducted in an intermittent-flow system by using a proportional diluter (Mount and Brungs, 1967) constructed to deliver 1 2/cycle/test aquarium at a dilution rate of 508. The average mumber of cycles was approximately 6.4/hour. This cycling rate provided approximately 3.0 volume additions in 24 hours. The surfactant stock solution (523,000 mg/i) was prepared by weighing 523 g of surfactant solution in a 1-1 volumetric flask and bringing to volume with deionized water. The stock solution was placed in a 4-2 glass reservoir attached to a "dipping bird" (Mount and Brungs, 1967). A 14.9 milliliter volume of stock solution was delivered into the mixing cell via the dipping bird during each diluter cycle. A Teflon solenoid valve, controlled by float switches, regulated seawater flow into the water cells. Mixing of various volumes of uncontaminated dilution seawater from the water cells with 001028 3 various volumes of stock solution in the test material cells produced the five concentrations of test material which were distributed to the appropriate test chambers. Test chambers were 30 centimeters (cm) x 30-cm x 9l-cm glass aquaria which contained approximately 50% of test solution or control seawater. Light was provided by two 3.7-m fluorescent bulbs suspended 46 cm above the test containers, providing approximately 1,100 lux incident to the water surface. Photoperiod was 12 hours of light and 12 hours of darkness. - Five concentrations of surfactant solution and a control were maintained. Selection of surfactant solution concentrations were based on the results of a 96-hour static test and a maximum test concentra- tion established by verbal authorization from Mr. Eric Reiner of the 3m Company. The nominal concentrations were 125, 250, 500, 1,000, and 2,000 ppm. To begin the test, the diluter cycled until all test chambers were filled with test solutions or control seawater. Ten fish were then placed into each duplicate test container. Survival of fish in all treatments was recorded at 24-hour intervals. The dissolved oxygen concentration and pi were measured in cach test container at 0 and 96 hours and in both controls, low, middle, and high test solutions after 24, 48, and 72 hours of exposure. The temperature and salinity were measured in both controls daily throughout the test. 3 The test was conducted 5-9 October 1981. 001029 4 Statistical analyses Based on the results of the test with the surfactant solution, 24-, 48-, 72-, and 96-hour LC50's and 95% confidence limits were calculated. The computer program estimated LCS0 values by three statistical methods; moving average angle analysis, probit analysis, or binomial probability. The method selected was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% mortality and the number of concentrations in which >03<100% mortality occurred (Stephan, 1977). The moving average angle method was used to calculate the 24-, 48-, and 72-hour LCS0's. The 96-hour LC50 was determined by the bionomial probability method. RESULTS AND DISCUSSION After 24 hours of exposure to the surfactant solution, mortality ranged from 0% in surfactant concentrations <1,000 ppm to 90% in the 2,000 ppm test concentration. The 24 hour LCS0 was 1,500 ppm with 95% confidence limits of 1,400-1,600 ppm (Tables 1 and 2). After 96 hours of exposure, mortality of fish ranged from 0% in test concentrations <1,000 ppm to 100% in the 2,000 ppm test concentra: tion (Table 1). The 96-hour LC50 was 1,400 ppm with 95% confidence limits of 1,000-2,000 ppm (Table 2). Physical or behavorial abnormalities were observed only in the 2,000 ppm test concentration. After 24 hours of exposure, the 2 surviving fish in 2,000 ppm were observed to be lethargic and remained so until their death. Salinity during the test ranged from 28-29 /oo. The temprature ranged from 22-249C; mean was 23#1C. The dissolved oxygen concentration remained >30% of saturation in all test containers throughout 001030 5 the exposure. The pi was 7.5-8.0 in all test containers throughout the exposure (Appendix A). Co 001031 6 REFERENCES EG&G Bionomics Marine Research Laboratory. 1980. Protocol for Flow-Through Toxicity Test with Fishes. 2 January 1981. 5 pp. Mount, D.I. and W. A. Brungs. 1967. A simplified dosing apparatus for fish toxicological studies. Water Research, 1:21-29. Stephan, C.E. 1977. Methods for calculating an LC50. ASTM, Aquatic Toxicology and Hazard Evaluation, .ASTM STP 634. F.L. Mayer and J.L. Hamelink, eds. pp. 65-84. 001032 7 TABLE 1. MaToerssttuarlcfioatncycteanonfttramstuoimlomunitscihoonagrse(8(g1Fi-unv1d/eu8nf)usasihnenteofrmloiocnwtiiatnlugs)cnoeanxtcpueornsatelrdatstieooanwsateorf. whole material. ConcNeonmtirnaatlion Control 125 250 500 1,000 2,000 SE (mg/%:ppm) Eom 0 0 0 0 o 90 MortRaetlity o 0 0 To 0 95 72(3)how o 0 0 o 95 96 How 0 o o o 100 -------- 001033 TABLE 2. 8 CtaolcaulsautrefdacLtCa5n0t'ssolfuotriomnumm(i8c1h-o1/g8s) (iFunndfulfuoswihnegternoactfiutruasl) seexapwoasteedr. Twheosltecmoantceernitarla.tions are given as nominal concentrations of Hour C50 (mg/%; ppm) 95% co(nmfgi/2d;epnpcme) limits 24 1,500 1,400-1,600 8 1,500 1,400-1,600 : 72 1,500 1,400-1,600 96 1,400 1,000-2,000 001034 PREPARED BY: AUDITED BY: 6. scott ward SGtu.dySbeiscecAtorDed. 5 20 OBamtae n \agl Alan 6. miller Yb, nt, Guattey assixence Galt 19Jadbaltee 5r8/) Raw data avait: Oley /9/ Preliminary report avait: s Ody /%/ Final report await: _J12idbso y REVIEWED AND APPROVED BY: Peter J. Shuba, Ph.D. Pas TecinioaT 1 Shee Foordinater 22 OebouDlatae y 001035 . Jn EG:G BIONOMICS MPaerrinseonteeseaHreon nLibrary An APPENDIX A oe. 3 i BEST COPY AVAILABLE | t[ea nr: yorcas iso SRRCT: ACUTE TONIEITY OATA SHEET CLIENT: 3M Company "PROJECT NO: R36 SPECIES: Fundulus heteroclitus TESTMATERIAL: Surfactant Solution (81-1/8 J wistoRy : wor w;_Q8-13=Ae-S_T a see pice 10:3__ ory COLLECTION/RECEIPT L0G FOR Ri DATA DATEMAINTENANCE/ACCLIMATION BEGAN: 30) Su harbec 19F1 covaorerminiowcns:_excellank - ure Smee: _uueslec b PORTAL)0 oe 4 wns agenEnTp esti: Q) x see mae 10, 3 or io in co FoR mad oATA Test comurions Orecnnry > [(JsTaric | TcnueemircoavLeianmas TOSERATIRE (*C) est swsten Wri [Ron[0800/\330| @%- | Rea Dass Es} Na oF MLS/COMAINER | CwOaNeTA0INTECeORsNtSC one vous (1) | soni oreo) [e@w- [Pe [heewlet) [ons Gm| mor ---- [m a Jafr [T e L PRoTocoL FOLLOIDT Prdacl TM sAcas3te ca b Cire ove: es okTEtS,T FAWGTEeiRbIAeLt r25 ACTIVdE auINdGbRs eEeDIsEeNT WHOLBUE1eF, "ATTESRoIAoL ny Test comenTATIOnS Gort eds co Souvenr/omiEn oo or cross ao)|wa[an] | WA A]||| coavion soos we an oe[on] 1TTT] FONT OF SOEAT RONED (ot) [o fwat wnfonfr unfurfa osJus | oSITIOW Cove (MEN AY GIAO PO ROO) Le od. > Six C sol c SN Me ok i oo jum Q s, a Fr , eo ess 9 -- eee r-- eg-- e -------- --_----_--_-- ii 001036 \EG:G BIONOMICS C1 [rms T wea J wm [wea 5 Gallo [L/L LRALLLALLaLL L, [Commoro cn. 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