Document wKB1wN2rnbvp79QzZeVygOYEE

-AH AS.G _ o 8 0 5 TO XICITY TO AQUATIC PLANTS TEST SUBSTANCE_______________________________________________ Identity: A mixture containing perfluorooctanesulfonate; may also be referred to as PFOS, FC-95, or as a component of FC-206C and 81-2/8. (1-Octanesulfonicacid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-, potassium salt, CAS # 2795-39-3) Rem arks: The test sample is FC-206C. Current information indicates it is a mixture of 0.73% PFOS, 76.09% water, 15% diethylene glycol butyl ether, 4% urea, 1.55% sodium octyl sulfate, 1.47% hydroxy foamer, 1.05% polyoxyethylene monooctylphenyl ether, 0.06% sodium lauryl sulfate, and 0.05% benzotriazole. The following summary applies to a mixture with incompletely characterized concentration o f impurities. Data may not accurately reflect the toxicity o f the fiuorochemicai compnent o f die test sample.. METHOD:______________________________________________________ Method: USEPA - 600/9-78-018 and ASTM-E-35.21 Draft No. 1. Type (test type): Acute Static GLP: No Y ear study perform ed: 1981 Species: Selenastrum capricomutum Source: Originally obtained from USEPA-ERL, Corvallis, Oregon, maintained in culture medium at the 3M Environmental Laboratory, St. Paul, MN. Elem ent basis: Algal cell counts (cells/ml), cell dry weights. Exposure period: 96 hours S tatistical Methods: Regression model. A nalytical m onitoring: Not given. Test Conditions: Algal Nutrient Medium : Nutrient medium per U.S. EPA 600/9-78-018. This nutrient medium provided all mineral nutrients essential for algal growth and also served as the diluent for all algal operations. The pH of this synthetic algal medium was adjusted to 7.5 prior to use in assays. Stock and te st solution preparation: Stock solutions were prepared in the algal standard test medium, however no further description was provided. 001686 Exposure vessels: 250 ml Erlenmeyers containing 50 ml test solution. Agitation: Shaken continuously at 100 rpm. Num ber o f replicates: 3 Initial cell loading: 1.0 x 104 cells/mL Num ber o f concentrations: five plus a blank control Lighting: 400 foot-candles from continuous cool-white fluorescent lighting. W ater Chemistry: pH range (0-96 hours): Not noted. Test tem perature range (0-96 hours): 23 + 2C (incubator) RESULTS_______________________________ ______________________ Nominal concentrations: Bk control, 280, 420, 6 5 0 ,1 ,0 0 0 , and 1,550 mg/L Elem ent values: 96-hour EC50 Cell-Dry weight: 345 (34 - 1,630) mg/L 96-hour EC50 Algal Cell Count: 379 (1 0 6 -1 ,1 3 7 ) mg/L Element values are based on nominal concentrations. Reversibility o f G rowth Inhibition: Aliquots of the 1,000 and 1,550 mg/L test solutions were diluted with algal medium and cultured for 10 days. Based on growth observed in the recovery phase, the effect on algal growth was found to be algistatic. Remarks: Testing was conducted on the mixture as described in the test substance remarks field. The values reported apply to that mixture and not the fluorochemical proportion alone. CONCLUSIONS___________________________________________________ The test substance exhibits a 96-hour EC50 cell count value of 379 (106 1,137) mg/L and a 96-hour EC50 based on cell dry weight of 345 (34 1,630) mg/L. This test substance was determined to be algistatic. Subm itter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY__________________________ ___________________ _ _ 001687 R eliability : Klimisch ranking = 2. This study meets the criteria for quality testing. However, the study lacks information on purity of the test substance and it lacks analytical confirmation of the amount of fluorochemical proportion in the solution. REFERENCES__________________________________________________ Test was conducted by the 3M Company, Environmental Laboratory, St. Paul, MN, Lab Request number 6812S, 1981. O T H E R _________________________________________________________ Last changed: 6/26/00 0016S TECHNICAL REPORT SUMMARY Date 6/23/81 Vy TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN . v* - . _________________. . . . . . . -- BEST C O P Y AVAILABLE (Im p ortan t -- I f report is printed on both sides o f paper, send tw oco p iesto TCC.) Division -- Environmental Engineering and Pollution Control (Envir. Lab) bept. lu m b er 0535 Project * Co ----. CaV'-- Report Title W\V. Project Num ber 9970012600 Report Number Algal Assay: Bottle Test (A:BT), Toxicity Testing FC-206C 051 To E. A. Reiner - 21-2W LR 6812 S A u th o r(s ) M. T. Elnabarawy - 2-3E Employe Number(s) 46981 N otebook Reference N o . o f Pages In c lu d in g Coversheet 5 SECURITY ( 3 Open O Closed 3M CHEMICAL i REGISTRY 1 New Chemicals Reported Yes S No KEYWORDS: (Select terms from 3M Thesaurus. Suggest other applicable terms.) EE & PC Div. (EnV . Lab) CURRENT OBJECTIVE: To determine the algal growth responses (ECr q ",_) following a 4-day exposure period to varying concentrations of FC-206C, followed by a 10-day recovery period. Algal Assay Bottle Test Toxicity REPORT ABSTRACT: (200-250 words) This abstract information is distributed by theTechnical Communications Cent alert 3M'ers to Company R&D. The algal acute 4-day EC5Q values obtained for freshwater green algae (SelenastrunrCapricornuturo) following exposure 1 FC-206C in triplicate experiments were 345 and 379 mg/1. These values represent the algal growth response as measurec by cell-dry weight and algal cell-count, respectively. No test material concentration was defined as the no-effect level. The algal biomass monitoring measurements following a 10-da: recovery period indicated insignificant algicidal effect at concentrations tested. Since the algal cells have, recoverei and resumed logarithmic growth when resuspended in fresh medium, the 4-day median growth response (EC5Q) is interpre' as an inhibitory effect. Information Liaison Initials:. \r\KrJ FC-206C/MTE Page Two June 23, 1981 BEST COPY AVAILABLE CONCLUSIONS In these triplicated 4-day EC50 algal assays, the calculated values for freshwater green algae (Selenastrum Capricornutum) were 345 and 379 mg/1. These values represent the algal growth response as measured by cell-dry weight and algal cell count, respectively. % No test material concentration was defined as the no-effect level. The algal biomass monitoring measurements following a 10-day recovery period indicated little or no algicidal effects at concentrations tested. Since the algal cells have recovered and . resumed logarithmic growth when resuspended in fresh medium, the 4-day median growth response (EC50) is interpreted as an inhibitory effect. INTRODUCTION In testing the possible effects of chemical substances on the aquatic environment, unicellular algae are recommended as a model sys-tem for evaluating the influence of chemicals on aquatic primary producers, plants, and phytoplankton. Toxic effects are tested on growing algal cultures that undergo cell-division during the test. Algal growth response include: a) no effect, b) stimulatory,. c ) inhibitory, d) algistatic, or algicidal. The test substance is a clear amber liquid, soluble in water at ambient room temperature. These bioassays performed on this material evaluated its potential algal toxicity and the data generated form the basis of this report. - MATERIAL AND EXPERIMENTAL DESIGN These algal assays consisted of a 4-day exposure period followed by a 10-day recovery period. The test protocol utilized for this study was modified after those described by USEPA - 600/9-78-018 and ASTM-E-35.21 Draft No. 1. (1), (2). Test Species: A culture of the freshwater green algae (Chlorophycee), Selenastrum Capricornutum of the order chlorococcales was obtained from USEPA - ERL, Corv., Oregon (February 13,-1981). The test algae are non-motile unicellular cells and having the appearance of a new moon. The algal culture was stored in the dark at 40C until use. 0-0169 FC-206C/MTE Page Three June 23, 1981 BEST COPY AVAILABLE Inoculum; r Algae from a 7-day old stock culture were used as inoculum to give a starting optimum inoculum level of green algae Selenastrum Capricornutum 1 x 10^ cells/ml. The use of a 7-10 day old stock culture will insure a sufficient amount of viable algal cells in the exponential growth phase. The initial algal cell count in the stock culture was determined with the aid of a hemocytometer (370,000 cells/ml). Algal Culture Medium; Standard culture and test medium was prepared as outlined in Attachment #1. This algal culture medium provided all nutrients essential for algal growth and also served as the diluent for all algal operations including the preparation of stock solutions. The pH of this synthetic algal medium was adjusted to 7.5 prior to use in assays. Culture Flasks; Each 250 ml erlenmeyer containing 50 ml of test solution .comprised a test flask. Culture flasks and all other glassware were specially prepared as described in Attachment #2. Autoclaved foam plugs were used as flask closures, allowing for free gas exchange. Range-Finding Test (Exploratory)-; The exploratory test consisted of determining the 4-day algal biomass in single flasks containing standard culture medium plus the test material at concentrations covering several cfders of magnitude; 0.1, 1.0, 10, 100 and 1,000 mg/1. Full-Scale Test (Definitive ); All definitive algal assays were carried out in triplicate using 250 ml erlenmeyer flasks containing 50 ml of test solution. The definitive test consisted of two culture stages; a 4-day exposure period followed by a 10-day recovery period. The purpose of the exposure (contact) period was to verify inhibitory effects. The recovery (subculture) stage provided information on algistatic/algicidal effects. The following FC-206C logarithmic concentration; 28.Q, 420, 650, 1,000 and 1,550 mg/1 were used to initiate the^l-day contact period. This dose range of dilutions was selected to bracket 4-day EC^q values predicted from preliminary ~ exploratory testing. Three flasks containing 100% fresh algal culture medium plus algal cells comprised the nontreated controls. The recovery stage was initiated by 5 * 001691 . FC-206C/MTE Page Four June 23, 1981 BEST COPY AVAILABLE subculturing (0.5 ml/50 ml) from each of the three replicates of the two highest test concentrations into flasks containing fresh algal medium. These two treatments induced inhibitory effects greater than 80% during the contact period. Fresh stock solutions of the test substance were prepared in the algal standard test medium. See Attachment #3. Test Conditions: All algal operations were carried out under sterile conditions, in order to avoid contamination with bacteria and other algae. Algal cultures were incubated in an environmental chamber under the following standard growth conditions: Temperature - 23 + 2C (70-77F) Fluorescent illumination - 400 ft.C. + 10% Free gas exchange - Continuous Platform Shaking 100 + 10 rpm. Algal Biomass Monitoring: A The algal growth response was measured by algal cell-dry weight (mg/1) and by cell count (cells/ml). Both growth measurements were made with algal cultures after the 4-day exposure period and only cell-count was made with the sulcultures after a 10-day recovery period. Both procedures of biomass measurements are briefly described in Attachment #4. EC50 values and 95% confidence limits were calculated utilizing 3M SIXCUR, a TRAC System for regression models of experimental data. TEST RESULTS The results of biomass measurements of the green algae Selenastrum Capricornutum used in these studies are self-explanatory and are detailed in data sheets attached. results of the definitive test are summarized in Table 1. The 001692 FC-206C/MTE Page Five June 23, 1981 BEST COPY AVAILABLE Table 1 Definitive Test - EC50 values, mg/1 with Selenastrum Capricornutum Type Test Algal Biomass Monitoring Cell-Dry Weight mg/1 Cell Count No./l ml 4-Day Exposure Period 345(34-1630)* 379(106-1137)* *95% Confidence limits. The data reported herein are based on studies performed by M. T. Elnabarawy. Accompanying data sheets "comprise original data. A copy of this report is filed in the Environmental Laboratory Archive System, Building 2-3E. It should be' noted that the reported EC50 values were evaluated on the basis of the calculated initial concentrations of FC-206C in test solutions. REFERENCES (1) Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Capricornutum Printz Algal Assay Bottle Test: Experimental design, application, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-600/9-78-018. 125 p. (2) ASTM--E-35.21i 1980. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 1. ooidM' ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (A A :8T ) roxic iTy tes^ s^ p y AVA|LAg ^ U . 2 _ i Type Test_ J d ___ 0<xy M aterial Tested F C E>pofotc <1 Lo t Ph y s i c a l D e s c r i p t i o n C\e^r AwAf.t Algal Species:Green Algae. Selenastrum capricornutum US' E ? A L^b Source Crtr,/A\\.f Date Rec 'd 'Z~ I$- 3 i Age of Ioocu Ium /<xC Test Container;250 ml Erlenmeyer flasks with foam plugs % Sample-to-Volume Ratio: ,XC> to ~bQ Initial Cell Loading; 10000 cells/wl Initial pH of Synthetic Algal Medium; 7.St 0.1 Test Conditions: Temperature - 23> 2C (70-77F) Fluorescent 11turn!nation H00 f t . c . t 10 % Free Gas Exchange - Continuous p la tfo rm - shaking a t 1 0 0 * 1 0 rpm. Date S tarted 6 ~ / ~ HI Date Completed Investigator tA .T - S ALGAL CELL DRY W E IG H T . MEAN VALUE In m g /l ( 0 TREATMENT EXPOSURE: U-DAY CONTACT PERIOD CONTROL___ 'l o ___ iLlft___ I-- 6 .^ . ... U ..... )oaO USP il SG RECOVERY: I0 -0 A Y SUBCULTURE PERIOD Hlf f il d it t i I iffffftc TREATMENT CONTROL 'IS O *-170 f,C X'oo \( f O NO EFFECTS/STIMULATQRV/l NHIBITORY Ai ftlS T A T IC /A I filCIQAI aHMBaaaaMmaMHH EXPOSURE U-DAY CONTACT PERIt I RECOVERY 10-PAY SiiBCllt TIIRF PFRi ttI :____ <4Q0tJQQQ__________ _____ Z HO. O _______ _ ^ Z P f . & o o _______ _______ i Z a , too Id 600 6 o OOQ W .o a o _ LXQ M J L / </<?; poo fcsdL u) LGfil"GBWTH O) Based on mean values o f each t r i p l i c a t e (2) Method o f c a lc u la tio n s ; ALGi t i l t t l i sets of fla s k s . R iC lPAI BEST COPY AVAILABLE 001694 ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA:BT) in RECOVERY STAGE DATA S U M M A R V jE O T P f ' l P V A X / A I I A R I E ALGAL CELL COUNT, C e l l s / m l (MEAN ^ U r Y A V A IL A d LC Fe TREATMENT I N I T I A L ALGAL CELL LOADING CONTROL 1 iooo 2 ir r o 3 Hooo F CoOO M 00 k 5 REF. TOX. F IN A L ALGAL CELL COUNT (2 ) 'i.H o .o o o l S~, o o o / ^ 0,000 -- . (1). Based on 0,5m1/50ml s u b c u lt u r e mede fro m *+-d ay e x p o s u r e c u l t u r e s .