Document NG0kbzJML7EZm9d9YjZVDG1pQ

M &SL& - 0 7 lf-lh TO XIC ITY 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-203C and L-5440. (1-Octanesulfonic acid, 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 3M production lot number was not noted. The test sample is FC-203C and is referred to by the testing laboratory as "81-1/8". Current information indicates it is a mixture of 1.47% test substance, 60% water, 30% diethylene glycol butyl ether, 3% sodium octyl sulfate, 3% hydroxy foamer, 10% polyoxyethylene monooctylphenyl ether, 0.12% sodium lauryl sulfate, and 0.05% benzotriazol. - The following summary applies to the test sample as a mixture o f the test substance in water solution with incompletely characterized concentration o f impurities. Data may not accurately relate toxicity o f dre test sample with that of the fluorochemical component. METHOD:________________________________________________________ Method: U S E P A - 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. Analytical m onitoring: pH and temperature. Test Conditions: Algal Nutrient Medium: Nutrient medium per EPA - 600/9-78-018. This nutrient 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 test solution preparation: Stock solutions were prepared in the algal standard test medium, however, no further description was provided. C C J038 Exposure vessels: 250 ml Erlenmeyers containing 50 ml test solution. A gitation: 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 Chem istry: pH range (0-96 hours): Not noted. Test tem perature range (0-96 hours): 23 + 2C (incubator) RESULTS______________________________ ___ ____________________ Nom inal concentrations: Bk control, 280, 420, 65 0,1 ,000 , and 1,550 mg/L ~ Elem ent values: 96-hour EC50 Cell-Dry weight: 408 (156 - 995) mg/L 96-hour EC50 Algal Cell Count: 420 (197 - 868) mg/L Element values are based on nominal concentrations. R eversibility o f G row th 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. Rem arks: Testing was conducted on the mixture of the test substance as described in the test substance remarks field. The values reported apply to that mixture and not the fluorochemical components alone. C O N C L U S IO N S ___________________________________________________ The test substance exhibits a 96-hour EC50 cell count value of 420 (197 868 mg/L) and a 96-hour EC50 based on cell dry weight of 408 (156 - 995) 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__________________________ ________________________ O O I (K? 9 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. There are also some errors (presumed typographical errors) in the report. The Technical Report cover sheet indicates a "40 day exposure" and page 2 indicates the algal culture was stored in the dark "at 40C". R E F E R E N C E S ____________________________________________________ Test was conducted by the 3M Company, Environmental Laboratory, St. Paul, MN, Lab Request number 6811S, 1981. O T H E R ____________________________________ Last changed: 6/26/00 001 0'Q 11b/ / technical report summary Date 6/12/81 ~ TO: TECHNICAL. COMMUNICATIONS CENTER -- 201-2CN ' (Important --I f report is printed on both sides of paper, send two copies to TCC.) Division 5 apt. Num ber Environmental Engineering & Pollution Control (Envir. Laboratory] 0535 Project Project N um ber Commercial Chemicals Report Title 9970012600 Report Number Algal Assay: Bottle Test(AA:BT), Toxicity Testing FC-203C 050 to E. A. Reiner - 21-2W LR #6811 S Author)) M. iTi. Elnabarawy - 2-3E Notebook Reference Employee Num ber!*) 46981 No. of Pages Including Coversheet 5 SECURITY . ( 3 Open KEYWORDS: (Select terms fro m 3 M Thesaurus. Suggest other applicable terms.) Closed 3M CHEMICAL ^ R E G IS T R Y New Chemicals Reported Yes S No CURRENT OBJECTIVE: To determine the algal growth responses (EC5q , ) following a 40-day exposure period to varying concentrations of FC-2( followed by a 10-day recovery period. EE & PC Div. (Env. Lab) Algal Assay Bottle Test Toxicity R EP O R T A B S TR A C T: (20 0-250 words) This abstract information is distributed by the Technical Communications Cent alert 3 M 'ers to Company R&O. The algal acute 4-day EC5Q values obtained for freshwater green algae (Selenastruin Capricornutum) following exposure to FC-203C in triplicate experiments were 408 and-420 mg/1 These values represent the algal growth response as measuri by cell-dry weight and algal cell-count, respectively. No test material concentration was defined as the no-effec level. The algal biomass,monitoring measurements following a 10-di recovery period indicated insignificant algicidl effext a concentrations tested. Since the algal cells have recover' and resumed logarithmic growth when resuspended in fresh r medmium, the 4-day median growth response (EC5Q) is interpreted as an inhibitory effect. LABLEB E S T C O P Y A W \| FC-203C/MTE Page Two June 23, 1981 CONCLUSIONS In these triplicated 4-day EC50 algal assays, the calculated values for freshwater green algae (Selenastrum Capricornutum) were 408 and 420 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 system 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, o) inhibitory, d) algistatic, or algicidal. The test substance is a clear ambr 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 (Chlorophyceae-), Selenastrum Capricornutum of the order chlorococcales was obtained rom USEPA - RL, 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.. FC-203C/MTE Page Three June 23, 1981 Inoculum: 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 the presence of 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. JThis 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 orders 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-203C logarithmic concentration: 280, 420, 650, 1,000 and 1,550 mg/1 were used to initiate the 4-day contact period. This dose range of dilutions was selected to bracket 4-day EC50 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 FC-203C/MTE Page Four June 23, 1981 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: 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 ;. r v ;. -k- V - . .* "'V' -7:: 001 FC-203C/MTE Page Five June 23, 1981 .v Table 1 *I 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 408(156-995)* 420(197-868)* *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-203C 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-35.2i. 1980. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 1. Type Test ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA :BT) TOXICITY TEST DATA SHEET --iJ_De^__ Sijporuce. S on LR Cfl 0 / | Js M a te ria l Tested P C - C Lo t Physical D escription O e< xr A l g a l Spec ies .-Green A lg a e . Selen astrum c a p rIc o rn u tu m u r e fA lal Source C o r v a \ \ ; s } Qre<^o.<v Da t e Rec1d 7 - /g - J / Age o f In o c u lu m ~7 <l<xy<;_____ Test C o n ta in e r;25Q ml Erlenmever fla s k s w ith foam plugs % Sample-to-Volume R a tio : 1Q Vo *^$"0 I n i t i a l C e ll Loading; 10000 c e ils An I I n i t i a l pH o f S y n th e tic A lg a l Medium: 7 . S t 0.1 Test Conditions: Temperature - 2 3 > 2 C (70-77F) F lu o resc en t Illu m in a tio n - 4oO 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 (j - I - %i Date Completed 6 - i T - T t In v e s tig a to r IA .T . E \h < x k a r <M ALGAL CELL DRY WEIGHT. MEAN VALUE In mg/1 ( 0 TREATMENT CONTRO! ____q j l s l .-6l Q I. OOP JL EXPOSURE: **-DAY CONTACT PERIOD SslJsL 5. a.q J JU -2 0 RECOVERY: 10-DAY SUBCULTURE PER100 L Gdrtteitr CPT ili MO EFFECTS/STlM lItATftft V / l HHI ft I TORY Al ClSTATIC/A l CICIDAI TREATMENT 'ONTROL . -- Cl 2 o . 4 x o ........ GS~0 i OOO t.fS o Oo c o O T ..... . . ^ . o o o . . ______ ___ __ I 0.000 loo. ooo . . --------------I S i + o o o . n>o. o o o .. __ : 1 , H Q ooo I O.OO e s (1) (2) () .............. .... ALm m u tk Cl ClDAI Oesed on mean values of each t r i p l i c a t e sets o f f la s k s . Method of calculations: BEST CO PY AVAILABLE to.m ENVIRONMENTAL LABORATORY ALGAL ASSAY: BOTTLE TEST (AA:BT) RECOVERY STAGE DATA SUMMARY ALGAL CELL COUNT, C e lls /tn l (MEAN VALUES) / / >s y c. TREATMENT a OS -o IN ITIA L ALGAL CELL LOADING (U CONTROL 1 / Ooo 2 590 3' k 5 REF. TOX. 7. ooo lo o Zoo: ' * (2) FINAL ALGAL CELL COUNT ' l 7 Oy O O O / H o . >oo 0 0 ,0 0 0 -- - - ( 1 ) . Based on 0.5m l/50m l su b cu ltu re made from k -day exposure c u lt u r e s . ( 2 ) . 10-day recovery c u ltu r e s .