Document OBjMwO09x1qd4rQqg0OJndNw

A R O rO S* TOXICITY TO AQUATIC PLANTS (e.g., Algae) TEST SUBSTANCE Identity: Perfluorooctanoic acid, ammonium salt; may also be referred to as PFOA ammonium salt, Ammonium perfluorooctanoate, PFO, FC116, FC-126, FC-169, orFC-143. (Octanoic acid, pentadecafluoro, ammonium salt, CAS # 3825-26-1 ) Remarks: The 3M production lot number was 37. The test sample is FC143. It's purity was not sufficiently characterized, though current information indicates it is a mixture of 96.5 -100% test substance and 0 - 3.5% C6, C 7 , and Cg perfluoro analogue compounds. METHOD: Method Followed: Modified (modeled) after those described by USEPA - 600/9-78-018; ASTM-E-35.23 Draft No. 2; OECD; A.G. Payne. Type (test type): Acute Static GLP: No Year study performed: 1981 Species: Selenastrum capricornutum. Source: USEPA - ERL, Corv., Oregon (July 14,1981). Analytical monitoring: Algal cell counts (cells/ml), cell dry weights, and temperature. Exposure period: 4, 7, 10, and 14 days Statistical Methods: EC50 values and 95% confidence limits were calculated utilizing 3M Sixcur, a linear regression model. Test orgamisms laboratory culture: Algae from a 7-day-old stock culture Algal Nutrient Medium: Sterile synthetic algal nutrient medium. This nutrient medium served as the diluent for all algal operations. The pH of this synthetic algal medium was adjusted to 7.50.1 prior to use in assays. Stock and Test Solution Preparation: A primary stock solution was prepared in algal medium at a concentration of 5 g/L. After mixing, the primary stock solution was diluted with algal medium to prepare the six test concentraions. Exposure vessels: Sterile 250 mL Erlenmeyer flasks containing 50 mL of test solution and stoppered with autoclaved foam plugs. Agitation: Continuous platform shaking at 100 + 10 rpm Number of replicates: three Initial algal cell loading: 1.0 x 104 cells/mL Number of concentrations: six plus negative control Nominal concentrations: Bk control, 100,180, 320, 560,1000, and 1800 mg/L Test conditions: Temperature: 232C (70-77F) Fluorescent illumination: 400 ft. candles 10% C03791 RESULTS Algal Growth Response EC50 values Exposure (Contact) Cell-Dry W eight(1) C e ll-C o u n t(2) Days mg/L (95% C.l.) mg/L (95% C.l.) 4 149 (57-340) 49 (28-75) 7 70 (34-118) 30 (21-40) 10 49(15-96) 27 (8-50) 14 73 (25-147) 43(14-81) (1) Growth response parameter; cell-dry weight (mg/L) , measured in triplicated sets of culture flasks. (2) Growth response parameter; cell-count (number cells / mL), measured in triplicated sets of culture flasks. Algal Growth Response EC10 and EC90 values based on Cell-Count (number of cells / mL) Exposure (Contact) Days 4 7 10 14 EC10 mg/L (95% C.l.) 5.3 (3-7) 3.3 (2-4) 2.9 (1-5) 5 (2-8) EC90 mg/L (95% C.l.) 624 (C.l. not calculated) 283 (150-590) 386 (C.l. not calculated) 307 (C.l. not calculated) Element values based on nominal concentrations. CONCLUSIONS Ammonium perfluorooctanoate exhibits a 14-day EC50 (cell count) value of 43 mg/L with a 95% confidence interval of 14 to 81 mg/L. DATA QUALITY Reliability: Klimisch ranking = 2. This study meets the criteria for quality testing at the time it was conducted. However, the study lacks information on purity of the test substance and actual measurements of the amount of test substance in solution. REFERENCES 3M Technical Report Summary, Multi-Phase Exposure / Recovery Algal Assay Test Method, Report Number 006, Project Number 9970030000, M. T. Elnabarawy, October 16, 1981. 003792 OTHER Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/24/00 t C03793 i n . TM ;--J*- , * TECHNICAL REPORT S U M M A R Y TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN (im p o rta n t -- i f rep o rt sp rin ted on both sides o f paper, send tw o copies to TCC J Division Environmental Laboratory - EE & PC - 2-3E Proiaet New Methods Development - FC-143, Lot 37 Raport Titla Multi-Phase Exposure/Recovery Algal Assay Test Method To D. Bacon/R. Bohon/^HSadHBW/A. Welter Author(t) i-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- M. T. Elnabarawy N o t*book Reference O at* 11/16/81 Dept. Numtwr 0535 Proiaet Number 9970030000 Report Number 006 Employee Number(s) 46981 No. of Paga Including Covershaet* SECURITY ^ 60 Open KEYWORDS: (Select terms from 3M Thesaurus. Suggest other applicable terms.) EE & PC-Div. (Env. Lab) Closed (Special Authorization) 3M CHEMICAL REGISTRY Naw Chemicals Reportad Ye. j Q No CURRENT OBJECTIVE: 1. To evaluate the algal growth response of freshwater green algae "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical FC-143, lot 37. 2. To develop an algal test protocol based on variable exposure or recovery periods to the test compound. REPORT ABSTRACT: (200-250 words) This abstract information is distributed by the Technical Communications Center l alert 3M'ers to Company R&D. In these triplicated four-phase exposure/recovery assays, the initial effects on organisms following varied exposures to FC-143 were assessed over several generations. Test results gave an indication of the possible effects on algal populations. Algal biomass was assessed quantitatively in terms of cell-dry weight (mg/1 ) and cell-count (No./ml). The following biomass indicators were measured and calculated for: A. Verification of inhibitory effects 1. Median growth response EC50 (mg/1) from varied exposures. 2 . ECjo and EC90 (mg/1 ) for same exposure periods. 3. No-effect levels B . Verification of algicidal effects 1. Resumption or absence of logarithmic growth in subcultures during varied periods of recovery. Informatici Initial: C03794 .FC-143/MTE Page Two November 16, 1981 CONCLUSIONS 1) Materials possessing an EC50 in the range of 10-100 mg/1 are considered to be slightly phytotoxic to algae and aquatic plant species. 2) The algal growth response in all subcultures following varied periods of recovery indicated minimal algicidal effects (death of algal cells) at concentrations tested. The algal cells recovered and resumed logarithmic growth when resuspended in fresh nutrient medium in the absence of the test material. The median growth response (EC50 ) was therefore interpreted as basically indicative of an inhibitory effect as opposed to an algicidal effect. In the former case, photosynthesis, cell-growth, and cell-division, are reduced whereas an algicidal effect causes direct cell-destruction and cell-death. 3) Exposure/recovery results indicated that extended exposures (e.g., >7 days) induced greater inhibition and consequently exhibited slower recovery rates in the subcultures. SUMMARY A four-phase exposure/recovery algal assay was developed to evaluate algal growth response of the freshwater unicellular green algae "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical, PC-143. This multiphase algal assay evaluated algal growth responses during four consecutive exposure/recovery combinations: 4/10, 7/7, 10/4, and 14 days. As is clearly indicated, the duration of exposure periods increased, while respective recovery periods decreased. The combined duration of each exposure/recovery phase was fixed at 14 days. Algal growth response was measured in terms of an increase in biomass as cell-dry weight (mg/1) and cell-count (No./ml). Dry-weight and cell-count results used for the calculation of ECs q 's (mg/1 ) were averages of triplicated sets of test culture flasks. In these triplicated multiphase multigeneration algal assays, the calculated EC50 values (mg/1 ) for freshwater green algae "Selenastrum capricornutum" are presented in Table 1. These values represent the median growth response (EC50 mg/1) following exposure to the test material for 4, 7, 10, and 14 days. The values of EC10 and EC90 (mg/1) for each of the exposure periods listed earlier were calculated in terms of cell-count and are presented in Table 2. No test concentrations were defined as a no-effect level. C03795 FC-143/MTE Page Three November 16, 1981 To verify algicidal effects, and to assess possible resumption of growth in the absence of the test material subcultures were established from each triplicated set of test culture flasks combined at the end of each exposure period. Algal recovery response was evaluated following respective recovery periods of 10, 7, and 4 days. 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 algal growth (aquatic primary producers), aquatic plants, and phytoplankton. Toxic effects are tested on growing algal cultures that undergo cell-division during the test. These growth responses may be: a) stimulatory, b) inhibitory and/or algicidal. MATERIAL AND EXPERIMENTAL DESIGN The test substance (FC-143, lot 37) is a fine white powder, soluble in water at ambient room temperature. The bioassays performed on this material evaluated its potential algal toxicity and the data generated form the basis of this report. The test protocol utilized for this study was modified after those described by USEPA - 600/9-78-018; ASTM-E-35.23 Draft No. 2; OECD; A. G. Payne. ( 1 ) , ( 2 ) , ( 3 ) , ( 4 ) . Test Species: A bacteria-free culture of the freshwater planktonic green algae (Chlorophyceae), Selenastrum capricornutum of the order chlorococcales was obtained from USEPA - ERL, Corv., Oregon (February 18, 1981). The test algae are non-motile unicellular cells, having the appearance of a new moon. The algal culture was stored in the dark at 4C. 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 to i0-day-old stock culture insured the presence of a sufficient number of viable algal cells in the exponential growth phase. The initial algal cell count in the stock culture was determined using a hemocytoraeter (277,000 cells/ml). G03796 FC-143/MTE Page Four November 16, 1981 Algal Nutrient Medium; Mineral (inorganic) standard nutrient medium for culturing and testing algae was prepared as outlined in Attachment #1. This nutrient medium provided all mineral 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+.1 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 used as flask closures, permitted free gaseous exchange to occur. Range-Finding Test (Exploratory): The exploratory test consisted of determining the 4-day algal biomass in triplicated flasks containing standard nutrient medium plus the test material at concentrations covering several orders of magnitude: 100, 250, 500, 750, 1,000, and 1500 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 assay consisted of four simultaneous exposure/recovery tests (4/10, 7/7, 10/4, and 14/0 days. The purpose of the exposure (contact) stage was to verify inhibitory effects. The recovery (subculture) stage evaluated the viability of growth-inhibited algae and verified algicidal effects. The following FC-143 logarithmic concentrations: 100, 180, 320, 560, 1000, and 1800 mg/1 were used to initiate the exposure stage. This dose range of dilutions was selected to bracket the 4-day EC50 values predicted from preliminary exploratory testing. Three flasks containing 100% fresh algal nutrient medium plus algal cells comprised the nontreated controls. A fresh stock solution of the test substance was prepared in the algal nutrient medium immediately prior to testing. The initial pH of this stock solution was 7.4. Procedure and steps for preparation of algal test flasks are outlined in Attachment #3. Each recovery stage was initiated by subculturing (0.5/50 ml) in the absence of the test material. Algal cells from each triplicated set of culture flasks were combined and then resuspended into fresh nutrient medium. Subculture methods are briefly described in Attachment #4. 003797 FC-143/MTE Page Five November 16, 1981 Test Conditions? All algal operations were carried out under aseptic conditions, in order to avoid contamination with bacteria and other algae. Algal cultures were maintained 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 appraised quantitatively by using either algal cell-dry weight (mg/1 ) and/or by cell counts (cells/ml). Both growth measurements were made with algal cultures after each of the exposure and recovery stages. Both procedures for algal biomass measurements are briefly described in Attachment #5. EC50 values and 95% confidence limits were calculated utilizing 3M SIXCUR, a TRAC System for regression models of experimentar 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 the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FC--143, lot 37, following exposure periods of 4, 7, 10, and 14 days are presented in Table 1. The values of EC10 and EC90 for each of the exposure periods listed above were calculated in terms of cell-count and are presented in Table 2. Recovery data and resumption of logarithmic growth in subcultures are summarized in Table 3. To illustrate the action of test material and to give an overview of what has happened in the exposure experiment, toxicity curves are shown in Figures 1 and 2. Utilizing the EC50 values, toxicity curves were constructed against time on semilog plots. The data reported herein are based on studies developed and performed by M. T. Elnabarawy and R. R. Robideau. Accompanying data sheets comprise original data. 003798 FC-- 143/MTE Page Six November 16, 1981 It should be noted that all the reported EC values were calculated on the basis of the initial concentrations of FC-143, lot 37, in test solutions at the beginning of the bioassay. REFERENCES (1) Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. The Selenastrum Capricornuturn Prints Algal Assay Bottle Test: xperimental design, application, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. EPA-600/9-78-018. 125 p. (2) ASTM-E-35.23. 1981. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 2. (3) OECD Guidelines for Testing of Chemicals (1981) Section 2, Effects on Biotic Systems, Test 201 "Algae, Growth Inhibition Test," Adopted May 12, 1981. (4) Payne, A. G. and R. H. Hall, 1979. A Method for Measuring Algal Toxicity and Its Application to the Safety Assessment of New Chemicals. ASTM STP #667, p. 171-180. 003799 TABLE 1 Algal Growth Response to FC-143, Lot 37 EC50's (mg/1) ) Exposure (Contact) Days Cell-Dry Weight(2) Cell-Count(3 ) 4 149(57-341) 49(28-75) 7 70(34-118) 30(21-40) 10 49(15-96) 27(8-50) 14 73(25-147) 43(14-81) (1) Method of EC50 calculation: 3M SIXCUR, a TRAC system for regression models of experimental data. (2) Growth response parameter; cell-dry weight (mg/1), measured in triplicated sets of culture flasks. (3) Growth response parameter; cell-count (no. cells/ml), measured in triplicated sets of culture flasks. (4) 95% Confidence Limits. C'k$ & Q Q Exposure (Contact) Pay 4 7 10 14 TABLE 2 Algal Growth Response to FC-143, Lot 37 ECi q 's and ECgg's (mg/1) Based on Cell-Count (no. celis/ml) ffglQ, 5.3(3-7) 3.3(2-4) 2.9(1-5) 5(2-8) aa 624(Mo Limits) 283(150-590) 386(No Limits) 307(60-No Limits) / C03801 TABLE 3 RecsjzatY Stage Data Summary Algal CellfCounty\ Cells/ml (MEAN VALUES)(D Initial Algal Cell Loading(2 >Final Algal Cell-Count(3) Treatment T H a y s 7 Days 16 Days 16 Daya i Days 4 Days Control 2,017 2,430 2,453 380,000 256,000 136,000 1 100 mg/1 643 580 587 372,000 226,000 48,000 2 180 mg/1 530 290 280 348,000 204,000 2 0 ,000 3 320 mg/1 383 273 380 300,000 214,000 2 0,000 4 560 mg/1 237 137 290 236,000 200,000 18,000 5 1,000 mg/1 57 60 103 310,000 174,000 18,000 6 1,800 mg/1 73 73 73 182,000 54,000 18,000 (1) Based on 0.$ ral/50 ml subcultures established from each triplicated sets of culture flasks combined. (2) Measured at the end of each exposure period. (3) Measured at the end of each recovery period. C03802 J4U . 3 4 ' L 4 . i U U l t - ^ L s L N o w ^ r i . ^iVQ "3'<11^04^3 . ATTACHMENT 1 NUTRIENT MEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) (Prepared separately with deionized water.) 1) 25.500 gm NaNO^ in 1liter Dl water. 2) 1.044 gm K^HPO^ In 1liter DI water. 3) 12.159 gm MgCl^* 6H2O in 1 liter Dl water. 4) 14.700 gm MgSO^*7H2O in 1 liter Dl water. 5) 4.410 gm C a C ^ 2H2O in 1 liter Dl water. 6) 15.000 gm NaHCO^ In liter Dl water. B. MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) (Combined in a single one-liter stock mix.) 1) 185.5 mg H3BO3 2) 415.6 mg MnCl2-4H2 0 3) 3.27 mg ZnCl2 4) 1.43 mg CoCl2* 6H20 5) 0.01 mg CuCl2 6) 7*26 mg Na2Mo0i+* 2H2O 7) 96 mg F e C ^ 8) 300 mg Na2EDTA2H20 * PREPARATION OF SYNTHETIC ALGAL NUTRIENT MEDIUM - Add one ml of each macronutrient stock solution plus one ml micronutrients stock mix per 1 liter of deionized water; - sonicate and then filter through 0.22 ion membrane; - adjust p.H to 7.5 0 . 1 ; - store in the dark at 4C. 03805 ATTACHMENT 2 PREPARATION OF ALGAL CULTURE FLASKS All flasks used In maintaining and testing algae were made of boros Idea te glass (KIMAX). - flasks were brushed Inside with a stiff bristle brush; - washed with non-pho*phate detergent (MICRO) and rinsed 3 times with tap water; - rinsed with a 10% HC1 solution; rinsed 3 times with tap water and 3 times with 0.1 , water; *' dried in an oven at 70C for 2 hours (placed inverted); o - and autoclaved with foam plugs Inserted at 121 C for 20 minutes. C03SQQ ATTACHMENT } WEWUATIMI Of ALCAL TEST TLACKS ALGAL FLASKS CONTROL ________________ __________ ___________ c o n t e n t s ____________________________ ALGAL MEDIUM TEST MATERIAL. ALGAL INOCULUM m TOTAL (n (ml) or (mgflfc____________ (ml)________ (ml) M-8.& K Io n e . 4 i.8 50 1tQOvw$. (SwajSOtiQ 2 ieo(q) 4T.Z- \ .0 vkS + 1 * 8 vhJP 1.8 * i.8 m 50 m 50 45.0 ..VKi 1-8 m 50 k SUC iZ>) H Z.Ip 1.8 - 50 5 IOOOvm^ (.50) 38,^ + 1O m i 1-8 - 50 6 \Qoo*$. (qo) 2 0 . & \& Mlfi + 1.8 . 50 7 + - 50 1) Determine Initial algal cell count in the stock culture. .2) Adjust pH of algal nutrient medium to 7*5 + 0 . 1 m l . 3) Hake up stock solutions with algal nutrient med ium as desired. A) Control flasks preparations: l* 1) Add inoculum (volume predetermined!; Initial cell loading of 1 x 10 cells/ml; 2) Add algal nutrient medium to bring volume to 50ml; swirl flasks, and place autoclaved foam plugs. B) Test flasks: 1) Add algal nutrient medium; 2) Add test material (dissolved In algal medium If required); 3) Swirl flasks; k ) Add algal inoculum; 5) Swirl flasks, and place autoclaved foam plugs. soyjumou i 5 ^ ELa C H \ =5 t5,ooo*2/\ooowt&). 03507 ATTACHMENT k SUBCULTURE (M0E>T5HOmD l/I5N0 ALGAL ASSAYS OBJECTIVE: To determine algal recovery response In absence of test substance. PROCEOURE: - Transfer 0.5ml from each of the triplicate culture flasks into Corning15m! sterile centrifuge tubes. .Centrifuge at 10,000 rpm for 5 minutes. - Decant supernatant liquid and save the precipitated algal cells. - Using the same centrifuge tube, resuspend algal cells in algal nutrient medium to a fixed volume; 15ml. * Mix thoroughly and decant the top !0m! of algal cejl suspension, and transfer the remaining volume (5ml) of algal cell suspension into a clean 250m! Erlenmeyer culture flask. r Using the same centrifuge tubes, transfer *5m! of fresh alga! nutrient medium into the above Erlenmeyer culture flask, to bring total volume to 50 ml. C03S08 ATTACHMENT 5 ALGAL BIOMASS MONITORING A. GRAVIMETRIC CELL DRY WEIGHT (1) The filter recommended is Millipore type BO with an 0.6 micrometer pore size. The method is as follows: - Dry filters for two hours at 70C in an oven; Cool filters in a desiccator containing desiccant, for at least two hours before weighing; - Filter a suitable measured aliquot of the culture under a vacuum or pressure not to exceed 8psi; - Rinse the filter funnel with D.l. water; - Dry the filter to constant weight at 70C, cool in a desiccator for two hours and weigh. Basic instrument used: Analytical Balance; Mettler ME 30. B. SPECTR0PH0T0METRIC DETERMINATION OF CHLOROPHYLL a (2) Basic instrument used; Stectrophotometer; Bausch & Lomb, Spectronic 20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEM0CYT0HETER COUNTING CHAMBER AND OPTICAL MICROMETER (Used to measure diameter ol* algal MCV) (31 (1) . Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. Selcnastrum caprlcornutum Printz Algal Assay Bottle Test: Experimental Design, Application, and Data Interpretation Protocol. Ecol. Res. Series EPA-600/9-78-018. Corvallis, Oregon, pp. 27. (2) . APHA-AWWA-WPCF (1975). "Standard Methods for the Examination of Water and Wastewater," (M. Franson, manag, ed.), Ufth edition, pp. 1030-1031. (3) . Brite-Line (R), American Optical Corporation, Buffalo, New York. 03809 * : $/' ..\ BWIRONHENTAL LABORATORY : ';^'<>y^- ALGAL ASSAY BOTTLE TEST (AA:BT) v p ^ / ^ - v - ' :v.; ALGAL GROWTH >E$PONSE to >0- - EC 50 W 2 / EXPOSUiIE (1) ). vs CELL-DRY WEIGHT CELL COUNT (wg/D (no. cells/mt) \4 8 . - 1 Csu>.e-m) ( Z P i , i -- 73,l'l ; M.7 i 3 4 - . l - U 7 .qy_ (zo.u.-sq.io') J 4q 27.1' > i f 4 > 7 - % Y . l9u7.-+fi.i) 'yz* r A4.?. NO. DAYS 10 7 A RECOVERtY (2) CELL-DRY WEIGHT (mg/l) CELL COUNT (no. ceiis/nl) SEQUENTIAL COMBINATIONS OF EXPOSURE/RECOVERY PER I QOS (PAYS)t - k + 10 77 10 + 4 1A + 0 (1) Productivity is based on percent growth reduct ion values indicate inhibitory response. (2) Productivity is based on percent growth reduct ion;values indicate recovery response. C03S10