Document LpoepznzpzrdEJXZ42NraGR77

TOXICITY TO AQUATIC PLANTS TEST SUBSTANCE_________________________________________________ Identity: N-ethylperfluorooctane sulfonamidoethanol; may also be referred to as N-EtFOSE Alcohol or FM-3422. (1-Octanesulfonamide, N-ethyl1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-N-(2-hydroxyethyl)-, CAS #1691-99-2) Remarks: Material is an off-white, waxy solid of uncharacterized purity: The limited water solubility of this material prohibited a definitive determination of the aquatic toxicity. 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 bioassay GLP: No Year study performed: 1981 Species/Strain/Supplier: Freshwater green algae, Selenastrum capricomutum. Obtained from USEPA - ERL, Corv., Oregon (July 14,1981). Analytical monitoring: Algal cell counts (cells/ml) and temperature. Exposure period: 4, 7,10, and 14 days Statistical Methods: EC50values and 95% confidence limits were calculated utilizing a linear regression model. Test Condition Remarks: Inoculum: Algae from a 7-day-old stock culture were used as inoculum to give a starting optimum inoculum level of green algae 1x104 cells/ml. The use of a 7 to 10-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 hemocytometer (338,000 cells/ml). Algal Nutrient Medium: Sterile synthetic algal nutrient medium. 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 7.50.1 prior to use in assays. Culture Flasks: Sterile culture flasks consisted of 250 ml Erlenmeyers containing 50 ml synthetic algal nutrient medium. Full-Scale Test (Definitive): 006420 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 exposure periods: 4, 7,10, and 14 days. The following N-EtFOSE Alcohol, Lot 716, logarithmic concentrations: 180, 320, 560,1000, and 1800 mg/L were used to initiate the exposure study (individual weights). Three flasks containing 100% fresh algal nutrient medium plus algal cells comprised the nontreated controls. 0.5 ml Ethanol /1 liter algal nutrient medium was used as a carrier solvent. 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: 232C (70-77F) Fluorescent illum ination: 400 ft. candles 10% Free gas exchange: Continuous Platform Shaking, 100 10 rpm. RESULTS_________________________________________________________ Testing ended with 4, 7,10, and 14-day calculated EC50 values greater than the solubility limit of the test material. EC50 calculated values were greater than 1800 mg/L (based on reduction in cell count). CONCLUSIONS__________________________________ :__________________ Calculated EC50 values indicate this material has insignificant toxicity to algae. DATA QUALITY____________________________________________________ R eliability: Klimisch ranking = 3. This study lacks information on purity of the test substance and actual measurements of the amount of test substance in solution. Testing was conducted at approximately 5 orders of magnitude above the solubility limit of the test substance. REFERENCES_____________________________________________________ 3M Technical Report Summary, Multi-Phase Algal Assay Test Method, New Methods Development - N-EtFOSE Alcohol, Lot 716, Report Number 008, Project Number 9970030000, M. T. Elnabarawy, December 30,1981. OTHER 00G421 Submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 Last changed: 5/18/00 006422 Form 6747-11-c TECHNICAL REPORT SUM M ARY Data 12/30/81 -T O : T E C H N I C A L C O M M U N IC A T IO N S C E N T E R 201-2C N (Im p o r ta n t -- I f re p o rt Is p r in te d on b o th sides o f paper, send tw o copies to T C C .) See " G uidelines fo r C o m pletio n o f Technical R e p o rt S u m m ary"availab le from In fo rm atio n Liaison o r TCC (3-5545). D ivision E n v i r o n m e n t a l La b o r a t o r y (EE & PC) P roject New Methods Development - FM-3422, R epo rt T itle Lot 716 Dept. Number 0535 Project Num ber 9970030000 Report Slum ber Multi-Phase Algal Assay Test Method To 008 D. Bacon/R. Bohon/E. A u th o r(s ) M. T. El n a b a r a w y N otebook Reference Reiner/A. Welter Em ployee Num berlsj 46981 N o. of Pages In clud ing Coversheet SECURITY KEYWORDS: Include Lab Code (Company Confidential) Closed (Special Authorization) 3M CHEMICAL REGISTRY ^ New Chemicals Reported Yes 5 Q No CURRENT OBJECTIVE: 1. To e v a l u a t e the algal g r o w t h r e s p o n s e of f r e s h w a t e r green algae "Selenastrum capricornutum" over several generations as they may be affected by exposure to the fluorochemical FM-3422, Lot 716. 2. To d e v e l o p an a lgal test p r o t o c o l based on v a r i a b l e exposure or r e c o v e r y p e r i o d s to the test compound. EE & PC Div. (EnV L a b ) REPORT ABSTRACT: This abstract information is distributed by the Technical Communications Center to alert 3M'ers to Company R&O. It is Company confidential material. In these trip l i c a t e d f o u r - p h a s e assays, the initial effects on organisms following varied exposures to FM-3422 were assessed over several generations. Test results gave an i n d i c a t i o n of the p o s s i b l e e f f e c t s on alga,l v p o p u l a t i o n s . Algal b i o m a s s was a s s e s s e d q u a n t i t a t i v e l y in terms of algal cell-count (No./ml), and the median growth . response EC50 (mg/1 ) was calculated information Ljpi f i tInitials: 006423 FM-3422/MTE Page Two December 30, 1981 CONCLUSIONS 1) M a t e r i a l s p o s s e s s i n g a n E C 50 g r e a t e r t h a n 1,000 m g / 1 a r e considered to be insignificantly phytotoxic to algae and aquatic plant species. 2) R e s u l t s i n d i c a t e d that e x t e n d e d e x p o s u r e s (e.g., >7 days) induced no greater reduction in algal cell counts. SUMMARY A four-phase 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, FM-3422. This multiphase algal assay evaluated algal growth responses during four c o n s e c u t i v e e x p o s u r e p e r i o d s : 4, 7, 10, and 14 days. A l g a l growth response was measured in terms of an increase in biomass as algal cell-count (No./ml). Cell-count results used for the c a l c u l a t i o n of E C s q 's (mg/1 ) w e r e a v e r a g e s of t r i p l i c a t e d s e t s of test culture flasks. In these triplicated multiphase algal assays, the calculated EC50 values (mg/1) for freshwater green algae "Selenastrum c a p r i c o r n u t u m " are p r e s e n t e d in T a b l e 1. T h e s e v a l u e s r e p r e s e n t the median growth response (EC50 m g / 1 ) following exposure to the test m a t e r i a l for 4, 7, 10, and 14 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 (FM-3422, Lot 716) is an off-white solid, with limited solubility 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). 006424 FM-3422/MTE Page Three December 30, 1981 Test Species: A bacteria-free culture of the freshwater planktonic green algae ( C h l o r o p h y c e a e ), S e l e n a s t r u m c a p r i c o r n u t u m of the o r d e r chlorococcales was obtained from USEPA - ERL, Corv., .Oregon (July 14, 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 1 0 -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 hemocytometer (338,000 cells/ml). Algal Nutrient Medium: Mineral (inorganic) standard nutrient medium for culturing and t e s t i n g algae was p r e p a r e d as o u t l i n e d in A t t a c h m e n t #1. 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+.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. A u t o c l a v e d f o a m plugs u s e d as f l a s k c l o s u r e s , p e r m i t t e d free gaseous exchange to occur. Full-Scale Test (Definitive): 4 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 s i m u l t a n e o u s e x p o s u r e tests: 4, 7, 10 and 14 days. The following FM-3422, Lot 716 logarithmic concentrations: 180, 320, 560, 1,000 and 1,800 mq/1 were used to initiate the exposure study. Three flasks containing 100% fresh algal nutrient medium plus algal cells comprised the nontreated controls. Procedure and steps for preparation of algal test flasks are o u t l i n e d in A t t a c h m e n t #3. 006425 FM-3422/MTE Page Four December 30, 198i 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: T e m p e r a t u r e - 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 algal cell counts (cells/ml). Procedures for algal b i o m a s s m e a s u r e m e n t s are b r i e f l y d e s c r i b e d in A t t a c h m e n t #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 the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FM-3422, Lot 716, f o l l o w i n g e x p o s u r e p e r i o d s of 4, 7, 10, and 14 d a y s are p r e s e n t e d in Table 1. E C 50 c a l c u l a t e d v a l u e s w e r e greater than 1,800 mg/1 , indicating insignificant toxic effects to algae. The data reported herein are based on studies developed and p e r f o r m e d by M. T. E l n a b a r a w y and R. R. R o b i d e a u . A c c o m p a n y i n g data sheets comprise original data. It should be noted that all the reported EC values w e r e calculated on the basis of the initial concen t r a t i on s of FM-3422, Lot 716, in test solutions at the beginning of the bioassay. 006426 FM-3422/MTE Page Five December 30, 1981 REFERENCES (1) Miller, W. E . , J. C. Greene, and T. Shiroyama. 1978. T h e Selenastrum Capricornutum Printz Algal Assay Bottle Test: Experimental design, application, and data interpretation. U.S. Environmental Protection Agency, Corvallis, Oregon. E P A - 6 0 0 / 9 - 7 8 - 0 18. 125 p. (2) A S T M - E - 3 5 . 23. 1981. Pro p o s e d S t a n d a r d Practice F o r Conducting Toxicity Tests with Freshwater and Saltwater Algae. D r a f t No. 2. (3) O E C D G u i d e l i n e s f o r T e s t i n g of C h e m i c a l s (1981) S e c t i o n 2, Effects on Biotic Systems, Test 201 "Algae, Growth Inhibition T e s t , " Adopted M a y 12, 1981. (4) Payne, A. G. and R. H. H a l l , 1979. A M e t h o d for M e a s u r i n g Algal Toxicity and Its Application to the Safety Assessment of New Chemicals. ASTM S T P #667, p. 171-180. < 006427 TABLE 1 Based Algal Growth Response to FM-3422, Lot 715 EC5 0 's (mg/lld) on Reducation In Cell C o u n t v 2 ) Exposure (Contact) Days 4 >1,800 7 >1,800 10 >1-, 8 00 14 >1,800 (1) M e t h o d of E C 50 c a l c u l a t i o n : 3M SIXCUR, a T R A C s y s t e m for regression models of experimental data. (2) A l g a l c e l l - c o u n t (No./ml) m e a s u r e d in t r i p l i c a t e d sets of culture flasks. 006428 ATTACHMENT 1 NUTR ENT MEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) (Prepared separately with deionized w a te r.) 1) 2 5 .5 0 0 gm NaNO^ in 1l i t e r Dl w a t e r . 2) 1.044 gm K2HPO^ in 1liter Dl water. 3) 12.159 gm MgCl2* 6H2O in 1 liter Dl water. 4) 14.700 gm MgSO^ 7H20 in 1 liter Dl water. 5) 4.410 gm CaCl2* 2H20 in 1 liter Dl water. 6) 15.000 gm NaHCOj in liter Dl water. B. MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) (Combined in a s in g le o n e - l i t e r s to c k m ix .) 1) 185.5 mg Hj BOj 2) 415.6 mg MnCl2*4H20 3) 3.27 mg ZnCl2 4) 1.43 mg CoC12*6h 20 5) 0.01 mg CuCl2 6) 7.26 mg Na^oOi**2H20 7) 96 mg FeC lj 8) 300 mg Na2EDTA*2H20 -;y P.R.E.P.A.R.A.TION1 OF 1SY'NTHETIC A.L..G.A.L. .NUT11RI1 ENT ME"iDIU1M - 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 urn membrane; - adjust pH to 7*5 0.1; - store in the dark at h.oC. 006429 ATTACHMENT ? PREPARATION OF ALGAL CULTURE FLASKS All flasks used In maintaining and testing algae were made of borosilicate glass (K1MAX). flasks were brushed inside with a stiff bristle brush; - washed with non-phosphate detergent (MICRO) and rinsed 3 times w ith tap water; - rinsed with a 10% HC1 solution; - rinsed 3 times with tap water and 3 times with 0.1. water; Q - dried in an oven at 70 C for 2 hours (placed inverted); - and autoclaved with foam plugs inserted at 121oC for 20 minutes. i 006430 ATTACHMENT 3 algal flasks CONTROL PREPARATION OF ALGAL TEST FLASKS 1 IN CHRONOLOGICAL ORDER ) alg al MEDIUM (ml) * * Vr xl ' l Ki t3 e ZT |k A j{ W O Tr > A ^ a *1| |1W0" CONTENTS + TEST MATERIAL ALGAL INOCULUM (mi) or (ml) 4 8 ,5 \.S s 33 1 IS O Ma$ 2 *>ZO VfijS. 3 v 5 ( i O Y^Pl ^ lO O O 5 IS c O iK 48. S 4 8 .S 48. S 4 8 .5 485 +4 + .Ito + Z3 + 50 40 + liS 1 .5 \-S + l.s + I.S S3 a - SB 6 ++- TOTAL (ml) 50 . 50 50 50 50 50 50 7+ - 50 1) Determine i n i t i a l a l g a l c e l l count in the stock c u l t u r e . 2) Adjust pH of algal nutrient medium to 7*5 + 0.1 ml. . 3) Make up stock solutions with algal nutrient medium as desired. '1 A) Control flasks,preparations: 1) Add inoculum (volume predetermined); initial ecu' loading of 1 x 10k 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; 006431 5) Swirl flasks, and place autoclaved foam plugs. l u O W t O U A W j WE.lftWTi> P L A C E D JMReCVL-V VM T O . * < 0 . 5 wJi E ^ V I A M C W \ A L 6 A U WUTR\0sTC MetXuid^j A> CAKR16JR. 5 D L V E U T . - WeMAdW"TOCcxXMTb 35,54;3l<> x*41, 2j5^ 55.6 558,000 c<=u.5/ mJQ. - A u h A k j 1W O C U U U M . V o l >u K E PJR Cl X.to4 egUfJuLtV S Q r t J ) - I H 8 ^ 1 , S * ^ 5 0 358.000 ATTACHMENT 3 ALGAL FLASKS CONTROL I 2 3 S lo O k PREPARATION OF ALGAL TEST FLASKS ( IN CHRONOLOGICAL OROER ) ____________________ __________________________bJwLl-- L u __________________________________________ CONTENTS alg al MEDIUM + TEST MATERIAL + ALGAL INOCULUM ae TOTAL (m l) (ml) or (mgjSAtttfl* (ml) (ml) 48.5 U S ' ss 50 + + m . 50 48.5 + + Z> + 1. 5" * - 50 - 50 m 50 5 + - 50 6 + m 50 7 + + - 50 1) Oetermlne initial aigal cell count in the stock culture. 2) Adjust pH of algal nutrient medium to 75 + 0.1 ml. V- 3) Make up stock solutions with alga) nutrient medium as desired. A) Control flasks.preparations: It 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; *0 Add algal inoculum; 006432 5) Swir) flasks, and place autoclaved foam plugs. * IkicivtouAkj - 'PuAcefc Cocluts iwrro Ouksus^E. V k A S J C S . >4 1 ,2 .5 > 3 3 *8 3 38 p o o - AunAk luocmuuwl Vouiw it PERVuASic. 336,000 1 n Vr ATTACHMENT H ALGAL BIOMASS MONITORING A. GRAVIMETRIC CELL DRY WEIGHT (1) The f i l t e r recommended is M i l l i p o r e type BD w ith an 0.6 micrometer pore size . The method is as follows: - Dry f i l t e r s f o r two hours a t 70C in an oven; - Cool filters in a desiccator containing desiccant, for at le a s t two hours b efore w eighing; - 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. SPECTROPHOTOMETR1C DETERMINATION OF CHLOROPHYLL a (2) Basic instrument used: Stectrophotometer; Bausch s Lomb, Spectronic 20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEM0CYT0METER COUNTING CHAMBER AND OPTICAL MICROMETER (Used to measure diameter of algal MCV). (3) .. (1) ; Miller, W. E., J. C. Greene, and T. Shiroyama. 1978. Selenastrum caprIcornutum 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.), 14th edition, pp. 1030-1031. (3) . B r i t e - L i n e ( R ) , American O p tic a l C o rp o r a tio n , B u f f a l o , New York. 006433