Document gaK1ZdrGGbZNRV216v8qdY6ja

A lU 76-o io G > - )3 T O X IC IT Y T O A Q U A T IC P L A N T S (E.G., A L G A E ) T E S T S U B S T A N C E __________________________________________________ Identity: Perfluorooctanesulfonate; m ay a lso be referred to a s P F O S or FC-95. (1-Octanesulfonicacid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, C A S # 2795-39-3) R e m a rk s field: The test substance is a white powder (3M Lot 583) of uncharacterized purity. T he following sum m ary is abbreviated due to the fact that this study h a s been superceded by a more recent test. M E T H O D ____________________________________________________________ M ethod: Test protocol utilized w as modified after those described by U S E P A - 600/9-78-018, 1978; A ST M -E -3 5 .2 3 ,1981; O E C D 201, 1979. ASTM STP #667 Type: Sem i-chronic GLP: No Y ear com pleted: 1981 S p e c ie s: Selenastrum capricomutum R E S U L T S ___________________________________________________________ ECso Values, mg/L (Cl) Exposure (contact) Cell-Dry W eight Days 4 115(18-65) 7 122 (41-366) 10 128(46-372) 14 146 (33-350) C e ll-C o u n t 82 (N o Conf. Limits) 99(19-398) 98 (16-431) 95 (12-455) R e m arks: The statistical program used w as questionable. D A T A Q U A L IT Y ____________________________________________________ Reliability: Klim isch ranking - 2. T his study satisfied criteria for quality testing at the time performed, but actual concentrations were not m easured. R esults were based on nominal concentrations. Additionally, sam ple purity w as not properly characterized. REFERENCES 001456 This study w as conducted by the 3M Com pany, Environm ental Laboratory, 1981. O T H E R ___________________________________________________ Subm itter: 3M Com pany, Environm ental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 L a st changed : 5/3/00 001457 P^6747uc TECHNICAL REPORT SUMMARY TO: TECHNICAL COMMUNICATIONS CENTER - 201-2CN Oat 12/16/81 (Important -- I f report is printed on both sides o f paper, send two copies to T C C JSe e "Guidelines for Completion o f Technical Report Sum m ary" available from Inform ation Liaison or T CC (3-5545). uivition Dept. N um ber Environmental Laboratory (EE & PC) Project 0535 ProjectN u m b e r ,, New Methods Development - FC-95, Lot 583 9970030000 oftn ap N u m b o r t Multi-Phase Exposure/Recovery Algal Assay Test Method__________ 007 A.8uPacnn/R- Bohon/E. M. T . Elnabarawv Notebook Reference Reiner/A. Welter____________________ fcmplovee N u m M i l 46981_________ No. of Pages In c lu d in g Coveratieet SE C U R IT Y >> KEYW ORDS: Includa tab Coda CT Open dotad (Company Confidantial) v (Spacial Authorization) 3M C H E M IC A L R E G IST R Y New Chamicali Reportad Yea 3 No C U R R E N T O B JE C T IV E : 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-95, Lot 583. \ 2. To develop an algal test protocol based on variable exposure or recovery periods to the test compound. EE & PC Div. (Env. Lab) R E P O R T A B S T R A C T : Thit abstract information it distributed hv tht Technical Communications Center to alert 3M 'ers to Company R& D. I In these triplicated four-phase exposure/recovery assays, the initial effects on organisms following varied exposures to FC-95 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. EC^o 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. Informational Initials:. 001458 FC-95/MTE Page Two December 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 most 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, pb'otosynthesis, cell-growth, and cell-division, are reduced whereas an algicidal effect causes direct cell-destruction and cell-death. 3) Results indicated that extended exposures (e.g., >7 days) induced no greater inhibition but slower recovery rates in the subcultures were noted. 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, FC-95. This multiphase algal assay evaluated algal growth responses during four consecutive exposure/recovery combinations!: 4 / 1 0 , 7/7, i o / 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. See test schematic: Phase Exposure (Day) Recovery (Day) Total N o . (Day) I4 II 7 III 10 IV 14 10 7 4 None 14 14 14 14 In these triplicated multiphase multiqeneration 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. 001459 FC-95/MTE Page Three December 16, 1981 The values of EC^o 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. 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-95, Lot 583) 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 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 (300,000 cells/ml). 001460 FC-95/MTE Page Four December 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. V* 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: 1, 5, 10, 50, 100, and 500 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, 1.0/4, 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-95, Lot 583 logarithmic concentrations: 26, 40, 61, 93, 145, and 225 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 7.3. Procedure and steps for preparation of algal test flasks ace 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. 001461 FC-95/MTE Page Five December 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 gaft, exchange - Continuous Platform Shaking 100 + 10 rpm. Algal Biomass Monitorings 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 experimental data. TEST RESULTS The results of biomass measurements of the green algae Selenastrum capricornutum used in these studies are selt-explanatory and are detailed in the attached data sheets. Calculated EC50 (mg/1) values indicating algal growth response to FC-95, Lot 583, following exposure periods of 4, 7, 10, and 14 days are presented in Table 1. The values of EC^o 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 H. T. Elnabarawy and R. R. Robideau. Accompanying data sheets comprise original data. 001462 FC-95/MTE Page Six December 16, 1981 It should be noted that all the reported EC values were calculated on the basis of the initial concentrations of FC-95, Lot 583, in test solutions at the beginning of the bioassay. 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-35.23.v 1981. Proposed Standard Practice For Conducting Toxicity Tests with Freshwater and Saltwater Algae. Draft No. 2. (3) OECD Guidelines for Testing of Chemicals (981) 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. 001463 TABLE 1 Algal Growth Response to FC-95, Lot 583 EC50's ( m g / l M 1 ) Exposure (Contact) Days Cell-Dry Weight Ce11-Count(3) 4 115{18-65)<4) 82(No Limits) 7 122(41-366) 99(19-398) 10 1 2 8 ( 4 6 - 3 7 2 ) 98(16-431) 14 146(33-350) 95(12-455) (1) Method of F.C50 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/ral), measured in triplicated sets of culture flasks. (4) 95% Confidence Limits. CC1464 Exposure (Contact) --gay--- 4 7 10 14 TABLE 2 Algal Growth Response to PC-95, Lot 583 Echo 's and ECgg's (mg/1) Based on Reduction in Cell-Count qc lQ 10 s/ 18 16 16 e c 9Q 173 179 % t 179 174 001465 TABLE 3 Recovery Stage Data Summary Algal Cell-Count/ Cells/ml (MEAN VALUES) ( D (1) Based on 0.5 ml/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. 001466 ATTACHMENT 1 NUTRIENT MEDIUM FOR FRESHWATER ALGAE A. MACRONUTRIENTS STOCK SOLUTIONS (CONCENTRATED) (Prepared separately with deionized water.) 1) 25.500 gm NaNO^ in I li t e r Dl water. 2) 1.044 gm I^HPO^ In I l i t e r Dl water. 3) 12.159 gm MgCljf 6h20 in 1 l i t e r Dl water. 4) 14.700 gm MgSOjf 7H2O in 1 l i t e r Dl water. 5) 4.410 gm C aC l^ 2H2O In 1 l i t e r Dl water. S/ 6) 15.000 gm NaHCO^ in li t e r 01 water. B. MICRONUTRIENTS STOCK SOLUTION (CONCENTRATED) V (Combined In a s in g le o n e -lit e r sto ck m ix.) ^ 1) 185.5 mg H3BO3 2) 415.6 mg MnCl2*4H20 3) 3 .2 7 m gZ n C l2 4) 1.43 mg CoC12* 6 h20 5) 0.01 mg CuCl2 6) 7.26 mg Na2Mo0tt* 2H20 7) 96 mg FeClj 8) 300 mg Na2EDtA-2H20 PREPARATION OF SYNTHETIC ALGAL NUTRIENT MEOIUM - Add one ml o f each macronutrient sto ck so lu tio n p lu s one ml m icronutrients stock mix per 1 lit e r o f deionized water; - sonicate and then f i l t e r through 0.22 jmi membrane; - adjust pH to 7*5 0 . 1 ; sto re In the dark a t h>oC* 001467 ATTACHMENT 2 PREPARATION OF ALGAL CULTURE FLASKS A ll f la s k s usad In m aintaining and t a s t in g a lgae were made o f b o ro slllc a ta g la s s (KIMAX). - fla s k s rare brushed Inside w ith a s t i f f b r i s t l e brush; - washed w ith non-phosphate detergent (MICRO) and rin se d 3 times with tap water; - rinsed w ith a 10% HC1 so lu tio n ; - rinsed 3 tim es w ith tap water and 3 times wtth 0.1. water; - dried in an oven a t 70C fo r 2 hours (placed in ve rte d ); o - and autoclaved w ith foam p lu gs In serted a t 12<4 C fo r 20 minutes. ** 001468 ATTACHMENT 3 PREPARATION OF ALGAL TEST FLASKS IN CHRONOLOGICAL ORDER t="C *fc5 kjcrrSSS ALGAL FLASKS CONTROL ALGAL MEDIUM (ml) H 6 .3 1toiHj^(L3wg&0idi) 24 0 e j^ 0U>) H lp .3 H 5 .Z,Z> 3 L > \ * p L U .S> 4 -3 . to t U .bs> H I. 15 5 145^ Ct2S) 3 *1 .1 5 6 2J5w ^.U .25 ) 3 1.0 7 CONTENTS + TEST MATERIAL + ALGAL INOCULUM (ml) or (rnq)H* (ml) + N ome + l. 'l * + Zj . o * l + 5 . 0 8 vh! + H.*1 4 + *1 .1 5 *4 + U. VS*4 + l.'-f +n + l.n + t*1 * + i.*l ++ \rt ++ TOTAL (ml) 50 50 50 50 50 50 50 50 1) Determine i n i t i a l a lg a l c e ll count in the stock cu ltu re . 2) A d ju st pH o f a lg a l n u trie n t medium to 7*5 + 0.1 ml. 3) Hake up sto ck s o lu t io n s w ith a lg a l n u trie n t medium as desired. A) Control fla sk s preparations: it 1) Add Inoculum ( volume predeterm ined!: i n i t i a l c e ll loading o f 1 x 10 c e lls /m l; 2) Add a lg a l n u trie n t medium to b rin g volume to 50ml; sw irl f la s k s , and place autoclaved foam p lu gs. B) Test fla s k s : 1) Add a lg a l n u trie n t medium; 2) Add te st m ate rial (d isso lv e d in a lg a l medium i f required); 3) Sw irl f la s k s ; it) Add a lg a l inoculum; 5) S w irl f la s k s , and place autoclaved foam p lu gs. * S t o c k s o u u m c k l * < 50 * * $ / (<oS O * m j / v o o O IklVTlAlJ .p H OF STO CK. SOULCnO N vt . o . E a d O hlX - 0*lo 5 * 5 001469 ATTACHMENT k SUBCULTURE METHOD IN ALGAL ASSAYS ! (0.5 ml/50 ml) OBJECTIVE: To determine a lg a l recovery response in absence o f te st substance. PROCEDURE: - T ra n sfe r 0.5ml from each o f the t r ip lic a t e cu ltu re fla s k s in to Corning-15ml ste r ile centrifuge tubes. - C e n trifu ge a t 10,000 rpm fo r 5 minutes. - Decant supernatant liq u id and save the p re c ip ita te d a lg a l c e l l s - U sin g the same cen trifu ge tube, resuspend a lg a l c e l l s in a lg a l n u trie n t medium to a fixe d volume; 15ml. - Mix thoroughly and decant the top 10ml o f a lg a l c e ll suspension and tra n sfe r the remaining volume (5ml) o f a lg a l c e ll suspension into a clean 250ml E rle n i^ ye r cu ltu re fla s k . - U sin g the same ce n trifu ge tubes, tra n sfe r k $ m ] o f fresh a lg a l n u trie n t medium in to the above Erlenmeyer cu ltu re fla s k , to b r in g to ta l volume to 50 ml. 001470 ATTACHMENT 5 ALGAL BIOMASS MONITORING A. GRAVIMETRIC CELL DRY WEIGHT (1) The f i l t e r recommended is Mi IIp o r e type BD with an 0.6 micrometer pore size . The method is as fo llo w s: - Dry f i l t e r s fo r two hours a t 70C in an oven; - Cool f i lt e r s In a desiccator containing desiccant, for at le a s t two hours before w eighing; - FiTtfer a su ita b le measured a liq u o t o f the cu ltu re under a vacuum or pressure not to exceed 8p st; - Rinse the f i l t e r funnel with 0.1. water.; - Dry the f i l t e r to constant weight at 70C, cool In a d e sic c a to r for two hours and weigh. Basic instrument used: A n a ly tic a l Balance; M e ttler ME 30. B. SPEtTROPHOTOMETRIC DETERMINATION OF CHLOROPHYLL a (2) Basic instrument used; Stectrophotcmeter; Bausch - Lomb, Spectronic 20. C. IMPROVED NEUBAUER 0.1 mm DEEP HEM0CYT0METER COUNTING CHAMBER ANO OPTICAL MICROMETER (Used to measure diameter of a lg a l MCVL- (31 (1) . M ille r , V. E . , J. C. Greene, and T. Shlroyama. 1978. Selenastrum capricornutum Printz A lg a l Assay B ottle Test: Experimental D esign, A p p lic a tio n , and Data In te rp re tatio n Protocol. Ecol. Res. S e rie s EPA-600/9-78-018. C o r v a llis , Oregon, pp. 27. ( 2 ) . APHA-AWVA-WPCF (1975). "Standard Methods fo r the Examination of Water and W a s t e w a t e r ( M . Franson, manag, e d .), l*th e d itio n , pp. 1030-1031* ( 3 ) . B rite -L in e (R ), American O ptical Corporation, B u ffa lo , New York. 001471 NO. 3 4 C 'L 2 1 0 DICTZO EN G RA PH PA PER SEMI-LOGARITHMIC BEST COPY AVAILABLED I E T Z S E N CORPORA* IO N M i . L IK I. < A. 2 C Y C L E S X ID D IV IS IO N S P E R INCH tw$) o to U L Ul 4 o o u a %i o o O BN&rq) A-LvdvxO}_ fc_x-pasRt: 001472 001473 N O . 3 4 C - L 2 1 Q D IC T Z B S N G R V i* n P.\*-R SrM I-CO G ARTH M IC BEST COPY AVAILABLEDIETZ O EN C n R * ' M A 3 , r V .