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CHRONIC TOXICITY TO FRESHWATER INVERTABRATES (Daphnia magna) TEST SUBSTANCE Identity: Perfluorooctanesulfonate; may also be referred to as PFOS or FC-95. (1-Octanesulfonic acid, 1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8heptadecafluoro-, potassium salt, CAS # 2795-39-3) Remarks: The test substance is a white powder. Sample was taken from 3M lot number 217. Sample was stored under ambient conditions prior to testing. Purity determined to be 90.49% by LC/MS, 1H-HMR, 19F-NMR and elemental analyses techniques. METHOD Method: OPPTS 850.1300, OECD Guideline 211, and ASTM Standard E 1193-87. Type: Semi-Static Life-Cycle Toxicity GLP: Yes Year completed: Study completed 1999. Report completed 2000 Species: Daphnia magna Supplier: In-house cultures, Wildlife International, Ltd., Easton, MD Analytical monitoring: PFOS measured on days 0, 2, 11, 14, 18, and 21. Exposure period: 21 days Statistical methods: Survival data was evaluated on first-generation daphnids, the number of live young and the length and dry weight of the surviving first-generation daphnids. Survival data were analyzed using Fisher's exact test. Reproduction and growth (length and dry weight) data were evaluated for normality using Shapiro-Wilk's test and for homogeneity of variance using Bartlett's test. Analysis of variance and Dunnett's test was used to identify treatment groups that were statistically significant in comparison to the negative control (p < 0.05). All statistical tests were performed using a personal computer with SPSS/PC Version 2.0 or "TOXSTAT Release 3.5" statistical software. Test organism age: < 24-hours old at test initiation Pretreatment: None Test conditions Dilution water: 0.45 pm filtered well water passed through a UV sterilizer to remove microorganisms and fine particles Dilution water chemistry (during the 4-week period immediately preceding the test): hardness: alkalinity: 124 (120-128) mg/L as CaC03 169 (164-172) mg/L as CaC03 pH: 8.2 (8.0-8.3) TOC: <1.0 mg/L ocio Conductivity: 329(315-340) pmhos/cm Ca/Mg ratio: 35/13.5 Na/K ratio: 21.3/6.62 Stock and test solution preparation: Primary stock solution was prepared in dilution water at 46 mg/L. It was stirred until all test substance was dissolved prior to use. After mixing, the primary stock solution was proportionally diluted with UV sterilized dilution water to prepare five additional stock solutions at nominal concentrations of 1.4, 2.9, 5.7, 11, and 23 mg/L. All test solutions appeared clear and colorless. Renewal rate: Every Monday, Wednesday and Friday. Exposure vessels: 250-mL plastic beakers containing approximately 200 mL test solution. The depth was approximately 5 cm. Number of replicates: 10 Number of test organisms per replicate: 1 Number of concentrations: 6 plus a negative control Feeding: Each test chamber was fed 0.3 mL of YCT (a mixture of yeast, Cerophyll, and trout chow at 1800 mg TSS/L) and 0.60 mL of Selenastrum capricornutum (3.5 x 107cells/mL) once daily. Lighting: Colortone 50 fluorescent lights. Intensity ranged from 329 - 383 lux at the water surface. Photoperiod of 16-hours light, 8hours dark with a 30-minute transition period. Water chemistry of new and old solutions during the study: Dissolved oxygen range (0 - 21 days): 8.3 - 8.9 mg/L (negative control exposure) 8.3 -9 .0 mg/L (12 mg/L exposure) 8.4 - 8.9 mg/L* (48 mg/L exposure) pH range (0 - 21 days) 8.1 - 8.4 (negative control exposure) 8 .2 -8 .5 (12 mg/L exposure) 8.4 - 8.5* (48 mg/L exposure) Test temperature range (0 -2 1 days) 1 9 .4 - 20.1C (negative control exposure) 1 9 .4 - 20.1 C (12 mg/L exposure) 19.4 - 19.5C* (48 mg/L exposure) * (Measurements discontinued at Day 3 due to 100% mortality. Element basis: Survival, reproduction and growth. Effect concentrations based on survival. CG10SG Method of calculating mean measured concentrations: arithmetic mean RESULTS Nominal concentrations: Negative control, 1.4, 2.9, 5.7, 11, 23, 46 mg/L Measured concentrations: <LOQ, 1.5, 2.9, 5.6, 12, 24, 48 mg/L Element value: 21-day NOEC = 12 mg/L 21-day LOEC = 24 mg/L 21-day MATC = 17 mg/L 2nd generation acute survival NOEC = 12 mg/L All element values based on mean measured concentrations Analytical Methodology: Analyses of test solutions were performed at Wildlife International Ltd. using high performance liquid chromatography with mass spectrometric detection (HPLC/MS). When determining the concentration of the test substance in the test solutions, the same and most prominent peak response for the test substance was used. No attempt was made to quantify on the basis of individual isomeric components. The LOQ (limit of quantitation) was 0.458 mg/L in this study. The mean procedural recovery of matrix fortifications analyzed concurrently during sample analysis was 104%. Measured values of new samples ranged from 94 to 121% of nominal. Measured values from the old solutions ranged from 90 to 108% of nominal values. PFOS was stable throughout the renewal periods. C010S7 Summary of analytical chemistry data: N om in al T e st C once n tra tio n , m g/L M easured D uplica te V a lu e s at 0, 2, 11, 14, 18, and 21 D ays, R e sp e ctive ly, m g/L M ean M easured C once ntra tion, m g/L P ercent o f N om inal N ega tive C ontrol A ll < LO Q 1.4 1 .7 8 , 1.72, 1.5 8 , 1.5 6, 1.3 8, 1.4 7, 1.3 6, 1.3 2 , 1.38, 1,43, 1.50, 1.45 . 1.5 107 2.9 3.2 0, 3.05, 3.01, 3.07, 2.75, 2.77, 2.85, 2.71, 2.79, 2.81, 2 .8 1 ,2 .8 2 2 .9 100 5.7 5 .9 7, 5.87, 5.65, 5.72, 5.63, 5.59, 5.36, 5.39, 5.58, 5.75, 5.24, 5.37 5 .6 98 11 1 1 .5 , 11.5, 1 1 .6 , 1 1 .8, 1 1 .3 , 1 1 .3, 1 1 .2, 1 1 .6 , 11 .8, 11.6, 11.5, 11.3 12 109 23 24 .2, 2 3 .1 ,2 4 .0 , 24.6, 22.8, 22.5, 23.6, 23.1, 24.8, 2 5 .0 , all d a p h n id s de ad a fte r 1 8 -d a ys e xp o su re 24 46 4 7 .3 , 4 8 .0 , 4 9 .1 ,4 9 .4 , all d a p h n id s d e a d a fte r 2 -d a y s 48 e x p o s u re 104 104 NOTE: Mean measured concentrations were determined from new (renewal solutions) and corresponding old solutions during each week of the test. Days 0, 11, and 18 are "new" and days 2, 24, and 21 are "old". Biological observations Survival: All surviving first generation daphnids appeared normal at test termination. Survival in the 24 and 48 mg/L treatments was statistically significantly different from the negative control group. Reproduction: Daphnids in the control and treatment groups <12 mg/L started producing neonates on Day 9. The Bonferroni t-test showed that reproduction was not significantly reduced in any treatment group <12 mg/L (p >0.05). The 24 and 48 mg/L treatment groups were not included in the statistical analysis of the reproduction data due to a statistically significant effect on survival. Growth: The Bonferroni t-test showed that mean length and dry weight in the treatment groups <12 mg/L were not significantly reduced in comparison to the negative control (p > 0.05). Second Generation Acute Exposure: After 48-hours of exposure, survival in the negative control was 95%. Survival in the 1.5, 2.9, 5.6, 12, and 24 mg/L treatment groups was 100, 100, 100, 90, and 0% respectively. Survival in the 24 mg/L treatment group was significantly different from the negative control (p < 0.05). C01GS3 Summary of Percent Mortality M ean M easured C o n ce n tra tio n , m g/L Day 7 D ay 14 N e g a tive C ontrol 0 0 1.5 0 0 29 0 0 5.6 0 0 12 0 10 24 70 90 48 100 100 D ay 21 0 10 10 10 10 100 100 Second Generation Mortality M ean M easured C o n ce n tra tio n s, m g/L T otal N um ber Exposed N ega tive C ontrol 20 N um ber A liv e after 4 8 -h o u rs C u m u la tive P e rce n t Dead 19 5 1.5 20 20 0 2 .9 20 20 0 5 .6 20 20 0 12 20 18 10 24 8 0 100 Summary of Length and Dry Weight of Surviving Individually-Exposed First-Generation Daphnids M ean M easured C o n ce n tra tio n , m g/L Number of S u rvivin g D aph nids T o tal Length, M ean D ry W e ig h t, M ean + S D , + SD, mm mg N ega tive C ontrol 1.5 2 .9 5 .6 12 24 48 10 4 .6 5 + 0.111 0 .6 9 5 + 0 .1 0 0 9 4 .6 6 + 0 .1 1 8 0.669 + 0.0623 9 4.6 2 + 0.100 0.724 + 0.110 9 4.61 + 0.124 0,727 + 0.0665 9 4 .5 9 + 0.102 0 .7 23 + 0.0661 0 .. 0 __ __ CCCS3 Reproduction M ean M easured C o n ce n tra tio n , m g/L Number of S u rvivin g D aph nids M ean Live Y oung / S u rvivin g A d u lt D aph nid (+ S D ) First D ay o f R eproduction N ega tive C ontrol 10 122 + 19.2 9 T otal N um b er o f D ead / Im m obile N eonates T otal N um b er o f A borted Eggs 00 1.5 9 142 + 24.7 9 0 0 2 ,9 9 136 + 17.9 9 0 0 5 .6 9 132 + 19.5 9 0 0 12 9 119 + 26.5 9 1 0 _2 4 0 11 10 0 48 0 None __ _ CONCLUSIONS There were no adverse effects on survival, reproduction or growth of Daphnia magna exposed to the test substance at concentration <12 mg/L for 21 days. Daphnia magna exposed to 24 and 48 mg/L had significantly reduced survival. Author and/or submitter: 3M Company, Environmental Laboratory, P.O. Box 33331, St. Paul, Minnesota, 55133 DATA QUALITY Reliability: Klimisch ranking 1. REFERENCES This study was conducted at Wildlife International, Ltd. Easton, MD at the request of the 3M Company. OTHER Last changed: 5/3/00 CCXOOQ