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AR226-2955 Du Pont HLO 235-91 DU PORT USE ONLY Study Title Static Acute Toxicity to the Freshwater Alga, Selenastrum capricornutum Authors Timothy J. Ward Robert L. Boeri Study Completed April 15, 1991 Performing Laboratory EnviroSystems Division Resource Analysts, Incorporated P.0. Box 2130 One Lafayette Road Hampton, New Hampshire 03842 Submitted To E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Sedicine Elktcn Road, Post Office Box 50 Newark, Delaware 19714 Haskell Laboratory Project ID Haskell Laboratory Outside Report No. 235-91 I I i i Page 1 of 20 ResourceAnalysts.Inc..SubsidiaryolMllllFOPE Company Sanitized. Does not cnn' T g r r'ni ontpany Sanzetf. Does not contain TSC CBi Du Pont HLO 235-91 I. GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was performed in compliance with Good Laboratory Practices as defined in 40 CFR part 792, and presented in the Federal Register, Thursday, August 17, 1989 (Toxic Substances Control Act [TSCA]; Good Laboratory Practice Standards; Final Rule). Light intensity was not measured daily during the study. Neither the Study Director nor the sponsor are aware of any circumstances that would affect the integrity of this study. Timothy'J/ Va/i Stydy Director EnviroSystems Study Number Page 2 of 20 Resource Analysts. In c. Subsdiaiy ot MIWPORE .Company Sanitized. Does not contain T P " Ou Pont HLO 235-91 II. QUALITY ASSURANCE STATEMENT Submitted by: Enviro5ysteB8 Division Resource Analysts, Incorporated P.0. Box 2130 One Lafayette Road Hampton, New Hampshire 03820 Certification: This study meets requirements of 40 CFR part 792. Data presented in this report were derived by methods and with materials identified in the section of the report entitled "Methods and Materials." The test was performed in accordance with EnviroSystems Protocol and the Product Registration Aquatic Toxicology Laboratory Standard Operating Procedures Manual. The toxicity test was performed by Jeanne Magazu, Robert Boeri, and Timothy Ward. All data transcribed from the raw data to the report were checked for accuracy, and all data were verified by Quality Assurance Auditors. Quality assurance audits were performed (and reported to management) on: Aspect Audited Audit Reported to Reported to Date Study Director Management Protocol In-life Draft report Final report 7/ 5/90 9/25/90 2/ 4/91 4/18/91 7/ 5/90 9/25/90 2/ 4/31 4/18/91 10/ 2/90 4/18/91 4/18/91 Quality Assurance Representative EnviroSystems Study Number Page 3 of 20 Resource Analysts. In c . Subsidiary of M1LLIPORE JSpfflpany Sanizsd. Dos noicoman T SC ,. J : Du Pont HLO 235-91 III. TABLE OF CONTENTS SECTION: I. Good Laboratory Practice Compliance Statement II. Quality Assurance Statement III. Table of Contents IV. Index of Tables V. Summary VI. Records VII. General Information VIII. Methods and Materials IX. Results X. Protocol Deviations XI. Signature Page XII. References APPENDIX: Appendix A. Water Quality Data from Toxicity Test PAGE 2 3 4 5 e 7 n 10 13 15 16 17 18 EnviroSystems Study Number Page 4 of 20 Resource Analysts, tnc. Subsidiaryof MHPORf Ou Pont HLO 235-91 IV. INDEX OF TABLES PAGE Table 1. Chemical characterization of a representative sample of deionized water used to prepare sterile enriched media used in the toxicity test with aQd `he freshwater alga Selenastrum capricornutum 11 Table 2. Cell growth data from the toxicity test with the freshwater alga Selenastrum capricornutum 14 Table A. 1. Temperature measured during toxicity test with the freshwater alga Selenastrum capricornutum 19 Table A. 2. pH values measured during toxicity test with the freshwater alga Selenastrum capricornutum 20 EnviroSystems Study Number Page 5 of 20 ResourceAnalysts. Inc.. Subsidiary onM1UPORE Company Sanitized. Does not contain TSA Du Pont RLO 235-91 V. SUMMARY An acute toxicity test was conducted with and the freshwater alga, Selenastrum capricornutua, to determine the effective concentrations (EC10, EC50, EC90). The test was conducted for E.I. duPont de Nemours and Company for 96 hours during September 21 to 25, 1990, at the EnviroSystems Division of Resource Analysts, Inc, in Hampton, Hew Hampshire. It was conducted" according to the protocol developed for EnviroSystems Study Number W M K K * The test substance was supplied by the sponsor. The test was performed under static conditions with five concentrations of test substance and a dilution water control with 3 replicates per level. The test was conducted at a temperature of 24 2C. The dilution water was sterile enriched media adjusted to a pH of 7.5. Nominal concentrations were: 0 (control), 315, 625, 1,250, 2,500, and 5,000 mg/L M K M t K K t Cell counts were made daily with a haemocytometer. Nominal concentrations were used for all calculations. Water quality parameters were within acceptable limits throughout the test. The test substance was not toxic to Selenastrua capricornutua at any tested concentration. Exposure of algae to the test substance for 96 hours resulted in a median effective concentration (EC50) of '>5,000 m g / L f i ^ B H B the highest tested concentration. EnviroSystems Study Number Page 6 of 20 ResourceAnalysts. !nc. SubsidiarycfiMiLPO^E Cpifiparfy' SanitizeS. Does not contain fSC A CD? VI. RECORDS Du Pont HLO 235-91 All records from those portions of this study conducted at Resource Analysts, Inc. and the final report will be maintained at the EnviroSystems Division of Resource Analysts, Inc., 1 Lafayette Road, Hampton, New Hampshire. EnviroSystems Study Number Page 7 of 20 Resource A n a ly st In c . Subsidiary of MtLLlPORE Du Pont HIiO 235-91 m . C O R R A L INFORMATION Material Tested: Medical Research Number: Haskell No.: 18,267 Synonyms : Submitter's Notebook Number: Purity: Contaminants: Composition: Sponsor: Submitted by: Du Pont Chemicals and Pigments E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 Du Pont Chemicals and Pigments E.I du Pont de Nemours and Company Jackson Laboratory Deepwater, New Jersey 08023 EnviroSystems Study Number Page 8 of 20 Resource Analysts. Inc Subsidiary of M iu [PORE Company Sanitized. Does not contain T SCA c tf Du Pont HLO 235-91 Study Schedule: Haskell Laboratory Test Code: Distribution: Study Initiation Date: August 6, 1990 Experimental Start Date: September 21, 1990 Experimental Completion Date: September 1990 Study Completion Date: April 15, 1991 25, EnviroSystems Study Number Page 9 of 20 Resource Analysts, me. Subsidiary of M4UBORE pm patY Sanitized, .Does not contain TSCA CB< TEST SUBSTANCE: Du Pont HLO 235-91 Till. METHODS ARD MATERIALS was delivered to EnviroSystems on June 18, 1990. It was contained in a glass bottle that was labelled with the following information: "H 18,267, 5-22-90, 412 grans gross". The test substance (a viscous, beige liquid) was shipped from E. I. duPont de Renours and Company, Stine-Haskell Research Center, P.0. Box 30, Elkton Road, Newark, Delaware. Prior to use the test aaterial was stored in the dark at 2-4C. A reserve sample (approximately 1.2 grams) will be archived at EnviroSystems for a minimum of 10 years, and unused test substance is returned to the sponsor.... .5#000 mg/L stock solution was prepared by combining 5.0_.g of test substance and dilution water and adjusting the mixture to 1,000 ml. T h ^ ^ ^ ^ ^ ^ s u b s t a n c e was tested as received and assumed to have a purity o f f l ^ m B ^ . DILUTION HATER: Hater used for acclimation of test organisms and for all toxicity testing was sterile enriched media (U.S. EPA, 1978; Resource Analysts, Inc. Standard Operating Procedure 01-02-1101) adjusted to a pH of 7.5. Results of chemical analysis of a representative sample of deionized water used to prepare the media are presented in Table 1. TEST ORGANISM. Algae used for the test was from a culture originally procured from the Culture Collection of Algae at the University of Texas at Austin and delivered to EnviroSystems on October 31, 1989. The culture was transferred to sterile enriched media identical to media used for this test and maintained for at least 14 days. TOXICITY TESTING: A screening test was conducted during August 9 to 13, 1990. Nominal concentrations of test substance were: 1, 10, 100, and 1,000 mg/L. After 96 hours t h ^ ^ r t ^ ^ ^ ^ c e l l s per ml was greater than the control value at 1,000 m g / L ^ ^ ^ ^ ^ H f e The definitive toxicity test was performed during September 21 to 25, 1990, according to the protocol developed for EnviroSystems Study The protocol is based on procedures of the U.S. Environmental Protection Agency (1985, 1987). The test was conducted at a target temperature of 24 2C with five concentrations of test material and a dilution water control. Nominal concentrations of were: 0 (control), 315, 625, 1,250, 2,500, and 5,000 mg/L. EnviroSystems Study Number Page 10 of 20 Pesource Analysts, In c. Subsidiary of KU.WOPE _, Tfiuie l. _. . , t Chemical eh .. Du Pont HLO 235-91 .enzation of representative staple of deionized eater used to prepare sterile enriched aedia used in the toxicity test w i t h g ^ B l ^ ^ a n d the freshwater alga Selenastrue capricornutum*2 Parameter Unit of Measurement Total organic carbon mg/L 0 hour 96 hour Total suspended mg/L solids 0 hour 96 hour Ammonia Aluminum mg/L as N mg/L Arsenic Boron mg/L mg/L Cadmium mg/L Chromium Copper Cobalt mg/L mg/L mg/L Fluoride Iron mg/L mg/L Lead mg/L Mercury Nickel - mg/L mg/L Silver Zinc mg/L mg/L Organochlorine and ug/L Organophosphorus Pesticides and PCBs Detection Limit 1 10 0.2 0.025 0.01 0.1 0.001 0.002 0.005 0.008 0.05 0.01 0.005 0.0003 0.008 0.005 0.005 5.0 Measured Value HD 11 HD 21 HD HD HD HD HD HD HD " HD HD 0.01 HD HD HD HD HD HD Notes: 1. 2. ND = Not detected at or above the detection limit. Total organic carbon and total suspended solids determination was conducted on a sample of dilution water collected from a control test vessel during this toxicity test (96 hour values represent analysis of the test organism in the dilution water). All other chemical data are the result of analysis of a representative sample of deionized water collected during March, 1990 as part of routine bi-annual water quality analysis. EnviroSystems Study Number Page 11 of 20 ResourceAnalyst n c . Sutwdiary oiMliffORf .Company Sanitized. Does no! contain T snn r> Du Pont HLO 235-91 Algae vas randomly and equally distributed among three replicates of each treatment at the rate of 10,000 cells/nl. The test vas performed in 250 si glass erlenmeyer flasks that contained 100 al of test solution. Test vessels vere randoaly arranged on a rotary shaker adjusted to 100 rpa. The test vessels vere placed in an incubator during the test (a random numbers table vas used to select the location of each vessel). A 24 hour light and 0 hour dark photoperiod vas automatically maintained vith cool-white fluorescent lights that provided a light intensity of 24 uEs-tm2 . The number of algal cells/ml in each test vessel and the occurrence of relative size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a baemocytometer. Cell counts were made and recorded daily during the 96 hour test. Temperature of the incubator was measured and recorded daily (thermometer number 2211), and pH (Beckman model pHI 12 meter; instrument number PRL-4) vas determined in each test vessel at the beginning and end of the test. STATISTICAL HETHODS: Results of the toxicity test could not be interpreted by standard statistical techniques because cell growth at the highest tested concentration was greater than 50% of control growth. Determination of EC10, EC50, and EC90 values were performed by the author using percent of control growth data and the nominal concentrations of the test substance. . EnviroSystems Study Numbe Page 12 of 20 Resource Analysts. Inc . Subsidiary of MlufPORE 5t>ntp&ny 'Sanitisted. Does no? contain T SC CBf IX. RESULTS Du Pont HLO 235-91 Water quality data generated by the acute toxicity test are presented 1?. Appendix X. Water quality throughout the test was within acceptable limits. The range of values measured in test vessels during tbe test were: incubator temperature - 23.2 to 24.2C, and pH - 7.5 to 9.0. Bl0JWlcal and water quality data generated by the acute toxicity test are presented in Table 2 and Appendix A, respectively. The 96 hour EC10, EC50, and EC90 values are >5,000 ag/L, the highest tested concentration. Ko effects (size differences, unusual cell shapes, colors, flocculations, adherence of cells to test containers or aggregation of cells) were noted. ' EnviroSystems Page 13 of 20 Resource Analysts. In c. Subsidiary a l MILUPORE Company Sanitized. Poos not contain T SC A CBI Du Pont BLO 235-91 Table 2 Cell growth data from the acute toxicity test with B a n d the freshwater alga, Seleaastrua caprieoroutua. Nominal Concentration (mg/L) rep. Number of Cells/ml x 10* (hour) 0 24 48 72 96 0 (control) 1 2 3 mean % control 10 10 10 10 10 315 1 10 2 10 3 10 mean 10 % control 100 625 1 10 2 1C 3 10 mean 10 % control 100 1,250 1 2 3 mean % control 10 10 10 10 100 2,500 1 2 3 mean % control 10 10 10 10 100 5,000 1 2 3 mean % control 10 10 10 10 100 56 61 54 57 100 60 49 52 54 95 52 48 54 51 89 53 51 49 51 89 46 57 53 52 91 58 47 52 52 91 238 212 246 232 1O0 209 227 230 222 96 198 212 226 212 91 204 221 240 222 96 218 212 227 219 94 222 241 210 224 97 945 1,070 1,325 1,113 100 1,212 938 1,110 1,087 98 1,070 1,083 962 1,038 93 924 981 1,042 982 88 1,221 989 1,014 1,075 97 845 910 885 880 79 1,678 1,929 1,842 1,883 100 1,750 1,825 1,803 1,793 95 1,821 1,800 1,862 1,828 97 1,760 1,740 1,826 1,775 94 1,912 1,716 1,844 1,824 97 1,861 1,840 1,962 1,888 1O0 EnviroSystems Study Number Page 14 of 20 Resource Anolysts. Ine. SubstoiY o<MUJPORE jtloffipany sanitized. Does not contain faC A CSs Z. PROTOCOL DEVIATIONS Du Pont HLO 235-91 No deviations were made from the protocol. EnviroSystems Study Number Page 15 of ?0 Resource Anolvsti me.. Subsidiary of MlUJfORE XX. SIGNATURE PAGE Du Pont HLO 235-91 Coauthor V 'f S 'h Inne P. Magaz Aquatic Toxicologist EnvirnSystems Study Nurabe Page 16 of 20 Resource Analysts, me. SubsktoY 0MKiPORt Company Sanitized. Does not contain To-A CBJ XII. REFEREHCES Du Pont HLO 235-91 O.S. EPA. 1978. The Seleaastruw eapricornutua Printz Algal Assay Bottle Test. EPA-600/9-78-018. Environmental Research Laboratory, Corvallis, Oregon. O.S. EPA. 1985. Guidelines; Final Section 797.1050. 40 CFR Part 797. Toxic Substances Control Act Test Rules. Federal Register, Friday, September 27, 1985. O.S. EPA. 1987. 40 CFR Part 797. Toxic Substances Control Act Test Guidelines; Amendments. Federal Register, Wednesday, May 20, 1987. Section 797.1050. . O.S. EPA. 1989. 40 CFR Part 792. Toxic Substances Control Act (TSCA); Good Laboratory Practice Standards; Final Rule. Federal Register, Thursday, August 17, 1989 EnviroSystems Study Number Page 17 of 20 Resource Anatysts. Inc.. Subsidiary of MILLIPORE lApaiw'Ss nltfced. D o e s r-ct contain T S C C D ! Dt! Pont HLO 235-91 Appendix A. VATER QUALITY DATA FROH TOXICITY TEST EnviroSystems S tu d y Number Page 18 of 20 Pesource Analysts. inc.. Subaoory al MiUIPOPf capricoroutua. Hour of Exposure 0 24 48 72 96 Temperature of Incubator,C 24.2 23.6 23.7 23.7 23.2 ' 1 | EnviroSystems Study Number Page 19 of 20 Pesource Analysts. In c. Subsidiary of MIUIPORE Company Sanitized. Does not contain TSCA Ci  Du Pout HLO 235-91 Table A.2. pH values measured during toxicity test with freshwater alga, Selenastrust capricornutum. and tbe Nominal concentration (mg/L) 0 fcontrol) 315 625 1,250 2,500 5,000 Replicate 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 pH Initial Final 7,5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 8.3 8.7 8.7 8.9 9.0 8.9 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 9.0 EnviroSystems Study Number Page 20 of 20 ResourceAnalysts. Inc.. SubsidiaryofMILUPORE ? contain T S C A O' 'Company Sanitised. Ooss ito?