1、American Petroleum Institute Tert-Amyl Methyl Ether (TAME)- Acute Toxicity to Daphnids (Daphnia magna) under Flow- through Conditions TSCA Guideline 5797.1 300 FEBRUARY 1995 TOXICOLOGY REPORT NUMBER 406 CAIS ABSTRACT NO. 42-1 51 9 I API TR*40b 95 Se 0732290 0554909 O99 I Ermmwmenta Parnmbrp One of t
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15、ND EXPERTISE DURING THIS STUDY AND IN THE PREPARATION OF THIS REPORT: - Richard Rhoden, Ph.D., Health and Environmental Sciences Department MBERS OF IlE -COJ .OGY EFFFSTS WORKGROUP Russell D. White, Ph.D., Chainnun, Chevron Research 95% confidence interval calculated by binomial probability. Springb
16、orn Laboratories, Inc. API TR*40b 95 P 0732290 0554923 696 Report No. 92-1 2-4545 Paae 9 of 67 1 .O INTRODUCTION The purpose of this study was to estimate the acute toxicity (EC50) of Tert-Amyl Methyl Ether (TAME) to daphnids (Daphnia magna) under flow-through conditions. The EC50 is defined as the
17、concentration of test material in dilution water which causes immobilization of 50% in the exposed test population after a futed period of time. This value is often used as a relative indicator of potential acute hazards resulting from the release of the test substance into aquatic environments. The
18、 study was initiated on 26 October 1992, the day the Study Director signed the protocol, and was completed on the day the Study Director signed the final report. The experimental phase of the 46-hour definitive toxicity test was conducted from 1 O - 12 December 1992 at Spring born Laboratories, Inc.
19、 (SLI), Environmental Sciences Division, Wareham, Massachusetts. All original raw data and the final report produced for this study are stored at su. 2.0 MATERIALS AND METHODS 2.1 Protocol Procedures used in this acute toxicity study followed those described in the SLI protocol entitled “Protocol fo
20、r Conducting a Flow-Through Acute Toxicity Test with Daphnia magna Following TSCA 5797.1 300“, SLI Protocol #: O91 192/TSCA 797.1 300 DM-FA and Protocol Amendment # 1 dated 15 December 1992 (Appendix I). The methods described in this protocol generally follow the standard procedures described in the
21、 EPNOTS guidelines for testing the effects of chemicals on daphnids and meet the TSCA guidelines as specified in the appropriate Registration Standard. This protocol is intended to meet premanufacture notice (UPMNII) registration requirements. 2.2 Test Material Two samples of Tert-Amyl Methyl Ether
22、(TAME) (CAS # 994-05-8), a clear liquid, were received from Experimental Pathology Labs, Inc., Herndon, Virginia. The first sample, Lot # 0251 462, was received at SLI on 17 August 1992 and was used to prepare exposure solutions during the preliminary exposure, analytical standards during the method
23、 validationhecovery study and to prepare Qualtty Control samples during the definitive exposure. The sample was identified Springborn Laboratories, Inc. API TR*406 95 O732290 0554922 522 W Report No. 92-124545 Page 10 of 67 by Aldrich Chemical Company to contain 98.8% active ingredient A.I. (Certifi
24、cate of Analysis, Appendix Il). The second sample, Lot # 07905Kz, was received at SLI on 2 November 1992 and was used to prepare exposure solutions during the definitive exposures. The sample was identified by Aldrich Chemical Company to contain 98.7% active ingredient A.I. (Certificate of Anaiysis,
25、 Appendix Il). Upon receipt at SLI, the samples of test material were stored in a dark, ventilated cabinet at room temperature (approximately 20 OC). Test concentrations are expressed as milligrams of test material (as active ingredient) per liter of test solution and are reported as mg A.I./L. At t
26、he request of the Study Sponsor, mass spectral analysis was conducted on the initial batch of TAME received at program initiation, and the additional batches received throughout the course of the program. The purpose of the mass spectral analysis evaluation was to determine test material integrity t
27、hroughout the duration of the program. Initial evaluation of test material (Le., lot # 0281482) was conducted on 3 December 1992. Following completion of the fiow- through acute toxicity test with mysids (SLI Report # 94-5-5269), spectral analysis was conducted on 20 July 1994 on each of the remaini
28、ng two lots (lot # 02814BZ and lot # 07905KZ). The spectral analysis conducted on 20 July 1994 on the two remaining lots in comparison to the initial spectral analyses of lot # 0281 482 established that negligible change in test material composition had occurred during storage at Springborn Laborato
29、ries, Inc. (Le., approximately 24 months). 23 Test Organisms The Daphnia magna used in this toxicity test were obtained from laboratory cultures main- tained at Springborn Laboratories, Inc., Wareham, Massachusetts. The culture water was prepared by fortifying well water based on the formula for har
30、d water (U.S. PA, 1975) and filtering it through an Amberlite XAD-7 resin column and a carbon filter. Two weeks prior to test initiation, this water had total hardness and total alkalinity as calcium carbonate (CaCOJ of 160 mg/L anri ? !.O mg/L, respectively, a pH of 8.1, a specific conductivity ran
31、ge of 400 to 500 pmhoslcm and a dissolved oxygen concentration of greater than 60% of saturation. The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of Springborn Laboratories, Inc. API TR*406 95 m 0732290 0554923 469 = Report No. 92-12-4545 Page 11 of 67 dark
32、ness. tight intensity in the culture area ranged from 32 - 46 footcandles (Invertebrate Culture Log, Vol. Vlll). A waterbath in the culture area was used to maintain the culture solution temperature at 20 * 2 OC. Daphnids were fed a combination of a trout food suspension and a unicellular green alga
33、e (Ankistrodesmus falcarus) once daily. The food solution contained 5.0 mg/mL trout food and approximately 4 X 1 O cells/mL of algae. Representative samples of the food source were analyzed for the presence of pesticides, PCBs and toxic metals (Appendix Ill). Food sources were considered to be of ac
34、ceptable quality since the total concentration of pesticides measured was less than 0.3 mghg (ASTM, 1985). 2.4 lest Dilution Water The dilution water used during this study was from the same source as the culture water described above and had a total hardness and total alkalinity (CaCO,) of 160 and
35、11 O mg/L, respectively, a pH range of 8.1 - 8.2 and a specific conductivity of 500 pmhos/cm (IWQ Log Book, Vol. 13). During holding and prior to use, the dilution water was continuously aerated. Representative samples of the dilution water source were analyzed for the presence of pesticides, PCBs a
36、nd toxic metals (Appendix IV). None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM standard practice (ASTM, 1985). In addition, representative samples of the dilution water source were analyzed monthly fo
37、r total organic carbon (TOC) concentrations. The results of these analyses demonstrated that the TOC concentration of the dilution water source ranged from 0.97 - 2.2 mg/L for the months of June - November 1992 (TOC and TSS master log, Volume I). Daphnid cultures are maintained in water from the sam
38、e source as the dilution water utilized in this study and have successfully suwived and reproduced over several generations. This, in combination with the previously mentioned analyses, confirms the acceptability of this dilution water for bioassays. 2.5 lest Conditions The toxicity test was c-,:icj
39、i!ded using an exposure system consisting of an intermittent- flow proportional diluter (Mount and Brungs, 1967) and a set of 12 exposure vessels. The test system was designed to provide five concentrations of the test material and a dilution water Springborn Laboratories, Inc. API TR*406 75 O732270
40、 0554724 3T5 Report No. 92-1 2-4545 Page 12 of 67 sontrol. Exposure vessels were maintained in an area illuminated with Duro-Test. Cool-White and Duro-Test. Vitalite fluorescent lights at an intensity of 45 - 70 footcandles. The photoperiod was the same as that of the culture area. Sudden transition
41、s from light to dark and vice versa were avoided. The test was conducted in a temperature controlled room and waterbath which were designed to maintain test solution temperatures at 20 f 2 OC. Two replicate vessels were established for each treatment level and the control. Exposure vessels were labe
42、led to identify the nominal test material concentration and designated replicate. 2.6 Test Concentrations Selection of nominal TAME concentrations for the 46-hour definitive flow-through toxicity test with daphnids was based on toxicity information developed at SLI through preliminary testing. 2.7 E
43、xposure Solution Preparation Prior to test initiation, a 50 mL Glenco* gas-tight syringe in conjunction with a Sage syringe pump (Model # 355) was calibrated to deliver 0.416 mUcycle of the test material (760 mg/mL) directly to the diluter systems chemical mixing chamber, which also received 0.456 L
44、 of dilution water per cycle. The mixing chamber was positioned over a magnetic stirrer which continuously mixed the contents of the mixing chamber and aided in the solubilization of the test material. The solution contained in the mixing chamber constituted the highest nominal treatment level (690
45、mg A.I./L) and was subsequently diluted (60% dilution factor) to provide the remaining nominal test concentrations (410, 250, 150 and 89 mg A.I./L). During each cycle of the diluter system, approximately 50 mL of exposure solution was delivered to each replicate test vessel. The system cycled approx
46、imately 216 times each day. The diluter system was calibrated prior to test initiation by measuring delivery volumes of toxicant and dilution water. During the study, visual checks of the diluter system and analysis of the exposure solutions for TAME concentration. were used to verify proper operati
47、on of the diluter system. The exposure system was in proper operation 9 days prior to test initiation to allow equilibration of the test material in the diluter apparatus and exposure vessels. Four glass Springborn Laboratories, Inc. API TR*:406 95 I 0732290 0554925 231 Report No. 92-12-4545 Page 13
48、 of 67 capillary tubes with an approximate length of five centimeter (cm) and a diameter of 1 -millimeter (mm) (inside diameter) were inserted through silicone stoppers in the mixing/splitting chambers of the diluter and into the test solution delivery tubes. This tubing sewed to restrict the flow o
49、f the test solutions, minimizing potentially stressful turbulence in the exposure vessels and provided equal distribution of the solutions to the replicate vessels. Each glass test vessel maintained a constant solution volume of 1.8 L and a solution depth of approximately 13 cm. Each replicate vessel received approximately 6.0 solution volume replacements per day. The function of the diluter system (e.g., flow rates, stock consumption) was monitored daily and a visual check was made twice daily to veri proper performance during the study period. 3.0 TEST PROCEDURES
