1、_ API TRr40 95 O732290 0555063 8T4 American Petroleum 11 Institute Health and Environmental Sciences Department Tert-Amyl Methyl Ether (TAME)- Acute Toxicity to Rainbow Trout (Oncorhynchus mykiss) Under Flow-through Conditions TSCA Guideline 5797.1 400 FEBRUARY 1995 TOXICOLOGY REPORT NUMBER 408 CAIS
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15、95 m 0732290 0555066 503 H ACKNOWLEDGMENTS THE FOLLOWING PEOPLE ARE RECOGNIZED FOR THEIR CONTRIBU- TIONS OF TIME AND EXPERTISE DURING THIS STUDY AND IN THE PREPARATION OF THIS REPORT: - Richard Rhoden, Ph.D., Health and Environmental Sciences Department CTS WORKGROUP Russell D. White, Ph.D., Chuimn,
16、 Chevron Research 95% confidence interval calculated by binomial probability. Since the 96-hour LC50 was not less than 50% of the 48-hour value, the study was not extended beyond 96-hours to determine the incipient LC50. Springborn Laboratories, lnc. API TR*408 95 0732290 0.555076 452 Report No. 93-
17、3-4682 Page 9 of 72 1 .O INTRODUCTION The purpose of this study was to determine the acute toxicity of Tert-Amyl Methyl Ether (TAME) to rainbow trout (Oncorhynchus mykiss) under flow-through conditions. The LC50 is defined as the concentration of the test material in dilution water which causes mort
18、ality of 50% in the exposed test population aiter a fixed period of time. This value is often used as a relative indicator of the potential acute hazards resulting from release of the test material into aquatic environments. The study was initiated on 14 October 1992, the day the Study Director sign
19、ed the protocol, and was completed on the day the Study Director signed the final report. The experimental phase of the 96-hour definitive flow-through toxicity test was conducted from 27 February - 3 March 1993 at Springborn Laboratories, Inc. (SLI), Environmental Sciences Division, Wareham, Massac
20、husetts. 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 test followed those described in the SLI protocol entitled “Protocol for Conducting a Flow-Through Acute Toxicity Test with Rain
21、bow Trout following TSCA 797.1 400, SLI Protocol #:O91 19mSCA 797.1 400 and Protocol Amendments #1 and #2 dated 1 O and 24 March 1993, respectively (Appendix I). The methods described in this protocol generally follow the standard procedures outlined in the EPNOTS guideline (S 797.1400) for acute to
22、xicity testing with fish (U.S. EPA, 1985, amended 1987). 2.2 Test Material Two samples of Tert-Amyl Methyl Ether (TAME) (CAS # 994-05-8), a clear liquid, were received from Experimental Pathology Labs, Inc., Herndon, Virginia. The first sample, Lot # 0281 4BZ, was received at SLI on 17 August 1992 a
23、nd was used to prepare analytical standards during the method validation/recovery study and to prepare Quality Control samples during the definitive exposure. The sample was identified by Aldrich Chemical to contain 98.8% active ingredient A.I. (Certificate of Analysis, Appendix II). The second samp
24、le, Lot # 07905KZ, was Springborn Laboratories, inc. API TR*408 95 = 0732290 0555077 399 W Report No. 93-34682 Page 10 of 72 received at SU on 2 November 1992 and was used to prepare exposure solutions during the preliminary and definitive exposures. The sample was identified by Aldrich Chemical to
25、Contain 98.7% active ingredient A.I. (Certificate of Anaiysis, 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)
26、per liter of test solution and are reported as mg A.I./L. At the 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
27、analysis evaluation was to determine test material integrity throughout the duration of the program. Initial evaluation of test material (Le., lot # 0281 462) was conducted on 3 December 1992. Following completion of the flow- through acute toxicity test with mysids (SLI Report # 944-5269), spectral
28、 analysis was conducted on 20 July 1994 on each of the remaining two lots (lot # 0281482 and lot # 07905K). The spectral analysis conducted on 20 July 1994 on the two remaining lots in comparison to the initiai spectral analyses of lot # 0281482 established that negligible change in test material co
29、mposition had occurred during storage at Springborn Laboratories, Inc. (Le., approximately 24 months). 3 Test Organisms Rainbow trout (Oncorhynchus mykiss) were selected as the test species since it is a recommended species (US. EPA, 1985) and a commonly used cold water fish in flow-through freshwat
30、er fish toxicity tests. The rainbow trout (SU lot #93B3) used during the study were obtained from Spring Creek Trout Hatchery, a commercial supplier located in Lewistown, Montana. Prior to testing, these fish were held in a 500-L fiberglass tank under a photoperiod of 16 hours light and 8 hours dark
31、ness. The well water which flowed into this holding tank was characterized as having total hardness and total alkalinity ranges as calcium carbonate (CaCOJ of 25 - 27 mg/L and 20 - 22 mg the dissolved oxygen concentration was measured with a YS1 Model #57 dissolved oxygen meter and probe and the dai
32、ly solution temperature was measured with a Brooklyn alcohol thermometer. Light intensity was measured with a General Electric Model 214 light meter. Test solution temperature was continuously monitored in one replicate (B) of the dilution water control solution using a Brooklyn Min/Max thermometer.
33、 3.4 Analytical Measurements Both replicate solutions of the high, middle and low treatment levels and the control were sampled and analyzed for TAME concentration prior to the start of the definitive exposure. Results of these pretest analyses were used to judge whether sufficient quantities of tes
34、t material were being delivered and maintained in the exposure aquaria to initiate the definitive test. During the in-life phase of the definKi8 study, water samples were removed from both replicate test solutions of each treatment level and the controls at - and 96-hours of exposure for analysis of
35、 TAME concentration. Each exposure solution sample was collected from the approximate midpoint of the aquarium with a volumetric pipet. In addition, three Quality Control (QC) samples were prepared at each sampling interval and remained with the exposure solution samples throughout the analytical pr
36、ocess. These QC samples were prepared in dilution water at TAME concentrations similar to the exposure concentration range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples. All samples were ana
37、lyzed for TAME using a gas chromatographic (GC) procedure according to the methodology described in Appendix V. A method validation study, conducted at SLI prior to the initiation of the definitive test, established an average recovery of TAME of 102 * 10% from hard reconstituted water. Springborn L
38、aboratories, Inc. API TRr408 95 = 0732290 0555082 75b = Report No. 93-3-4682 Page 15 of 72 4.0 STATISTICS The mean measured concentrations (O- and 96-hour analysis) and the corresponding mortalit)r data derived from the definitive test were used to estimate the median lethal concentration (LC50) and
39、 95% confidence interval at each 24-hour interval of the exposure period. The LC50 is defined as the concentration of the test material in dilution water lethal to 50% of the test animal population at the stated exposure interval. if at least one test concentration caused mortality of greater than o
40、r equal to 50% of the test population, then a computer program (Stephan, 1982, personal communication) was used to calculate the LC5O values and 95% confidence interval. Additionally, the data was evaluated to estimate an incipient LC50. The incipient LC50 is defined as the concentration that is let
41、hal to 50% of a test population when exposure to the test substance is continued until the mean increase in mortality does not exceed 10% in any concentration over a 24-hour period. Since the 96-hour LC50 was not less than 50% of the estimated 48-hour LC50, the duration of the exposure was not conti
42、nued to determine the incipient LC50 value (US. PA, 1987). Three statistical methods were available in the computer program: moving average angle analysis, probit analysis, and nonlinear interpolation with 95% confidence interval calculated by binomial probability. Moving average angle and probit an
43、alyses yield statistically sound results only if at least two concentrations produce a mortality of between O and 100% of the test organism population. The selection of reported LC50 values and 95% confidence interval was based upon an examination of the data base and the results of the computer ana
44、lysis. Selection criteria included the establishment of a concentration-effect relationship (mortality), the number of concentrations causing partial responses, and the span of responses bracketing the LC50 value. if two or more statistical methods produced acceptable results, then the method which
45、yielded the smallest 95% confidence interval was selected. The No-Observed-Eff ect Concentra- tion (NOEC) during the 96-hour exposure period was also determined. The NOEC is defined as the highest concentratirw tested at and below which there were no toxicant-related mortalities or physical and beha
46、vioral abnormalities (e.g., lethargy, loss of equilibrium, darkened pigmentation) , with respect to the control organisms. Springborn Laboratories, Inc. API TR*4Q8 95 U 0732290 0555083 692 Report No. 93-3-4682 Page 16 of 72 5.0 RESULTS 5.1 Preliminary Testing Prior to initiating the definitive study
47、, several preliminary range-finding studies were conducted at SLI. During these preliminary studies, the test material was delivered to the test aquaria using conventional delivery methods in a Benoit-style diluter (.e., mixing chamber, chemical cells). Test vessels were not covered during any of th
48、e exposure. In three separate tests, rainbow trout were exposed to nominal concentrations of TAME ranging from 570 - 15, 11 00 - 89 and 1700 - 130 mg A.I./L After 120 hours of exposure, no toxicant related mortality or sublethal effects were observed among fish exposed to 570 - 15 mg A.I./L treatmen
49、t levels. Following 72 hours of exposure at treatment levels of 1 100 - 89 mg A.I./L, 10% was observed among fish exposed to the highest treatment level. Surviving fish at this treatment exposure concentration (1 1 O0 mg A.I./L) were described as exhibiting darkened pigmentation and complete loss of equilibrium. During the third exposure, mortality of 80% was observed among fish exposed to the 1700 mg A.I./L treatment level following 24-hours of exposure. No mortality or sublethal effects were noted among organisms exposed to the remaining test concentratio
