23 Concentrations of TAME measured in replicate A$ test solutions during the 48-hour flow-through exposure of daphnids Daphniamagna.. 24 Mean measured concentrations tested, correspondin
Protocol
Procedures used in this acute toxicity study followed those described in the SLI protocol entitled "Protocol for Conducting a Flow-Through Acute Toxicity Test with Daphnia magna
The protocol outlined in TSCA 5797.1 300, along with SLI Protocol #: O91 192/TSCA 797.1 300 DM-FA and its Amendment # 1 from December 15, 1992, adheres to the EPNOTS guidelines for assessing the impact of chemicals on daphnids It complies with the TSCA guidelines specified in the relevant Registration Standard and is designed to fulfill the premanufacture notice (UPMNII) registration requirements.
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 #
On August 17, 1992, sample 0251 462 was received at SLI and utilized for preparing exposure solutions during preliminary exposure, analytical standards for method validation recovery studies, and Quality Control samples for definitive exposure.
Aldrich Chemical Company reported that the first sample contained 98.8% active ingredient A.I (Certificate of Analysis, Appendix II) The second sample, Lot # 07905Kz, was received at SLI on November 2, 1992, and was utilized to prepare exposure solutions for the definitive exposures, with an active ingredient A.I content of 98.7% as identified by Aldrich Chemical Company (Certificate of Analysis).
Upon arrival at SLI, the test material samples were stored in a dark, ventilated cabinet at approximately 20°C The test concentrations are measured in milligrams of active ingredient per liter of test solution, reported as mg A.I./L.
At the request of the Study Sponsor, mass spectral analysis was performed on the initial batch of TAME and subsequent batches throughout the program to assess test material integrity The initial evaluation of lot # 0281482 took place on December 3, 1992 Following the flow-through acute toxicity test with mysids, spectral analysis of the remaining two lots (lot # 02814BZ and lot # 07905KZ) was conducted on July 20, 1994 The comparison of these analyses with the initial evaluation of lot # 0281482 indicated that there was negligible change in the test material composition during the approximately 24 months of storage at Springborn Laboratories, Inc.
Test Organisms
Daphnia magna used in the toxicity test were sourced from laboratory cultures at Springborn Laboratories, Inc in Wareham, Massachusetts The culture water was created by fortifying well water according to the hard water formula established by the U.S EPA in 1975, followed by filtration through an Amberlite XAD-7 resin column and a carbon filter Prior to the test, this water maintained total hardness and total alkalinity measured as calcium carbonate (CaCO₃).
160 mg/L anri ? !.O mg/L, respectively, a pH of 8.1, a specific conductivity range 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
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Report No 92-12-4545 Page 11 of 67 darkness tight intensity in the culture area ranged from 32 - 46 footcandles (Invertebrate Culture
In the culture area, a waterbath was utilized to maintain the temperature of the culture solution at 20 ± 2 °C Daphnids were fed daily with a mixture of trout food suspension and unicellular green algae (Ankistrodesmus falcarus), consisting of 5.0 mg/mL of trout food and approximately 4 × 10^6 cells/mL of algae Samples of the food source were analyzed for pesticides, PCBs, and toxic metals.
Food sources were considered to be of acceptable quality since the total concentration of pesticides measured was less than 0.3 mghg (ASTM, 1985)
The dilution water utilized in this study was sourced from the same location as the culture water, exhibiting a total hardness of 160 mg/L and total alkalinity (CaCO₃) of 110 mg/L, with a pH range of 8.1 to 8.2 and a specific conductivity of 500 µmhos/cm (IWQ Log Book, Vol 13) Prior to use, the dilution water was continuously aerated, and representative samples were analyzed for pesticide presence.
PCBs and toxic metals were not detected at toxic concentrations in any analyzed water samples, aligning with ASTM standard practice Monthly analyses of the dilution water source revealed total organic carbon (TOC) concentrations ranging from 0.97 to 2.2 mg/L between June and November 1992 Daphnid cultures, maintained in the same dilution water, have thrived and reproduced over multiple generations, further confirming the suitability of this water for bioassays.
The toxicity test was conducted using an exposure system that included an intermittent flow proportional diluter and a set of 12 exposure vessels This system was specifically designed to deliver five different concentrations of the test material along with dilution water.
The exposure vessels were kept in a well-lit area using Duro-Test Cool-White and Vitalite fluorescent lights, maintaining an intensity of 45 to 70 footcandles The photoperiod matched that of the culture area, ensuring gradual transitions between light and dark The tests were conducted in a temperature-controlled room and water bath, keeping the test solution temperatures at 20 ± 2 °C Each treatment level and control had two replicate vessels, which were clearly labeled to indicate the nominal test material concentration and replicate designation.
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
Before starting the test, a 50 mL Glenco gas-tight syringe, paired with a Sage syringe pump (Model # 355), was calibrated to deliver 0.416 mUcycle of the test material (760 mg/mL) directly into the diluter system's chemical mixing chamber, which also received 0.456 L of dilution water per cycle The mixing chamber, placed over a magnetic stirrer, continuously mixed the contents to enhance the solubilization of the test material The solution in the mixing chamber represented the highest nominal treatment level (690 mg A.I./L) and was subsequently diluted by a factor of 60% to achieve the remaining nominal test concentrations of 410, 250, 150, and 89 mg A.I./L.
The diluter system delivered approximately 50 mL of exposure solution to each replicate test vessel during each cycle, operating around 216 times daily Prior to the test, the system was calibrated by measuring the delivery volumes of both toxicant and dilution water Throughout the study, visual inspections and TAME concentration analyses of the exposure solutions ensured the proper functioning of the diluter system To facilitate the equilibration of the test material, the exposure system was operational for nine days before the test began.
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Report No 92-12-4545 Page 13 of 67 capillary tubes with an approximate length of five centimeter (cm) and a diameter of 1 -millimeter
Silicone stoppers were used to insert tubing with an inside diameter of (mm) into the mixing and splitting chambers of the diluter, directing the test solutions into delivery tubes This tubing was designed to restrict the flow of test solutions, thereby minimizing turbulence in the exposure vessels and ensuring equal distribution to replicate vessels Each glass test vessel maintained a consistent solution volume of 1.8 L and a depth of approximately 13 cm.
Each replicate vessel underwent around 6.0 solution volume replacements daily The diluter system's performance, including flow rates and stock consumption, was monitored on a daily basis, with visual inspections conducted twice a day to ensure proper functionality throughout the study period.
The test was initiated when 10 daphnids (S 24 hours old) were impartially selected and introduced to each replicate exposure vessel (20 per treatment level and the control)
The immobilization of daphnids was recorded at 3, 6, 24, and 48 hours during the exposure period, with daphnids deemed immobile if no movement, aside from minor appendage activity, was observed after gentle prodding Additionally, biological observations, including any abnormal behavior or appearance of the test organisms, as well as the physical characteristics of the test solutions, such as precipitate or surface film, were documented at the start and at each time interval It is important to note that daphnids were not fed throughout the 48-hour definitive exposure.
Dissolved oxygen concentration, temperature, and pH levels were recorded daily in both replicate vessels for each treatment level and the control throughout the exposure period Additionally, total hardness, total alkalinity, and specific conductance were assessed at the start of the test in one replicate vessel for each treatment level and the control solution.
The measurements presented in this report utilized the EDTA titrimetric method, while total alkalinity concentrations were determined through potentiometric titration, reaching an endpoint at pH 4.5.
In 1985, a Jenco Model 601 A pH meter with a combination electrode was utilized to measure pH levels Specific conductivity was assessed using a Yellow Springs Instrument Company (YSI) Model #33 salinity, conductivity, and temperature meter The dissolved oxygen concentration was determined with a YSI Model #57 dissolved oxygen meter, while daily solution temperature was recorded using a Fisher alcohol thermometer Continuous temperature monitoring was conducted in one replicate.
(B) of the 690 rng A.I./L test solution (nominal) using the Omega Data Acquisition System (ODAS) tight intensity was measured with a General Electric type 214 light meter
Prior to the definitive exposure, both replicate solutions of high, middle, and low treatment levels, along with the control, were sampled and analyzed for TAME concentration The results from these pretest analyses determined if adequate TAME quantities were delivered to the test vessels and if the appropriate test concentrations were maintained During the in-life phase of the definitive study, water samples were collected from each treatment level and the control at 0 and 48 hours for TAME concentration analysis, using a volumetric pipet from the midpoint of the test vessel.
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.
Exposure Solution Preparation
Before starting the test, a 50 mL Glenco gas-tight syringe and a Sage syringe pump (Model # 355) were calibrated to deliver 0.416 mUcycle of the test material (760 mg/mL) to the diluter system's chemical mixing chamber, which also received 0.456 L of dilution water per cycle The mixing chamber, placed over a magnetic stirrer, continuously mixed the contents to aid in solubilizing the test material The solution in the mixing chamber represented the highest nominal treatment level (690 mg A.I./L) and was diluted by a factor of 60% to create the remaining nominal test concentrations of 410, 250, 150, and 89 mg A.I./L.
The diluter system delivered approximately 50 mL of exposure solution to each replicate test vessel during each cycle, operating around 216 times daily Prior to the test, the system was calibrated by measuring the delivery volumes of both toxicant and dilution water Throughout the study, visual inspections and TAME concentration analyses of the exposure solutions ensured the diluter system's proper functioning The system was confirmed to be operational for nine days before the test began, allowing for the equilibration of the test material in both the diluter apparatus and exposure vessels.
Provided by IHS under license with API
Report No 92-12-4545 Page 13 of 67 capillary tubes with an approximate length of five centimeter (cm) and a diameter of 1 -millimeter
Silicone stoppers were used to insert tubing with an inside diameter of (mm) into the mixing and splitting chambers of the diluter, directing the test solutions into delivery tubes This tubing was designed to restrict the flow of test solutions, thereby minimizing turbulence in the exposure vessels and ensuring equal distribution to replicate vessels Each glass test vessel held a constant solution volume of 1.8 L, with a solution depth of approximately 13 cm.
Each replicate vessel underwent around 6.0 solution volume replacements daily The diluter system's performance, including flow rates and stock consumption, was monitored on a daily basis, with visual inspections conducted twice a day to ensure proper functionality throughout the study period.
Test Initiation
The test was initiated when 10 daphnids (S 24 hours old) were impartially selected and introduced to each replicate exposure vessel (20 per treatment level and the control).
Test Monitoring
The immobilization of daphnids was recorded at 3, 6, 24, and 48 hours during the exposure period, with daphnids deemed immobile if no movement, aside from minor appendage activity, was observed after gentle prodding Additionally, biological observations, including any abnormal behavior or appearance of the test organisms, as well as the physical characteristics of the test solutions, such as precipitate or surface film, were documented at the start of the test and at the specified time intervals It is important to note that daphnids were not fed throughout the 48-hour definitive exposure.