Acute Toxicity of Fire-Retardant and Foam-Suppressant Chemicals to Hyalella azteca (Saussure)
Materials and Methods
Culture
This study was conducted at the Yankton Field Research Station (YFRS) of the Columbia Environmental Research Center, Columbia, Missouri, USA. Hyalella azteca were intensively cultured based on established methods [11,12]. The culture media (hardness 725 mg/L as CaCO3, alkalinity 518 mg/L as CaCO3, pH 8.72) were replenished with YFRS well water to replace evaporation losses. The culture aquaria were placed in a temperature-controlled water bath with the temperature maintained at 20 ± °C. An artificial substrate of nylon coiled-base web material (3M, St. Paul, MN, USA) was placed in the bottom of each aquarium. A layer of hard maple leaves was scattered over the artificial substrate. Maple leaves were soaked in well water for at least 30 d to soften them, to remove tannins, and to initiate periphyton growth. Once weekly, a handful of TetraMin ®, flake food was crushed and scattered over the water surface for the amphipods to feed on ad libitum. The culture was provided with a 16:8-h light: dark photoperiod.
Dilution water
Standardized soft (hardness 41 mg/L as CaCO3, alkalinity 32 mg/L as CaCO3, pH 7.45) and hard (hardness 162 mg/L as CaCO3, alkalinity 111 mg/L as CaCO3, pH 8.36) waters were used in tests with amphipods [13]. The water was reconstituted in blending tanks by adding the appropriate amounts of reagent grade salts to deionized water. All dilution water was analyzed for general chemical characteristics according to standard methods [14] prior to use to verify adherence to established methods [13].
Test chemicals
The fire-retardant chemicals tested were Fire-Trol LCG-R (FT LCG-R), Fire-Trol GTS-R (FT GTS-R), and Phos-Chek D75-F (PC D75-F), and the foam-suppressant chemicals were Phos-Chek WD-881 (PC WD-881) and Silv-Ex.
All are proprietary products; therefore, proportional compositional analyses were not available. Consequently, all test concentrations and subsequent mean lethal concentrations (LC50s) were based on a 100% active formulation. A general listing of the components of the chemicals is given in Appendix 1. Test chemicals were obtained from the U.S. Forest Service, Intermountain Fire Sciences Laboratory, Missoula, Montana, USA.
Acute toxicity testing
Acute toxicity tests conducted with H. azteca were based on established methods [13]. Mature H. azteca (0.677 ± 0.239 mg dry weight) were tested in 96-h static acute toxicity tests with separate waterbome test chemicals. In each test, 10 animals were exposed to each of eight or nine toxicant concentrations with a 60% dilution factor between each concentration, in addition to exposure to a control treatment. One control exposure was used for each chemical and each water quality tested.
Three days prior to testing, the appropriate number of mature amphipods were transferred by pipetting them into an acclimation vessel containing approximately 4 L of culture water. At the time of transfer, the amphipods were fed crushed Tetra-Min ® flake food ad libitum. The amphipods were provided with gentle aeration during acclimation. Acclimation began 48 h prior to testing and was accomplished by removing 50% of the culture water and replacing it with an equal quantity of dilution water twice daily.
During preliminary testing in our lab and in reports by others [15], H. azteca exhibited cannibalism when 10 animals were placed in a single test vessel. To prevent cannibalistic behavior, amphipods were tested in a single-animal per exposure-vessel system. Test apparatuses were constructed from a 20- × 26-cm plexiglass sheet with 20 holes, 3.8 cm in diameter, to hold 30-ml disposable plastic cups. The plexiglass was supported by four 75-cm long brass screws and covered with a 20- × 26-cm plexiglass sheet to prevent evaporation of the test solution. The test concentration was prepared in 2-L beakers containing 1 L of dilution water. Glass volumetric pipettes were used to transfer 20 ml of test solution into each cup. Each test concentration was randomly assigned to either half of five stands and a control was included for each chemical tested. One animal was placed in each cup with 10 cups per test concentration. Transfer of the animals was accomplished by holding a pipette with an animal below the surface and allowing the animal to swim out of the pipette.
The number of affected amphipods in each test vessel was monitored at 24-h intervals. The effect criterion for H. azteca was death as defined by the American Society for Testing and Materials (ASTM) [13].
Dissolved oxygen, pH, and ammonia were measured in the control and low, medium, and high test chemical solutions at 0, 48, and 96 h. Because there was not enough chemical solution in the exposure vessels, dissolved oxygen and pH were measured directly in the test solution remaining in the 2-L beakers, which were covered with plastic wrap to simulate the covered exposure vessels. Ammonia concentrations were analyzed in 100 ml of the solution remaining in the 2-L beakers. When the tests were terminated, the exposure water was pooled and a sample was taken for ammonia analysis. Temperature was measured daily in the waterbath.
The size of H. azteca was determined gravimetrically. After test termination, control animals were placed in predried and preweighed 43-mm aluminum weigh boats and placed in a 60°C oven for 24 h until a constant weight was attained.
Ammonia analysis
Total ammonia concentrations, measured as nitrogen (NH3-N) concentrations, were taken with an Orion 95-12 electrode (ATI Orion, Boston, MA, USA) using a Fisher Accumet model 610 pH meter and were adjusted for temperature and pH to determine the concentrations of un-ionized ammonia. A regression equation was determined for each test to allow the prediction of the NH3-N concentration that would be present at the time the test was initiated for the 96-h LC50 concentration. This equation was determined by regressing the NH3-N values in the low, medium, and high test concentrations at the time the test was initiated against the corresponding 96-h LC50 for the test chemical. The un-ionized ammonia concentration in the 96-h LC50 cannot be predicted because the pH of the solution is unknown.
Statistical analysis
The moving-average angle method was used to calculate the 96-h LC50 and 95% confidence intervals [16]. Regression analyses for the ammonia data were calculated using Lotus 1-2-3 and Statistical Analysis System programs [17]. The standard error of the difference was calculated to determine significant differences (p > 0.05) and rank order between the LC50 for each set of tests [18]. All LC50 values are expressed as nominal concentrations of the fire-control chemical.
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