Acute Toxicity of Fire Control Chemicals to Daphnia magna (Straus) and Selenastrum capricornutum (Printz)
Materials and Methods
Culture
D. magna were cultured following established methods recommended by the American Society for Testing and Materials (ASTM) (ASTM, 1984, 1992b). Culture medium was well water at the Yankton Field Research Station, South Dakota (hardness, 264-284 mg/liter as CaCO3; alkalinity, 166 mg/liter as CaCO3; pH 8.56).
S. capricornutum cells were obtained from the University of Texas Algal Collection (Austin, TX; strain UTEX 1648), and were transferred aseptically to ASTM algal assay medium (continuous photoperiod 4519 ± 182 lumens/m2; 26 ± 1°C, pH 7.5 ± 0.1), following established methods (Miller et al., 1978; ASTM, 1992a). Initial algal culture was inoculated 2 weeks prior to test initiation. Subsequent cultures were inoculated every 4 days to maintain log-growth phase.
Dilution Water
ASTM soft (hardness 42 mg/liter as CaCO3; alkalinity 32 mg/liter as CaCO3; pH 7.60) and hard (hardness 162 mg/liter as CaCO3; alkalinity 112 mg/liter as CaCO3; pH 8.23) waters were used in tests with daphnids (ASTM, 1989). The water was reconstituted in blending tanks by adding the appropriate amount of salts to reverse osmosis purified and deionized water. All dilution water was analyzed prior to use to determine adherence to ASTM guidelines (ASTM, 1989).
Algal acute toxicity tests were conducted in ASTM algal assay medium (ASTM, 1992a). Medium was prepared by pipetting aliquants of nutrient stock solutions into deionized water.
Test Chemicals
Three non-foam retardant and two foam suppressant chemicals were tested. All are proprietary products, therefore no compositional analysis is available; a listing of chemical components is given in Table 1. The non-foam retardants were Fire-Trol GTS-R1, Fire-Trol LCG-R, and Phos-Chek D75-F. The foam suppressants included Phos-Chek WD-881 and Silv-Ex. These fire retardant test chemicals were obtained from the USDA Forest Service, Intermountain Fire Sciences Laboratory (Missoula, MT).
Acute Toxicity Testing
D. magna. Acute toxicity tests with Daphnia magna were conducted following established methods (ASTM, 1989). Daphnids (<24-hr old) were tested in 48-hr static acute toxicity tests. In each test, 10 animals were exposed to each of eight or nine toxicant concentrations with a 60% dilution factor between test concentrations plus a control treatment. Tests were conducted in 250-ml beakers containing 200 ml of dilution water. Test chemicals were added directly or by pipetting from a stock solution prepared with deionized water. The number of affected daphnids in each test vessel was monitored at 24-hr intervals. An EC50 based on immobilization was determined at 24 and 48 hr of exposure. Dissolved oxygen (YSI [Yellow Springs, OH] Model 58 DO meter) and pH (Orion [Boston, MA] Model SA250 pH meter) were monitored in the control, low, medium, and high test concentrations at 0 and 48 hr of exposure.
S. capricornutum. Acute toxicity tests with S. capricornutum were conducted following established methods (ASTM, 1992a). S. capricornutum in log- growth phase was used in 96-hr static acute toxicity tests with separate waterborne test chemicals. Algae tests were conducted using six toxicant concentrations with a 60% dilution factor between test concentrations plus a control treatment. Tests were conducted in 250-ml Erlenmeyer flasks containing 125 ml ASTM algal assay medium (pH 7.5 ± 0.1) inoculated with 2.0 × 104 cells/ml. Each treatment had three replicates. Test chemicals were added by pipetting from a stock solution prepared with deionized water.
The effect criterion for algal tests was inhibition of cell reproduction as indicated by chlorophyll a production. The 96-hr IC50 was calculated using results from chlorophyll a analysis as standing crop. At test termination, each flask was sampled, chlorophyll a extracts were prepared in vitro with 90% aqueous acetone buffered with MgCO3, and fluorometric analysis was conducted using a Turner Designs Model 10-AU fluorometer (APHA, 1989).
Ammonia Analysis
Ammonia analysis was conducted on low, medium, and high test concentrations and the control treatments of each test. Because the test volumes were low in test vessels with daphnids, identical test vessels without daphnids were prepared for ammonia analysis and held in the same manner as for tests with daphnids. Analyses of ammonia concentrations were conducted at 0-hr and 48-hr for the daphnid tests and 0-hr and 96-hr for the algal tests. Ammonia was measured in the samples collected from test vessels containing algal cells. Analyses of ammonia concentrations were conducted at 0 and 48 hr for the daphnid tests and 0 and 96 hr for the algal tests. Concentrations were determined with an Orion 95-12 ammonia electrode attached to a Fisher Acumet model 610 pH meter reading out relative millivolts. A regression equation was determined for each set of daphnid tests to allow prediction of ammonia as nitrogen (NH3-N) at the 48-hr EC50. This equation was determined by regressing the NH3-N values in the low, medium, and high treatments against the corresponding concentrations of the test chemical.
Nitrate and Nitrite Analysis
Nitrate and nitrite concentrations were determined colorimetrically in water samples for exposures conducted after tests with daphnids and algae were conducted. Nitrate concentrations were determined using a cadmium reduction procedure and standard additions method, and nitrite concentrations were determined using a ferrous sulfate procedure and standard additions method (Hach 1992). Analysis was conducted on 48-hr EC50 concentrations of the non-foam fire retardants Fire-Trol GTS-R, Fire-Trol LCG-R, and Phos-Chek D75-F; foam suppresants were not tested. Each test solution was retained in 250-ml beakers to simulate the conditions under which ammonia concentrations were determined during the actual exposure.
Statistical Analysis
The moving average-angle method (Peltier and Weber, 1985), using a computer program prepared by the Environmental Monitoring and Support Laboratory of USEPA (Cincinnati, OH), was utilized to calculate the EC50, IC50, and their 95% confidence intervals for each species. In tests with no partial mortality, the binomial method was used to calculate the EC50 and the upper 95% confidence limit was the lowest test concentration with 100% mortality and the lower 95% confidence limit was the highest test concentration with 0% mortality. All EC50 and IC50 values are expressed as nominal concentrations of the fire control chemical. The standard error of the difference (Zar, 1974; Sprague and Fogels, 1977) was calculated to determine significant differences (P = 0.05) between the EC50 or IC50 for each set of tests. Regression analyses for the ammonia data were calculated using Lotus 1-2-3 and Statistical Analysis System programs (SAS, 1990).
1References to trade names, commercial products, or manufacturers do not imply or constitute government endorsement or recommendation for use.
Previous Section -- Introduction
Return to Contents
Next Section -- Results
