Northern Prairie Wildlife Research Center
Table 1. Life Stages and Mean Weight (g) and Total Length (mm) and range (in Parentheses) of Fathead Minnow Tested with Five Firefighting Chemical Formulations in ASTM Soft and Hard Water
|Life stage||Water type||Age||Weight||Total length|
aDays postfertilization at test initiation.
bPooled weight of 10 eggs.
cDays posthatch (DPH) at test initiation.
dPooled weight of 10 fry.
eN = 10.
fN = 20 (combined hard and soft control treatments).
Three fire retardants and two foam suppressants used as test chemicals were received from the USDA Forest Service, Intermountain Fire Sciences Laboratory, Missoula, Montana. All test concentrations and subsequent LC50s were based on a 100% active formulation because the formulations are proprietary products and the proportional composition of the formulations was unknown.
Fire-Trol GTS-R (FT GTS-R; lot 84FT232) is a long-term, dry fire-retardant formulation manufactured by Chemonics, Inc. It is composed of ammonium sulfate, diammonium phosphate, guar gum thickener, spoilage inhibitor, corrosion inhibitor, and iron oxide as a red coloring agent (Chemonics, 1992a).
Fire-Trol LCG-R (FT LCG-R; lot 91FT11) is a long-term, liquid fire-retardant formulation manufactured by Chemonics, Inc. It is composed of ammonium polyphosphate, attapulgite clay thickener, corrosion inhibitor, and iron oxide as a red coloring agent (Chemonics, 1992b).
Phos-Chek D75-F (PC D75-F; lot 2468762A) is a long-term, dry fire-retardant formulation manufactured by Monsanto Company. It is composed of ammonium sulfate, ammonium phosphate, guar gum thickener, orange coloring agent, and other additives (Monsanto, 1991).
Phos-Chek WD-881 (PC WD-881; lot 3616836A, batch 18227) is a short-term, liquid fire-suppressant foam formulation produced by Monsanto Company. Its formulation consists of a mixture of anionic surfactants, alcohol, foam stabilizers, and inhibiting additives (hexylene glycol) (Monsanto, 1990).
Silv-Ex (lot 75451, batch US6203) is a short-term liquid fire-suppressant foam formulation manufactured by Ansul Fire Protection. It contains anionic surfactants, alcohol, and solvents (diethylene glycol monobutylether) (Ansul, 1991).
Test solutions were prepared with concentrated chemicals by directly adding appropriate amounts of FT GTS-R, FT LCG-R, and PC D75-F to the test vessel. FT GTS-R and PC D75-F were mixed two to three times for 40 - 50 sec at 1000 - 1200 rpm using a three-blade, polyethylene stirrer attached to an electric drill. This mixing was usually sufficient to bring the test formulation into solution for a short time, but after 24 hr of exposure some inert ingredients (guar gum thickener and colorants) had precipitated. FT LCG-R was mixed in a similar fashion; however, it was mixed only once for 40 - 50 sec to prevent foaming. PC WD-881 and Silv-Ex were added by preparing stock solutions in deionized water and then pipetting an appropriate aliquot to each test vessel. Both PC WD-881 and Silv-Ex were gently hand mixed with teflon or glass stir rods to prevent foaming.
Static acute toxicity tests were conducted in standardized reconstituted hard and soft water (ASTM, 1989). Dilution water was prepared by adding appropriate amounts of reagent grade mineral salts (CaSO4·2H2O, MgSO4, NaHCO3, and KCl) to known amounts of purified water. Purified water was prepared by passing well water through a water softener, reverse-osmosis unit, and deionizer. After mixing and aeration of the salts in the purified water, chemical characteristics of dilution water were determined according to standard methods (APHA, 1989; USEPA, 1979). Water chemical parameters for dilution water are presented in Table 2.
Table 2. Mean Measured Water Quality Characteristics and Standard Deviation (in Parentheses) of Test Water Used in Tests with Four Life Stages of Fathead Minnow Exposed to Five Firefighting Chemical Formulations in ASTM Soft and Hard Water.
|pH||7.5 (0.0)||8.2 (0.1)|
|Conductivity (µmhos/cm at 25°C)||163 (3)||544 (12)|
|Hardness (mg/liter as CaCO3)||42 (2)||163 (2)|
|Alkalinity (mg/liter as CaCo3)||33 (1)||112 (1)|
|Calcium (mg/liter)||7 (10)||28 (1)|
|Magnesium (mg/liter)||6 (1)||23 (1)|
|Chloride (mg/liter)||1 (0)||3 (0)|
|Sulfate (mg/liter)||39 (3)||175 (19)|
|Note. N = 4.|
Static acute tests were conducted according to standard methods (ASTM, 1989). Each test consisted of exposing groups of 10 fish to a series of seven or eight toxicant concentrations, and one control exposure. There was a 60% dilution factor between each toxicant concentration. One control exposure was used in each set of life stage tests and in each water quality tested.
Eggs, fry, 30-DPH (days post-hatch), and 60-DPH juveniles were tested in 3.9-liter glass test vessels containing 3 liter of dilution water. During the testing of eggs and fry, test organisms were confined in 0.47-liter glass jars with the bottom removed and replaced with Nytex screen fixed in place with silicone. These hatching cups were suspended by latex tubing from glass rods so that eggs or fry were in the test solution but the cup rim was above the waterline.
Eggs were tested at 20 ± 1°C to ensure that the egg life stage would be maintained through the test. Temperature was maintained at 25 ± 1°C for fry and juveniles by immersion of the test vessels in temperature-controlled waterbaths.
Egg acclimation was begun immediately upon arrival to both temperature and dilution water and testing was begun 24 hr later. Fry were also acclimated over a 24-hr period prior to testing but were fed brine shrimp twice during the first 12 hr of acclimation. Juveniles were acclimated over a 96-hr period and were not fed during acclimation.
Eggs and fry were randomly transferred by glass pipet from the acclimation chamber to plastic weigh boats filled with dilution water to minimize handling stress. Excess water was then decanted from the weigh boats and test organisms distributed to a randomized test vessel. Juveniles were randomly distributed two at a time to randomized test vessels using a small dip net.
Mortality and abnormal behavior were monitored and recorded at 24-hr intervals during tests and dead fish were removed at those intervals. Eggs and fry were examined with a 30X dissecting microscope for the presence of a heart beat, which was the criterion for mortality in these tests. Cessation of opercular movement was the criteria for mortality in juveniles.
At the conclusion of each test, all control eggs or fish were weighed and total length measured. Mean weight and mean total length of life stages tested are given in Table 1.
Dissolved oxygen and pH were measured and recorded in the control, low, medium, and high concentrations of each test with live fish present at 0, 48, and 96 hr of exposure. Dissolved oxygen was measured with a YSI model 58 dissolved oxygen meter and probe with a standard membrane. pH was measured with an Orion SA250 portable pH meter with a Ross pH electrode and automatic temperature compensation electrode.
Ammonia was measured in 100-ml samples of the control, low, medium, and high test concentrations containing live fish. Concentrations were determined with an Orion 95-12 ammonia selective electrode attached to a Fisher Accumet model 610 pH meter reading relative millivolts. A regression equation was determined for each set of tests to allow prediction of ammonia as nitrogen (NH3-N) at 96-hr LC 50; regression equations are given in Gaikowski (1994). Equations were determined by regressing the NH3-N estimates for the low, medium, and high concentrations of the chemical being tested. The 0-hr NH3-N estimates were used for data analysis to minimize the influence of ammonia contribution from test fish or volatization of ammonia contributed by the formulation. Un-ionized ammonia concentrations were derived by determining the estimated NH3-N concentration from the regression equation coefficients and the 96-hr LC50 concentration because actual NH3-N and pH measurements could not be performed at the calculated 96-hr LC 50 concentration. The percentage of un-ionized ammonia was estimated by using the high and low measured pH recorded at test initiation. The two percentage values were then used to determine the un-ionized ammonia range.
Nitrate and nitrite concentrations were determined colorimetrically using a cadmium reduction procedure and standard additions method (Hach, 1992). Each sample was analyzed in duplicate. Standard additions to nitrate and nitrite samples were 0.2, 0.4, and 0.6 ml nitrate-nitrogen standard solution (100 mg/liter as NO3--N) and 0.4, 0.6, and 0.8 ml nitrite-nitrogen standard solution (100 mg/liter as NO2--N). Each standard addition was analyzed in duplicate. Analysis was conducted on concentrations of three fire-retardant chemicals: FT GTS-R, FT LCG-R, and PC D75-F; foam suppressants were not tested. Chemical was added to test vessels matching those used in actual tests at 25°C, but without fish. Fire-retardant solutions were prepared to simulate exposure at the 96-hr LC50 concentrations of the fry life stage. A regression equation was determined and was used to calculate the nitrate and nitrite concentrations at the 96-hr LC50 for the egg, 30-DPH, and 60-DPH life stages.
Most LC50 values and 95% confidence intervals were calculated using the moving-average angle method (Peltier and Weber, 1985). This method requires at least two concentrations with >50% mortality above the LC50. In tests with less than two concentrations above the LC50, the binomial distribution method was used (Peltier and Weber, 1985). In tests with no partial mortality, 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 LC50 values are expressed as nominal concentrations of the firefighting chemical. The Friedman test (Conover, 1980) was used to rank the test chemicals from most to least toxic and life stages from most to least sensitive. Significant differences (P = 0.05) were determined with Friedman's multiple comparison test. The standard error of the difference as described by Sprague and Fogels (1977) and Zar (1974) was used to determine statistical differences (P = 0.05) between individual LC50 values. The Wilcoxon signed rank test (Conover, 1980) was used to determine the overall difference between water type on the toxicity of the test chemicals.
2References to trade names, commercial products, or manufacturers do not imply or constitute government endorsement or recommendation for use.