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Culture

This study was conducted at the Yankton Field Research Station (YFRS) of the Columbia Environmental Research Center, Columbia, Missouri, USA. Table 1 lists for each life stage of rainbow trout (Oncorhynchus mykiss) the age at test initiation, lot number, mean weight, and mean total length. Fish were cultured at YFRS in aerated well water (hardness 290-1100 mg/L as CaCO₃, alkalinity 207-275 mg/L as CaCO₃, pH 7.7-8.1). Two lots of rainbow trout were obtained from Ennis National Fish Hatchery, Ennis, Montana, USA, as eyed eggs. Both lots of rainbow trout were of the same strain (McConaughy), to minimize potential variation in sensitivity [21].

Table 1. Life stages of rainbow trout tested with five fire-fighting chemical formulations in ASTM^a soft and hard reconstituted water.

^aASTM = American Society for Testing and Materials.
^bDays post median hatch day (dph) to test initiation.
^cMeasurements are mean values (with range in parentheses) of control fish.
^dAverage of two pools of 10 eggs each.
^eVolume displacement; d = diameter.
^fN = 20 (pool of hard and soft control treatments).
^gN = 20.
^hN = 30.

The life stages tested are defined as follows: eyed eggs with distinct eye spots, embryo-larvae (tests were initiated with eyed eggs that hatched during testing), sac fry (alevins possessing a definite yolk sac), swim-up fry (fry had absorbed most of their yolk sac and were actively swimming in search of food), and juveniles 60 and 90 days post hatch (dph)(the 90-dph fish looked like small adults with parr marks). The sac-fry life stage was exposed only to Fire-Trol LCG-R. This exposure was conducted to retest the exposure of embryo-larvae to the retardant which did not produce an acceptable LC50 in the first test.

Dilution water and test chemicals

Static acute toxicity tests were conducted in standardized reconstituted hard and soft water [22]. Dilution water was prepared by adding appropriate amounts of reagent grade salts (CaSO₄2H₂O, 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 de-ionizer. After mixing and aeration of the salts in the purified water, chemical characteristics of the dilution water were determined according to standard methods [23,24], and are presented in Table 2.

Table 2. Water quality characteristics (mean with SD in parentheses) of the water used in acute toxicity test with five life stages of rainbow trout exposed to five fire-fighting chemical formulations in ASTM^a soft and hard water.

Chemical formulations used as test chemicals were received from the U.S. Forest Service, Intermountain Fire Sciences Laboratory, Missoula, Montana, USA, on November 20, 1992. All test concentrations and subsequent LC50s were based on a 100% active formulation because the proportional compositional analyses are not available for public disclosure.

The following five chemical formulations were tested. Fire-Trol GTS-R (FT GTS-R; lot number 84FT232) is a long-term, dry fire-retardant formulation manufactured by Chemonics Industries,Phoenix, AZ, USA. It is composed of ammonium sulphate, diammonium phosphate, guar gum thickener, spoilage inhibitor, corrosion inhibitor, and iron oxide as a coloring agent [16].

Fire-Trol LCG-R (FT LCG-R; lot number 91FT11) is a long-term, liquid fire-retardant formulation also manufactured by Chemonics Industries. It is composed of ammonium polyphosphate, attapulgite clay thickener, corrosion inhibitor, and iron oxide as a coloring agent [17].

Phos-Chek D75-F (PC D75-F; lot number 2468762A) is a long-term, dry fire-retardant formulation manufactured by Monsanto, Ontario, CA, USA. It is composed of ammonium sulfate, ammonium phosphate, guar gum thickener, orange coloring agent, and other additives [25].

Phos-Chek WD-881 (PC WD-881; lot number 3616836A, batch 18227) is a liquid, fire-suppressant foam formulation produced by Monsanto. Its formulation consists of a mixture of anionic surfactants, foam stabilizers, and solvents (hexylene glycol) [18].

Silv-Ex (lot number 75451, batch US6203) is a liquid fire-suppressant foam formulation manufactured by Ansul Fire Protection, Marinette, WI, USA. Silv-Ex contains anionic surfactants, stabilizers, inhibitors, and solvents (diethylene glycol monobutylether) [19].

Test methodology

Rainbow trout eyed eggs, embryo-larvae, and sac-fry life stages were acclimated to the test water and temperature over a 48-h period prior to testing. All other life stages were acclimated over a 96-h period. Fish were not fed during acclimation.

Static acute toxicity tests followed the guidelines outlined by the American Society for Testing and Materials (ASTM) [22]. Each test consisted of exposing groups of 10 fish to a series of seven or eight toxicant concentrations. There was a 60% dilution factor between each toxicant concentration. One control exposure was used in each set of life-stage tests and for each water quality tested. Temperature was maintained at 12 ± 1°C by immersion of test vessels into temperature-controlled waterbaths.

Test solutions were prepared by directly adding appropriate amounts of FT LCG-R, FT GTS-R, or PC D75-F to the test vessel. Fire-Trol GTS-R and PC D75-F were mixed two to three times for 40-50 s with a three-blade, polyethylene stirrer attached to an electric drill at 1,000-1,200 rpm. This mixing was usually sufficient to bring the test formulation into solution for a short time, but after 24 h of exposure some test material, probably thickener and some colorant, had precipitated. FT LCG-R was mixed in a similar fashion but was mixed only once for 40-50 s per test vessel. Phos-Chek WD-881 and Silv-Ex were added by preparing stock solutions in deionized water and then using a pipette to place an appropriate aliquot to each test vessel. Both PC WD-881 and Silv-Ex were hand-mixed with Teflon or glass stir rods to prevent excessive foaming.

Eyed eggs, embryo-larvae, sac fry, and swim-up fry were tested in 3.9-L glass test vessels containing 3.0 L of dilution water. During testing with eyed eggs and sac fry, test organisms were confined in 0.47-L glass jars with the bottom removed and replaced with stainless-steel screen fixed in place with silicone. The hatching cups were suspended by latex tubing from glass rods in the test vessels so that eyed eggs or sac fry were in the test solution but the cup rim was above the waterline. Tests conducted with 60-dph juveniles were conducted in 19.6-L glass test vessels containing 15 L of dilution water. The 90-dph juveniles were tested using two replicate 19.6-L test vessels each containing 15 L of dilution water and five fish each. Replicate vessels were used in the 90-dph juveniles to maintain loading densities of less than 0.8 g/L [22].

Eyed eggs, sac fry, or swim-up fry were randomly transferred from the acclimation chamber to plastic weigh boats filled with dilution water by glass pipet to minimize handling stress. Excess water was decanted from the weigh boats, and the test organisms were distributed to randomized test vessels. 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-h intervals during tests, and dead fish were removed at those intervals. Whitening of eyed eggs was the criterion for mortality in these tests. Embryo larvae and sac fry were examined with a x30 dissecting microscope for the presence of a heart beat, which was the criterion for mortality in these tests. Cessation of opercular movement was the criterion for mortality in tests with swim-up fry, 60- and 90-dph juveniles.

At the conclusion of each test, all control eggs or fish were weighed and total length was measured, except in the embryo-larvae tests in which control alevins were discarded prior to measurement. Weight and total length of each life stage tested are summarized in Table 1.

Dissolved oxygen and pH were measured and recorded in the control, low, medium, and high test concentrations, with live fish present at 0, 48, and 96 h of exposure. Dissolved oxygen was maintained at greater than 40% saturation, as recommended by the ASTM [22], in all but one test. Rainbow trout 60-dph juveniles exposed to FT LCG-R in soft water had a dissolved oxygen of 31.1% saturation at 96 h, probably because seven fish had died in this concentration during the preceding 24 h. The live fish in this test showed no overt signs of oxygen deprivation. Eight tests had dissolved oxygen readings below 60% saturation at 48 h (range, 42.4-59.7%), but none of the fish in these tests showed overt signs of oxygen stress. Temperature was measured daily in the waterbath.

Ammonia was measured in 100-ml samples of the control, low, medium, and high test concentrations containing live fish at 0, 48, and 96 h of exposure. 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 the 96-h LC50; regression equations are given in Gaikowski [26]. Equations were determined by regressing the NH₃-N estimates for the low, medium, and high test concentration using initial (0-h) NH₃-N estimates against the appropriate test concentrations of the chemical being tested. The 0-h 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 calculated by determining the NH₃-N concentration from the regression equation coefficients and the 96-h LC50 concentration. The percentage of un-ionized ammonia was estimated by using the high and low pH recorded at test initiation. The two percentage values were then used to determine the NH₃ range.

Nitrate and nitrite concentrations were determined colorimetrically using a cadmium reduction procedure and standard additions method [27]. 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/L as NO₃^--N) and 0.4, 0.6, and 0.8 ml nitrite-nitrogen standard solution (100 mg/L 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 but without fish. Fire-retardant solutions were prepared to simulate exposure at the 96-h LC50s of the swim-up fry life stage, except PC D75-F in soft water, which replicated the LC50 of the 90-dph life stage juveniles.

A regression equation was determined for tests conducted with rainbow trout and chinook salmon (Oncorhynchus tshawytscha) tested at 12°C (n = 4). The regression equation was used to calculate the nitrate and nitrite concentrations at the 96-h LC50 for the eyed egg, embryo-larvae, 60- and 90-dph life stages. A regression equation could not be derived for PC D75-F.

Statistical analysis

Most 96-h LC50 values and 95% confidence intervals were calculated using the moving-average angle method [28]. In tests with less than two concentrations above the LC50, the binomial distribution method [28] was used. In tests with no partial mortality, the upper 95% confidence limit was assumed to be the lowest test concentration with 100% mortality and the lower 95% confidence limit was assumed to be the highest test concentration with 0% mortality. All LC50 values are expressed as nominal concentrations of the fire-control formulation.

Formulations were ranked according to toxicity by the Friedman test [29]. The standard error of the difference as described by Sprague and Fogels [30] and Zar [31] was used to determine statistical differences between LC50 values. Values were considered to be significantly different at p ≤ 0.05. Comparisons and rankings of tests in which an LC50 value could not be determined due to insufficient mortality were accomplished by assuming the highest concentration tested in that series was less than the LC50. These LC50 values are therefore reported as greater than the highest concentration tested.