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Effects of the Herbicide Atrazine on Ambystoma tigrinum Metamorphosis: Duration, Larval Growth, and Hormonal Response

Material and Methods


Eyed-eggs of tiger salamanders were collected from a semipermanent wetland in Stutsman County, North Dakota, USA, on 17-18, 1995. Eggs were delivered double bagged, in double coolers, with ice packs between the inner and outer cooler, by air to Yankton Field Research Station, Yankton, South Dakota, on May 19, 1995, for testing. The eggs were removed from plant detritus and placed in a Heath incubator at 13°C. Well water quality for egg incubation was pH 7.59, hardness as CaCO3 1,000 mg/L and alkalinity as CaCO3 286 mg/L. Larval culture well water quality was pH 7.51 - 8.27, hardness as CaCO3 770 - 1,050 mg/L and alkalinity as CaCO3 268 - 283 mg/L.

Dilution Water

Tests were conducted in American Society for Testing and Materials (ASTM 1989) very hard reconstituted water (hardness and alkalinity as CaCO3 280 - 320 mg/L and 225 - 245 mg/L, respectively, and pH 8.0 - 8.4). Reconstituted water was prepared by adding calcium sulfate, magnesium sulfate, potassium chloride, and sodium bicarbonate to deionized water in polyethylene tanks equipped with a recirculating pump to mix and aerate the water (Hamilton et al. 1989). Test water was not prepared with reagent grade salts, as suggested by ASTM (1989), because of the large volume of water needed. Water quality characteristics, including chloride, calcium, sulfate, total hardness, and total alkalinity, were measured for each tank of dilution water according to methods of American Public Health Association (APHA 1989). Magnesium concentrations were calculated using calcium and hardness values, conductivity (corrected to 25°C) was measured with a Yellow Springs Instruments (YSI) Model 31 conductivity bridge with a cell constant of K = 1.0, and pH was determined with an Orion Model 901 Ionalyzer using a Ross combination electrode. (Use of trade names throughout this article does not imply endorsement by the Federal Government.)

Test Chemical

The chemical used in the static renewal chronic test was atrazine (2-chloro-4-ethylamino-6-isopropylamine-s-triazine) obtained from Ciba-Geigy Corporation, Greensboro, North Carolina. Test solutions were prepared by pipetting appropriate aliquots of stock solution into 55-gallon drums of reconstituted water. Stock solutions were prepared daily by dissolving atrazine in reagent grade acetone then diluted with deionized water. Control solutions were prepared by pipetting appropriate elicits of 35% acetone stock solution into a 55-gallon drum of reconstituted water. Control and test solutions were thoroughly mixed prior to use.

Toxicity Testing

A static renewal test was conducted following procedures outlined by the ASTM (1989). The test exposed 102 individually housed salamander larvae to two concentrations of atrazine, 75 or 250 µg/L, plus a control for 86 days (n = 34 for each treatment). An additional 102 larvae were treated in the same way, but plasma from these larvae was used in a separate analysis which will be reported elsewhere. We used all 204 larvae in growth and metamorphic timing analyses. Larvae were assigned to one of two groups: those that would be sampled at stage 2 (early) and those that would be sampled at stage 4 (middle) of metamorphic climax (Norman 1985). Larvae were tested individually in 7.6-L polyethylene containers covered with screened lids, containing 4.0 L of test solution. Tests were initiated when larval snout to vent length was 30 mm (Mean of X = 32.58 ± 0.46 mm). Larvae were allowed to gradually acclimate to test water over a period of 2 d before atrazine was introduced; test water was renewed daily during acclimation. After acclimation, test solution was renewed every second day by removing one-half of the solution and adding an equal volume of fresh solution. Larvae were fed three red worm pieces daily, supplemented with brine shrimp naupii. Mortality was recorded daily and metamorphic stages were determined using tail-fin measurements as described by Norman (1985). Upper and lower tail-fin measurements were taken every third day until the animal neared the next metamorphic stage, at which time measurements were taken daily. Salamanders were killed and blood samples were collected in capillary tubes and centrifuged; then plasma stored frozen at -25°C until analyzed. Before sacrifice each salamander was weighed and measured; plasma corticosterone measures obtained in this fashion cannot be considered basal, but because all larvae were handled in the same way, we consider the concentrations to be comparable among treatment groups.

Ambient test temperature ranged from 19.0° to 26.0°C. Water quality parameters (see "Dilution water" above for list) in exposure vessels for both studies were measured weekly during the study. Dissolved oxygen was measured weekly in exposure vessels before and after renewal with a YSI Model 58 oxygen meter. The photoperiod simulated conditions in North Dakota during mid-June to mid-July.

Toxicant Analysis

Test water was analyzed to verify the concentration of atrazine in the test vessels. Subsurface grab samples were collected in acid-cleaned 16 x 125 mm glass culture tubes with Teflon-lined screw caps from 12 arbitrarily chosen test vessels at day 1, 7, 15, 28, 42, 56, 70, and 84, and refrigerated until analysis. Samples were analyzed in triplicate utilizing Ohmicron's Enyzme-Linked Immunosorbent Assay tests for atrazine.

Radioimmunoassay for Corticosterone and Thyroxine

Plasma concentrations of corticosterone were assayed with techniques modified from the method of Wingfield and Farner (1975) as reported in Fivizzani et al. (1986) and Gratto-Trevor et al. (1991). Variations of these methods have been used to quantify plasma steroid hormones in different vertebrates from fish to mammals. Plasma samples (100-200 µl) were brought up to a final volume of 400 µl with distilled water, and corticosterone was extracted with 5 ml of dichloromethane. The organic phase was removed and dried under a stream of nitrogen. The extraction efficiency for this method in previous studies has been between 80% and 95%.

The extract was suspended overnight in phosphate-buffered saline-gelatin assay buffer. Duplicate 200-µl aliquots of the resuspended extract were assayed with a highly specific antibody from Endocrine Sciences, Tarzana, California (B3-163), and radioactive corticosterone (1,2,6,7 -3H) from New England Nuclear, Boston. Chromatographic separation of the samples was not performed as this antibody is highly specific with a cross-reactivity of 3.3% with desoxycorticosterone and 1% or less with all other steroids. To validate the use of this assay for Ambystome tigrinum, a pooled sample of plasma from this species was serially diluted and was found to be parallel to the standard curve. Additionally, a plasma volume from A. tigrinum was stripped of endogenous steroids via multiple charcoal treatments, and exogenous corticosterone was added in a known concentration to verify the accuracy of the assay. The assay continued overnight at 4°C, and dextran-coated charcoal followed by centrifugation was employed to separate the bound from the free fractions. Aliquots of the bound fraction were counted on a Beckman LS-6800 liquid scintillation counter. Plasma values were compared against a series of corticosterone standards from Sigma Chemical Company. These methods have been previously determined to allow for detection of corticosterone concentrations as low as 1.5 pg/assay tube (between 30 and 60 pg/mL plasma depending on plasma sample size). The intra-assay coefficient of variation was 8.7%.

Plasma thyroid hormone concentrations were determined according to the methods discussed in Hoffnagle and Fivizzani (1990) utilizing solid-phase radioimmunoassay kits (Micromedic Systems, Horsham, Pa.). The cross-reactivity of this assay system was 1.3% with triiodothyronine and less than 0.1% with other similar amino acids. All samples were analyzed in a single assay to eliminate the problem of interassay variability. Standards were prepared by adding known quantities of plasma thyroxine to tiger salamander plasma previously charcoal-stripped of all endogenous thyroid hormones. Duplicate 5-µl undiluted samples and standards were assayed, and the bound fraction counted with a Beckman 5500 gamma counter. Estimates of sample plasma thyroxine content were determined via a comparison of each sample with a graph of the complete series of standards.

Statistical Analysis

We used ANOVA techniques to assess the effects of atrazine concentration (0, 75, or 250 µg/L), stage (2 or 4), and their interaction on plasma corticosterone concentration, plasma thyroxin concentration, days-to-stage, weight, and snout-vent length of larval tiger salamanders. Because the salamanders were allocated to individual containers and then randomly assigned to one of the six stage-by-dose combinations, we considered each individual salamander to be an independent replicate and conducted the ANOVA as a simple two-way factorial (Kirk 1982).

We tested for differences in the change in the response variable from stage 2 to stage 4 among the three atrazine treatments by using specified contrasts statements (Kirk 1982). We isolated differences among least squares means (SAS 1989) for significant main effects and interactions using Fisher's protected least significant difference value (Milliken and Johnson 1984). We used the general linear models procedure of SAS (1989) to conduct the ANOVA analyses and isolate differences among least squares means for significant effects in the ANOVA, and the ESTIMATE statement for conducting the specified contrasts. All statistical tests were conducted at the 0.05 significance level unless stated otherwise.

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