Endosulfan Exposure Disrupts Pheromonal Systems in the Red-Spotted Newt: A Mechanism for Subtle Effects of Environmental Chemicals

Environmental Health Perspectives, July, 2001 by Daesik Park, Steven Hempleman, C., Catherine R. Propper

Plasma collection and measurement of plasma 17[Beta]-estradiol. To collect plasma, trunk blood was collected in heparinized tubes and stored on ice until centrifugation at 1,200 x g for 15 min. After centrifugation, the plasma was removed and stored at -80 [degrees] C until the steroids were extracted and assayed by radioimmunoassay (RIA) (27).

To measure recovery efficiency during steroid extractions, we incubated plasma with 800-1,000 cpm of the tritiated analog of the steroid for 1 hr. Plasma was then extracted with diethyl ether, dried, and resuspended in RIA buffer. We measured 17[Beta]-estradiol using rabbit estradiol antiserum. The antiserum showed cross-reactive binding to 5[Alpha]-dihydrotestosterone (3%), estrone (6%), and estriol (16%). Six other steroids showed no cross-reactivity. Standards ranged from between 0.98 and 1,000 pg/tube, and all tubes were incubated in 5,000 cpm [2,4,6,7,16,17-.sup.3H]estradiol (Amersham Life Science, Buckinghamshire, UK). Serial dilutions of extracted plasma showed parallelism with a standard curve. The intra-assay coefficient of variation was 7.7%, sensitivity was 1.2 pg/tube, and specific binding was 24.6%. Data obtained were analyzed by one-way ANOVA (23).

Results

Effects of endosulfan on nonreproductive female behaviors, visual selection of females by males, and ovary and oviduct weight. Endosulfan-exposed females did not exhibit any nonreproductive behavioral deficits. General activity (two-way ANOVA, p = 0.53), amount of food consumed (Kruskal-Wallis test, p = 0.42), and surfacing to breathe (Kruskal-Wallis test, p = 0.51) did not differ among control and treated females. Also, there were no significant differences in ovary/body mass and oviduct/body mass among groups (one-way ANOVA, p = 0.37 and 0.55, respectively). Furthermore, the visual selection of females by males was not different between control and exposed females (Table 1).

Table 1. Visual mate selection tests demonstrated that
endosulfan exposure did not affect visual female

               Endosulfan treatment   Median number
Test set (n)          (ppb)           stayed with male   p-Value(a)

1 (30)                 0.0                   14            0.094
                       0.5                   16
2 (28)                 0.0                   15            0.701
                       1.0                   15

(a) Wilcoxon signed-rank test.

Effects of endosulfan on olfactory selection of females by males. Males less frequently selected water containing odor sources from low-concentration-treated females than water from control females (Table 2). Males did not show any preferences between control and high-concentration-treated females or between low- and high-concentration--treated females (Table 2).

Table 2. Olfactory mate selection tests demonstrated that males select
control females over those exposed to 5 ppb endosulfan.

               Endosulfan treatment   No. chosen
Test set (n)          (ppb)            by male     p-Value(a)

1 (34)                  0                25          < 0.01
                        5                 9
2 (31)                  0                17            0.360
                       10                14
3 (33)                  5                15            0.364
                       10                18

(a) One-tailed binomial test.