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Amphibian populations can be affected adversely by multiple biotic and abiotic stressors that together can contribute to their local and global decline. We focused on the combined effects of food limitation, drying conditions, and exposure to possibly the most abundant and widely used herbicide in the world, atrazine. We used a factorial design to evaluate the effects of exposure to four ecologically relevant doses of atrazine (approximate measured doses: 0, 4, 40, and 400 g/L), two food levels (limited and unlimited food), and two hydroperiods (presence or absence of a dry down) on the survival, life history, and behavior of the streamside salamander, Ambystoma barbouri, from the embryo stage through metamorphosis. In general, food and atrazine levels did not interact statistically, and atrazine affected dependent variables in a standard, dose-dependent manner. Exposure to 400 g/L of atrazine decreased embryo survival and increased time to hatching. Drying conditions and food limitation decreased larval survival, while 400 g/L of atrazine only reduced larval survival in one of the two years tested, suggesting that the lethality of atrazine may be condition dependent. Sublethal effects included elevated activity and reduced shelter use associated with increasing concentrations of atrazine and food limitation. The larval period was lengthened by food limitation and shortened by 400 g/L of atrazine. Drying conditions accelerated metamorphosis for larvae exposed to 0 and 4 g/L of atrazine but did not affect timing of metamorphosis for larvae exposed to 40 or 400 g/L of atrazine. Food limitation, drying conditions, and 400 g/L of atrazine reduced size at metamorphosis without affecting body condition (relationship between mass and length), even though feeding rates did not differ significantly among atrazine concentrations at any time during development. This suggests that high atrazine levels may have increased larval energy expenditures. Because smaller size at metamorphosis can lower terrestrial survival and lifetime reproduction, resource limitations, drying conditions, and environmentally realistic concentrations of atrazine have the potential to contribute to amphibian declines in impacted systems.

Abstract-Agricultural contaminants may be contributing to worldwide amphibian declines, but little is known about which agrichemicals pose the greatest threat to particular species. One reason for this is that tests of multiple contaminants under ecologically relevant conditions are rarely conducted concurrently. In this study, we examined the effects of 37-d exposure to the agrichemicals atrazine (4, 40, and 400 g/L), carbaryl (0.5, 5, and 50 g/L), endosulfan (0.1, 1, and 10 g/L for 31 d and 0.1, 10, and 100 g/ L for the last 6 d), and octylphenol (5, 50, and 500 g/L) and to a solvent control on streamside salamanders (Ambystoma barbouri) in the presence and absence of food. We found that none of the agrichemicals significantly affected embryo survival, but that hatching was delayed by the highest concentration of octylphenol. In contrast to embryos, larval survival was reduced by the highest concentrations of carbaryl, endosulfan, and octylphenol. Growth rates were lower in the highest concentrations of endosulfan and octylphenol than in all other treatments, and the highest concentration of endosulfan caused respiratory distress. Significantly more carbaryl, endosulfan, and octylphenol tanks had larvae with limb deformities than did control tanks. Refuge use was independent of chemical exposure, but 10 g/L of endosulfan and 500 g/L of octylphenol decreased larval activity. Systematically tapping tanks caused a greater activity increase in larvae exposed to 400 g/L of atrazine and 10 g/L of endosulfan relative to solvent controls, suggesting underlying nervous system malfunction. Hunger stimulated a decrease in refuge use and an increase in activity, but this response was least pronounced in larvae exposed to the highest concentration of any of the four agrichemicals, possibly because these larvae were the most lethargic. More studies are needed that concurrently examine the effect of multiple contaminants on amphibians so we can better identify effective mitigating measures.

Acetylcholinesterase (AChE) activity has traditionally been monitored as a biomarker of organophosphate (OP) and/or carbamate exposure. However, AChE activity may not be the most sensitive endpoint for these agrochemicals, because OPs can cause adverse physiological effects at concentrations that do not affect AChE activity. Carboxylesterases are a related family of enzymes that have higher affinity than AChE for some OPs and carbamates and may be more sensitive indicators of environmental exposure to these pesticides. In this study, carboxylesterase and AChE activity, cytochrome P4501A (CYP1A) protein levels, and mortality were measured in individual juvenile Chinook salmon (Oncorhynchus tshawytscha) following exposure to an OP (chlorpyrifos) and a pyrethroid (esfenvalerate). As expected, high doses of chlorpyrifos and esfenvalerate were acutely toxic, with nominal concentrations (100 and 1 μg/l, respectively) causing 100% mortality within 96 h. Exposure to chlorpyrifos at a high dose (7.3 μg/l), but not a low dose (1.2 μg/l), significantly inhibited AChE activity in both brain and muscle tissue (85% and 92% inhibition, respectively), while esfenvalerate exposure had no effect. In contrast, liver carboxylesterase activity was significantly inhibited at both the low and high chlorpyrifos dose exposure (56% and 79% inhibition, respectively), while esfenvalerate exposure still had little effect. The inhibition of carboxylesterase activity at levels of chlorpyrifos that did not affect AChE activity suggests that some salmon carboxylesterase isozymes may be more sensitive than AChE to inhibition by OPs. CYP1A protein levels were ∼30% suppressed by chlorpyrifos exposure at the high dose, but esfenvalerate had no effect. Three teleost species, Chinook salmon, medaka (Oryzias latipes) and Sacramento splittail (Pogonichthys macrolepidotus), were examined for their ability to hydrolyze a series of pyrethroid surrogate substrates and in all cases hydrolysis activity was undetectable. Together these data suggest that (1) carboxylesterase activity inhibition may be a more sensitive biomarker for OP exposure than AChE activity, (2) neither AChE nor carboxylesterase activity are biomarkers for pyrethroid exposure, (3) CYP1A protein is not a sensitive marker for these agrochemicals and (4) slow hydrolysis rates may be partly responsible for acute pyrethroid toxicity in fish.

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