https://www.waterboards.ca.gov/water_issues/programs/tmdl/records/region_2/2008/ref2426.pdf

Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates.

Amphibian populations are declining globally at an alarming rate. Pesticides are among a number of proposed causes for these declines. Although a sizable database examining effects of pesticides on amphibians exists, the vast majority of these studies focus on toxicological effects (lethality, external malformations, etc.) at relatively high doses (parts per million). Very few studies focus on effects such as endocrine disruption at low concentrations. Further, most studies examine exposures to single chemicals only. The present study examined nine pesticides (four herbicides, two fungicides, and three insecticides) used on cornfields in the midwestern United States. Effects of each pesticide alone (0.1 ppb) or in combination were examined. In addition, we also examined atrazine and S-metolachlor combined (0.1 or 10 ppb each) and the commercial formulation Bicep II Magnum, which contains both of these herbicides. These two pesticides were examined in combination because they are persistent throughout the year in the wild. We examined larval growth and development, sex differentiation, and immune function in leopard frogs (Rana pipiens). In a follow-up study, we also examined the effects of the nine-compound mixture on plasma corticosterone levels in male African clawed frogs (Xenopus laevis). Although some of the pesticides individually inhibited larval growth and development, the pesticide mixtures had much greater effects. Larval growth and development were retarded, but most significantly, pesticide mixtures negated or reversed the typically positive correlation between time to metamorphosis and size at metamorphosis observed in controls: exposed larvae that took longer to metamorphose were smaller than their counterparts that metamorphosed earlier. The nine-pesticide mixture also induced damage to the thymus, resulting in immunosuppression and contraction of flavobacterial meningitis. The study in X. laevis revealed that these adverse effects may be due to an increase in plasma levels of the stress hormone corticosterone. Although it cannot be determined whether all the pesticides in the mixture contribute to these adverse effects or whether some pesticides are effectors, some are enhancers, and some are neutral, the present study revealed that estimating ecological risk and the impact of pesticides on amphibians using studies that examine only single pesticides at high concentrations may lead to gross underestimations of the role of pesticides in amphibian declines.

/pdf/pesticide-mixtures-endocrine-disruption-and-amphibian-3pzlrg8b62.pdf

Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact?

Amphibians and agricultural chemicals: Review of the risks in a complex environment

Greater than 70% of the world's amphibian species are in decline. We propose that there is probably not a single cause for global amphibian declines and present a three-tiered hierarchical approach that addresses interactions among and between ultimate and proximate factors that contribute to amphibian declines. There are two immediate (proximate) causes of amphibian declines: death and decreased recruitment (reproductive failure). Although much attention has focused on death, few studies have addressed factors that contribute to declines as a result of failed recruitment. Further, a great deal of attention has focused on the role of pathogens in inducing diseases that cause death, but we suggest that pathogen success is profoundly affected by four other ultimate factors: atmospheric change, environmental pollutants, habitat modification and invasive species. Environmental pollutants arise as likely important factors in amphibian declines because they have realized potential to affect recruitment. Further, many studies have documented immunosuppressive effects of pesticides, suggesting a role for environmental contaminants in increased pathogen virulence and disease rates. Increased attention to recruitment and ultimate factors that interact with pathogens is important in addressing this global crisis.

/pdf/the-cause-of-global-amphibian-declines-a-developmental-mlzabcxp9m.pdf

The cause of global amphibian declines: a developmental endocrinologist's perspective

ObjectiveThe biological effects of the herbicide atrazine on freshwater vertebrates are highly controversial. In an effort to resolve the controversy, we conducted a qualitative meta-analysis on th...

A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians.

Tadpoles of the African clawed frog (Xenopus laevis) were exposed to sublethal concentrations of atrazine (0, 40, and 320 microg/L) and nitrate (0, 37, and 292 mg/L) from feeding stage to metamorphosis. A 3 x 3 factorial design was used to identify both single and interactive effects. At metamorphosis, tadpole weight, snout-vent length (SVL), and hematocrit were determined. Mean mortality was greater in tanks receiving 320 microg/L atrazine; nitrate had no effect on mortality. Significant differences for all mean traits at metamorphosis occurred among atrazine treatments; higher atrazine exposure increased time to metamorphosis and decreased weight, SVL, and hematocrit. Nitrate treatments were not significantly different. Significant interaction tests between atrazine and nitrate occurred for weight and SVL at metamorphosis; the specific type of interaction varied among treatments. Assuming an additive mixture model, at low atrazine (40 microg/L), the addition of 37 mg/L nitrate produced SVL values less than expected (a synergistic effect) while the addition of 292 mg/L nitrate yielded SVL values greater than expected (an antagonistic effect). A similar response was noted for tadpoles in the 320-microg/L atrazine treatments. These results indicate that environmentally realistic concentrations of atrazine exert a negative impact on amphibian metamorphosis. Also, this study suggests that mixtures of agricultural chemicals, even if sublethal, may exert negative and not necessarily consistent mixture effects.

Effects of sublethal concentrations of atrazine and nitrate on metamorphosis of the African clawed frog

Two species of fishes common to the Great Plains of North America (the western mosquitofish [Gambusia affinis] and the sand shiner [Notropis ludibundus]) were used to examine the relationship between individual time-to-death (TTD) in 96-h toxicity tests and average fluctuating asymmetry (FA). Previous studies have indicated that FA, the random deviation from bilateral symmetry in metric traits, is increased in organisms that develop in stressful and/or marginal environments. This association has led to the prediction that FA may be useful as a bioindicator. For example, if an individual's FA is negatively related to stress resistance, a population that has comparatively high values of FA may be more susceptible to further stressors. Adult fish used in these experiments were captured from natural populations in Kansas and acclimated to laboratory conditions for 1 month before use in toxicity tests. Each species was separately exposed to two insecticides: parathion, an organophosphate, and lindane, an organochlorine. For each TTD test, 200 individuals were exposed over a 96-h period to an LC70 concentration. Dead individuals were removed every 3 h. Individuals that survived the test were sacrificed for comparison to those that died. Measurements for seven external morphologic characters were obtained for each individual and used to estimate average FA. Three of four statistical tests showed a significant negative regression between TTD and average FA, indicating that individuals with smaller FA survived longer in the test conditions. These results support the use of FA as an indicator of susceptibility to pesticides.

Higher fluctuating asymmetry as a measure of susceptibility to pesticides in fishes

Differential genotypic tolerance of two species of fish native to the Great Plains of North America (the western mosquitofish [Gambusia affinis] and the sand shiner [Notropis ludibundus]) was examined in 96-h acute exposures to the pesticides lindane and parathion. Although a significant amount of data is available indicating that heavy metals cause selective genotypic mortality in aquatic organisms, very few studies have focused on the effects of pesticides on population genetic structure. Fish used in this experiment were captured from field populations in Kansas, USA, and acclimated to laboratory conditions for 1 month before the acute toxicity tests. Two hundred individuals of each species were exposed, in two separate experiments, to estimated LC70 concentrations of each pesticide for 96 h. Dead individuals were removed at 3-h intervals allowing for comparison of genotypic times-to-death. Individuals of each species were electrophoretically examined at two polymorphic loci. Phenotypes of mosquitofish were determined for phosphoglucose mutase (PGM) and malic enzyme (ME). Phenotypes of sand shiners were determined for PGM and aspartate aminotransferase (AAT). Three of eight statistical tests (two species × two loci × two pesticides) indicated significant differences among survival curves of genotypic classes. Genotypes of ME and PGM of mosquitofish exhibited significantly different survivorship functions in the parathion and lindane time-to-death tests, respectively. Genotypes of AAT of sand shiners exhibited significantly different survivorship curves when exposed to lindane. Given certain experimental limitations, these results support a continued examination of population genetic characteristics as biomarkers of environmental contamination.

Karen Brown Sullivan

Papers

Effects of sublethal concentrations of atrazine and nitrate on metamorphosis of the African clawed frog

Higher fluctuating asymmetry as a measure of susceptibility to pesticides in fishes

Differences in survival functions of mosquitofish (Gambusia affinis) and sand shiner (Notropis ludibundus) genotypes exposed to pesticides