Fish short-term reproduction assay with atrazine and the Japanese medaka (Oryzias latipes).

Breeding groups of Japanese medaka (Oryzias latipes) were exposed to atrazine at measured concentrations of 0.6, 5.5, and 53 µg/L for 35 d. Evaluated endpoints included survival, fecundity, fertility, growth (weight and length), behavior, secondary sex characteristics (anal fin papillae), gonad histopathology, and hepatic vitellogenin. No statistically significant effects of atrazine exposure on survival and growth of medaka were noted during the test, and mean survival was ≥97.5% in all treatment groups on day 35. No significant effects of atrazine exposure on reproduction were observed. The number of mean cumulative eggs produced in the negative control and the 0.6, 5.5, and 53 µg/L treatment groups was 7158, 6691, 6883, and 6856, respectively. The mean number of eggs per female reproductive day was 40.9, 38.2, 40.2, and 39.2, respectively. There were also no dose-dependent effects on mean anal fin papillae counts among male fish or expression of vtg-II in males or females. In addition, atrazine exposure was not related to the developmental stage of test fish, with testes stages ranging from 2 to 3 in all groups and ovaries ranging from stage 2 to 2.5. Overall, exposure to atrazine up to 53 µg/L for 35 d did not result in significant, treatment-related effects on measured endpoints related to survival, growth, or reproduction in Japanese medaka. Environ Toxicol Chem 2017;36:2327-2334. © 2017 SETAC.

Effects of pulsed atrazine exposures on autotrophic community structure, biomass, and production in field-based stream mesocosms.

The authors performed a multiple-pulsed atrazine experiment to measure responses of autotrophic endpoints in outdoor stream mesocosms. The experiment was designed to synthetically simulate worst-case atrazine chemographs from streams in agricultural catchments to achieve 60-d mean concentrations of 0 µg/L (control), 10 µg/L, 20 µg/L, and 30 µg/L. The authors dosed triplicate streams with pulses of 0 µg/L, 50 µg/L, 100 µg/L, and 150 µg/L atrazine for 4 d, followed by 7 d without dosing. This 11-d cycle occurred 3 times, followed by a recovery (untreated) period from day 34 to day 60. Mean ± standard error 60-d atrazine concentrations were 0.07 ± 0.03 µg/L, 10.7 ± 0.05 µg/L, 20.9 ± 0.24 µg/L, and 31.0 ± 0.17 µg/L for the control, 10-µg/L, 20-µg/L, and 30-µg/L treatments, respectively. Multivariate analyses revealed that periphyton and phytoplankton community structure did not differ among treatments on any day of the experiment, including during the atrazine pulses. Control periphyton biomass in riffles was higher immediately following the peak of the first atrazine pulse and remained slightly higher than some of the atrazine treatments on most days through the peak of the last pulse. However, periphyton biomass was not different among treatments at the end of the present study. Phytoplankton biomass was not affected by atrazine. Metaphyton biomass in pools was higher in the controls near the midpoint of the present study and remained higher on most days for the remainder of the study. Ceratophyllum demersum, a submersed macrophyte, biomass was higher in controls than in 20-µg/L and 30-µg/L treatments before pulse 3 but was not different subsequent to pulse 3 through the end of the present study. Maximum daily dissolved oxygen (DO, percentage of saturation) declined during each pulse in approximate proportion to magnitude of dose but rapidly converged among treatments after the third pulse. However, DO increased in controls relative to all atrazine treatments during the last 17 d of the experiment, likely a result of metaphyton cover in the pools. Finally, atrazine significantly limited uptake of PO4(3-) and uptake and/or denitrification of NO3(-) but only during pulses; percentage of dose removed from the water column was >85% for P and >95% for N after pulse 3 through the end of the present study. Collectively, only DO and metaphyton biomass differed at the end of the present study and only slightly. Some other endpoints were affected but only during pulses, if at all. The high levels of primary production and accumulation of algal biomass in all streams suggest that effects of pulses of atrazine at the concentrations used in the present study appear transient and likely do not represent ecologically significant adverse outcomes to periphyton, phytoplankton, and aquatic macrophytes, particularly in agricultural streams subjected to high nutrient loads.

Effects of atrazine on egg masses of the yellow-spotted salamander (Ambystoma maculatum) and its endosymbiotic alga (Oophila amblystomatis).

Embryonic growth of the yellow-spotted salamander (Ambystoma maculatum) is enhanced by the presence of the green alga Oophila amblystomatis, in the egg capsule. To further assess potential impacts of herbicides on this relationship, A. maculatum egg masses were exposed to atrazine (0-338 µg/L) until hatching (up to 66 days). Exposure to atrazine reduced PSII yield of the symbiotic algae in a concentration-dependent manner, but did not significantly affect visible algal growth or any metrics associated with salamander development. Algal cells were also cultured in the laboratory for toxicity testing. In the 96-h growth inhibition test (0-680 µg/L), ECx values were generally greater than those reported for standard algal test species. Complete recovery of growth rates occurred within 96-h of transferring cells to untreated media. Overall, development of A. maculatum embryos was not affected by exposure to atrazine at concentrations and durations exceeding those found in the environment.

Assessing temporal and spatial variation in sensitivity of communities of periphyton sampled from agroecosystem to, and ability to recover from, atrazine exposure.

Lotic systems in agriculturally intensive watersheds can experience short-term pulsed exposures of pesticides as a result of runoff associated with rainfall events following field applications. Of special interest are herbicides that could potentially impair communities of primary producers, such as those associated with periphyton. Therefore, this study examined agroecosystem-derived lotic periphyton to assess (1) variation in community sensitivity to, and ability to recover from, acute (48h) exposure to the photosystem II (PSII)-inhibiting herbicide atrazine across sites and time, and (2) attempt to determine the variables (e.g., community structure, hydrology, water quality measures) that were predictive for observed differences in sensitivity and recovery. Periphyton were sampled from six streams in the Midwestern U.S. on four different dates in 2012 (April to August). Field-derived periphyton were exposed in the laboratory to concentrations of atrazine ranging from 10 to 320µg/L for 48h, followed by untreated media for evaluation of recovery for 48h. Effective quantum yield of PSII was measured after 24h and 48h exposure and 24h and 48h after replacement of media. Inhibition of PSII EC50 values ranged from 53 to >320µg/L. The majority of periphyton samples (16 out of 22) exposed to atrazine up to 320µg/L recovered completely by 48h after replacement of media. Percent inhibition of effective quantum yield of PSII in periphyton (6 of 22 samples) exposed to 320µg/L atrazine that were significantly lower than controls after 48h ranged from 2% to 24%. No distinct spatial or temporal trends in sensitivity and recovery potential were observed over the course of the study. Conditional inference forest analysis and variation partitioning were used to investigate potential associations between periphyton sensitivity to and ability to recover from exposure to atrazine. Although certain environmental variables (i.e., proximity of high flow/velocity events and dissolved solutes) were significantly associated with sensitivity to atrazine, recovery was not significantly associated with any variables, which is predicted by the rapid reversible binding at PSII. Consistent and rapid recovery of effective quantum yield of PSII across sites and sampling dates indicates that acute exposure to atrazine is unlikely to adversely affect function of these communities in their current state in intensive agroecosystems.

Atrazine and its degradates have little effect on the corticosteroid stress response in the zebrafish.

The present study examined the effects of atrazine on basal and forced swimming induced changes in whole body cortisol content in adult zebrafish. Zebrafish were exposed to graded concentrations of atrazine or the atrazine degradates deisopropylatrazine (DIA), deethylatrazine (DEA) and diamino-s-chlorotriazine (DACT) for up to 10 days. Some fish were sampled for the measurement of whole body cortisol levels under basal conditions while others were sampled after being subjected to a 20 min swimming challenge in order to quantify stress induced cortisol levels. In one experiment, zebrafish were subjected to two bouts of forced swimming 3h apart to test whether prior atrazine exposure affects the ability of the fish to respond appropriately to a repeated stressor. The results demonstrated that controls not exposed to atrazine and zebrafish exposed to atrazine or the atrazine degradates at nominal concentrations of up to 100 µg/L consistently exhibited increased whole body cortisol content in response to the swimming challenge. Separate analyses revealed few changes in basal or stress induced cortisol levels following atrazine exposure. Overall, these data suggest that atrazine and some of its degradates at the concentrations tested have minimal effects on the cortisol mediated stress response in the zebrafish.

Spatial and temporal variation of algal assemblages in six Midwest agricultural streams having varying levels of atrazine and other physicochemical attributes.

Potential effects of pesticides on stream algae occur alongside complex environmental influences; in situ studies examining these effects together are few, and have not typically controlled for collinearity of variables. We monitored the dynamics of periphyton, phytoplankton, and environmental factors including atrazine, and other water chemistry variables at 6 agricultural streams in the Midwest US from spring to summer of 2011 and 2012, and used variation partitioning of community models to determine the community inertia that is explained uniquely and/or jointly by atrazine and other environmental factors or groups of factors. Periphyton and phytoplankton assemblages were significantly structured by year, day of year, and site, and exhibited dynamic synchrony both between site-years and between periphyton and phytoplankton in the same site-year. The majority of inertia in the models (55.4% for periphyton, 68.4% for phytoplankton) was unexplained. The explained inertia in the models was predominantly shared (confounded) between variables and variable groups (13.3, 30.9%); the magnitude of inertia that was explained uniquely by variable groups (15.1, 18.3%) was of the order hydroclimate>chemistry>geography>atrazine for periphyton, and chemistry>hydroclimate>geography>atrazine for phytoplankton. The variables most influential to the assemblage structure included flow and velocity variables, and time since pulses above certain thresholds of nitrate+nitrite, total phosphorus, total suspended solids, and atrazine. Time since a ≥30 µg/L atrazine pulse uniquely explained more inertia than time since pulses ≥ 10 µg/L or daily or historic atrazine concentrations; this result is consistent with studies concluding that the effects of atrazine on algae typically only occur at ≥30 µg/L and are recovered from.

Effects of atrazine in fish, amphibians, and reptiles: an analysis based on quantitative weight of evidence.

A quantitative weight of evidence (WoE) approach was developed to evaluate studies used for regulatory purposes, as well as those in the open literature, that report the effects of the herbicide atrazine on fish, amphibians, and reptiles. The methodology for WoE analysis incorporated a detailed assessment of the relevance of the responses observed to apical endpoints directly related to survival, growth, development, and reproduction, as well as the strength and appropriateness of the experimental methods employed. Numerical scores were assigned for strength and relevance. The means of the scores for relevance and strength were then used to summarize and weigh the evidence for atrazine contributing to ecologically significant responses in the organisms of interest. The summary was presented graphically in a two-dimensional graph which showed the distributions of all the reports for a response. Over 1290 individual responses from studies in 31 species of fish, 32 amphibians, and 8 reptiles were evaluated. Overall, the WoE showed that atrazine might affect biomarker-type responses, such as expression of genes and/or associated proteins, concentrations of hormones, and biochemical processes (e.g. induction of detoxification responses), at concentrations sometimes found in the environment. However, these effects were not translated to adverse outcomes in terms of apical endpoints. The WoE approach provided a quantitative, transparent, reproducible, and robust framework that can be used to assist the decision-making process when assessing environmental chemicals. In addition, the process allowed easy identification of uncertainty and inconsistency in observations, and thus clearly identified areas where future investigations can be best directed.

Assessment of periphyton, aquatic macrophytes, benthic communities, and physical habitat in midwestern United States streams coinciding with varying historical concentrations of atrazine.

The objectives of this pilot study were to: (1) characterize periphyton and benthic communities using standard collection methods in six Midwest watersheds with varying historical levels of atrazine (low range, medium range and upper range); (2) qualitatively assess presence of aquatic vascular plants at each site; (3) assess and compare physical habitat at each study site in order to evaluate how physical habitat structure may influence the biological communities and (4) analyze the periphyton and benthic macroinvertebrate community data (i.e., series of metrics) among sites to evaluate possible differences or similarities among sites with different historical atrazine exposures. Five of the eight physical habitat metrics (including total physical habitat score) were different among the six study sites. There appeared to be no substantial difference in the structure of periphtyon communities at the six Midwest sites based on 9 of 12 metrics. For the three metrics that showed differences among sites-percentage of sensitive diatoms, percent Achnanches minutissima and percent motile diatoms - there was no consistent pattern with previous degrees of atrazine exposure and the scoring of these metrics. There were also no statistical differences in aquatic macrophyte spatial coverage among the six study areas. Thus, based on the spatially and temporally limited periphyton and aquatic macrophyte data, varying historical atrazine exposure was not associated with impact on resident plant communities (the target receptor group for atrazine). All 10 benthic community metrics showed significant differences among the six Midwest sites. Although no consistent pattern existed with varying historic levels of atrazine, benthic communities at one site with lower historical levels of atrazine were of higher quality than the other five sites. However, this one site also had a higher quality habitat compared to the other sites which was most likely the reason for this benthic condition.

Response of the green alga Oophila sp., a salamander endosymbiont, to a PSII-inhibitor under laboratory conditions.

In a rare example of autotroph-vertebrate endosymbiosis, eggs of the yellow-spotted salamander (Ambystoma maculatum) are colonized by a green alga (Oophila sp.) that significantly enhances salamander development. Previous studies have demonstrated the potential for impacts to the salamander embryo when growth of the algae is impaired by exposure to herbicides. To further investigate this relationship, the authors characterized the response of the symbiotic algae (Oophila sp.) alone to the photosystem II (PSII) inhibitor atrazine under controlled laboratory conditions. After extraction of the alga from A. maculatum eggs and optimization of culturing conditions, 4 toxicity assays (96 h each) were conducted. Recovery of the algal population was also assessed after a further 96 h in untreated media. Average median effective concentration (EC50) values of 123 µg L(-1) (PSII yield), 169 µg L(-1) (optical density), and 299 µg L(-1) (growth rate) were obtained after the 96-h exposure. Full recovery of exposed algal populations after 96 h in untreated media was observed for all endpoints, except for optical density at the greatest concentration tested (300 µg L(-1) ). Our results show that, under laboratory conditions, Oophila sp. is generally less sensitive to atrazine than standard test species. Although conditions of growth in standard toxicity tests are not identical to those in the natural environment, these results provide an understanding of the tolerance of this alga to PSII inhibitors as compared with other species.

Assessing sensitivity and recovery of field-collected periphyton acutely exposed to atrazine using PSII inhibition under laboratory conditions.

Periphyton communities are an integral component of freshwater ecosystems and the desire to include data from toxicity testing with these organisms for ecological risk assessment is growing. This study developed sampling, storage, and exposure methods for the consistent and effective characterization of acute response and recovery of field-derived periphyton to photosystem II (PSII) inhibiting herbicides, particularly atrazine. Pulse amplitude modulated fluorometry was used to assess PSII quantum yield. For the method development phase, periphyton samples were collected from lotic and lentic systems in the Guelph, Ontario, Canada area during the summer of 2011. Following method development, native periphyton communities from three agricultural streams from the midwestern U.S. were sampled and exposed to atrazine (10-320 µg/L) and assessed for inhibition of PSII quantum yield (from 2 up to 24 h) and subsequent recovery upon cessation of exposure (up to 48 h post-exposure). Sensitivity to atrazine (EC10 and EC50 values) varied slightly (typically less than twofold difference) by site, date of sampling, and exposure interval. Only the highest initial test concentrations (160 or 320 µg/L) demonstrated greater than ~5% inhibition at 48 h post-exposure; however all other test concentrations recovered to within 5% of control levels, typically within 24 h. The rapid physiological recovery of periphyton communities upon atrazine removal supports the conclusion that acute exposure will not likely result in significant or sustained impacts on either structure or function of periphyton in lotic ecosystems. For ecological risk assessment, this suggests the current approach of relying on direct effects data for the most sensitive single species alone may result in overly conservative estimates of potential effects, especially for complex communities of primary producers.

Seasonal synchronicity of algal assemblages in three Midwestern agricultural streams having varying concentrations of atrazine, nutrients, and sediment.

Numerous studies characterizing the potential effects of atrazine on algal assemblages have been conducted using micro- or mesocosms; however, few evaluations focused on in situ lotic algal communities, potentially confounding risk assessment conclusions. This exploratory study, conducted at several sites in the midwestern United States where atrazine is commonly used, presents in situ observations of native algal communities relative to atrazine exposure and other parameters. Planktonic and periphytic algae from three streams in three Midwestern states, having historically differing atrazine levels, were sampled over a 16-week period in 2011 encompassing atrazine applications and the summer algal growth period at each site. Changes in abundance, diversity, and composition of algal communities were placed in the context of hydrological, climatic, and water quality parameters (including components sometimes present in agricultural runoff) also collected during the study. Diatoms dominated communities at each of the three sites and periphyton was much more abundant than phytoplankton. As expected, significant variations in algal community and environmental parameters were observed between sites. However, correspondence analysis plots revealed that patterns of temporal variation in algal communities at each site and in periphyton or phytoplankton were dominated by seasonal environmental gradients. Significant concordance in these seasonal patterns was detected among sites and between phytoplankton and periphyton communities (via procrustes Protest analysis), suggesting synchronicity of algal communities across a regional scale. While atrazine concentrations generally exhibited seasonal trends at the study watersheds; no effects on algal abundance, diversity or assemblage structure were observed as a result of atrazine pulses. This lack of response may be due to exposure events of insufficient concentration or duration (consistent with previously reported results) or the composition of the algal assemblages present. This was in contrast to the effects of elevated flow events, which were associated with significant changes in periphyton abundance, diversity and assemblage.

Recovery of photosynthesis and growth rate in green, blue-green, and diatom algae after exposure to atrazine.

We evaluated the recovery of photosynthesis and growth rate in green (Pseudokirchneriella subcapitata), blue-green (Anabaena flos-aquae), and diatom (Navicula pelliculosa) algae after pulsed exposure to atrazine. Subsequent to a grow-up period of 24 to 72 h to establish requisite cell density for adequate signal strength to measure photosystem II (PSII) quantum yield, algae were exposed to a pulse of atrazine for 48 h followed by a 48-h recovery period in control media. Photosynthesis was measured at 0, 3, 6, 12, 24, and 48 h of the exposure and recovery phases using pulse amplitude modulation fluorometry; growth rate and cell density were also concomitantly measured at these time points. Exposure to atrazine resulted in immediate, but temporary, inhibition of photosynthesis and growth; however, these effects were transient and fully reversible in the tested species of algae. For all three algal species, no statistically significant reductions (p ≤ 0.05) in growth rate or PSII quantum yield were detected at any of the treatment concentrations 48 h after atrazine was removed from the test system. Effects at test levels up to the highest tested exposure levels were consequently determined to be algistatic (reversible). Both biochemically and physiologically, recovery of photosynthesis and growth rate occur immediately, reaching control levels within hours following exposure. Therefore, pulsed exposure profiles of atrazine typically measured in Midwestern U.S. streams are unlikely to result in biologically meaningful changes in primary production given that the effects of atrazine are temporary and fully reversible in species representative of native populations.

Investigating the impact of chronic atrazine exposure on sexual development in zebrafish.

Atrazine (ATZ) is a selective triazine herbicide used primarily for preemergent weed control in corn, sorghum, and sugar cane production. It is one of the most widely used herbicides in North America. Some research published over the last decade suggests that chronic exposure to environmentally relevant ATZ concentrations can adversely impact gonadal development and/or sexual differentiation in amphibians and fish, while other studies report no effect, or moderate effects. As a result, contrasting conclusions have been published regarding the potential effects of the herbicide ATZ on aquatic species. Two near-identical 4-month studies in 2009 (Study I) and 2010 (Study II) were performed investigating the potential for chronic ATZ exposure to affect zebrafish (Danio rerio) sexual development and differentiation. Zebrafish were chronically exposed to 0, 0.1, 1, 10 µM ATZ or 1 nM 17ß-estradiol (E2). Fish were histologically examined to assign gender and to evaluate potential impacts of E2 or ATZ on gonadal development. Exposure to E2 consistently resulted in a significantly higher proportion of female fish to normal male fish when compared to unexposed fish (both studies). In both studies, ATZ exposure did not significantly influence the percentage of female or male fish when compared to unexposed fish. A greater percentage of abnormally developed male fish and fish lacking differentiated gonadal tissue was observed in Study II E2 exposures but not in ATZ exposures. Together, these studies indicate that long-term exposure to ATZ at or above environmentally relevant concentrations does not significantly impact zebrafish gonadal development or sexual differentiation.

Recovery of duckweed from time-varying exposure to atrazine.

The purpose of the present study was to evaluate the recovery of duckweed (Lemna gibba L. G3) after being removed from multiple duration exposures to the herbicide atrazine. Consequently, L. gibba were exposed under various scenarios to atrazine at nominal concentrations ranging from 5 to 160 µg/L and durations of 1, 3, 5, 7, 9, and 14 d under static-renewal test conditions. Exposures were followed by a recovery phase in untreated media for either 7 or 14 d. The 3-, 5-, 7-, 9-, and 14-d median effective concentration (EC50) values were >137, >137, 124, >77, and >75 µg/L, respectively, based on mean growth rate. No clear effect trends were apparent between exposure duration and the magnitude of effective concentrations (EC50s or EC10s). No phytocidal effects of chlorosis or necrosis were identified for any treatment scenario. Nearly all L. gibba plants transferred from treatment groups of different exposure scenarios to media without atrazine during the recovery phase had growth rates that demonstrated immediate recovery, indicating effects were phytostatic in nature and reversible. Only the 1- and 5-d exposure scenarios had growth rates indicating marginally prolonged recovery at the higher concentrations (160 µg/L; additionally, at 40 µg/L for the 5-d exposure). Time to recovery, therefore, was found to be largely independent of exposure duration except at the highest concentrations assessed. Based on growth rate by interval, all treatments demonstrated recovery by the final assessment interval (days 5-7), indicating complete recovery in all exposure scenarios by 7 d, consistent with the mode of action of atrazine.

Influence of light intensity on the toxicity of atrazine to the submerged freshwater aquatic macrophyte Elodea canadensis.

Light intensity can have a profound influence on the degree of phytotoxicity experienced by plants exposed to photosystem II (PSII) inhibiting herbicides. This relationship was evaluated in the submerged aquatic macrophyte Elodea canadensis exposed to three different concentrations of atrazine (510, 1000 and 2000 µg a.i./L) plus an untreated control at three different light intensities (0, 500 and 6000 lx) under static-renewal conditions for 14 days. Under 500 lx light intensity, control plants demonstrated a rapid increase in shoot length but minimal increase in dry shoot weight, suggesting limited photosynthesis. Based on shoot-length and biomass, growth was not affected by any atrazine exposure relative to controls under dark conditions (0 lx). Under low-light conditions at 500 lx, exposures to 510, 1000 and 2000 µg a.i./L atrazine significantly decreased net shoot lengths by 34%, 38% and 35%, respectively, relative to corresponding (500 lx) controls. However, atrazine exposure under this light condition did not significantly decrease biomass (dry shoot weight). Compared to 6000 lx, only approximately 8% of photosynthetically active radiation (PAR) was measured under 500 lx intensity, indicating that minimal PAR was available for photosynthesis. Under optimal light conditions (6000 lx), net shoot lengths significantly decreased in the treated atrazine groups by 48%, 51% and 68%, and net dry shoot weights (biomass) were significantly decreased by 79%, 81% and 91%, respectively, relative to corresponding (6000 lx) controls. These data show that under low light conditions, atrazine-induced effects on dry shoot weight (biomass) are dependent on available PAR and active photosynthesis.

Effects of 17 beta-estradiol exposure on Xenopus laevis gonadal histopathology.

The natural estrogen 17 beta-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 microg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 microg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p<0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment.

Survival and iono-regulatory performance in Atlantic salmon smolts is not affected by atrazine exposure.

This study was conducted to determine the potential effects of atrazine exposure on survival and physiological performance in Atlantic salmon (Salmo salar) during the period of smoltification. This study involved two separate experiments in which juvenile Atlantic salmon were exposed to atrazine for a four day period in freshwater after which the fish were transferred to 50% seawater for two days and then to 100% seawater for five more days. The nominal concentrations of atrazine tested (1, 10 and 100 microg/L) were representative of and exceeded the levels measured in the North American freshwater environment. After seven days in seawater, fish were weighed, bled for the determination of plasma electrolyte levels, euthanized and samples collected for the determination of gonadosomatic index, muscle water content and gill Na+/K+-ATPase activity. Measured atrazine concentrations during the freshwater exposure period were 76-99% of nominal levels. There were no mortalities attributed to atrazine exposure. There were also no statistically significant differences in body weight, plasma sodium, potassium, magnesium and chloride levels, muscle water content or gill Na+/K+-ATPase activity between control and atrazine treated fish. Measurement of testis and ovary weights showed that there were no treatment effects on relative gonad size in male or female fish. These studies have shown that short term exposure to atrazine during the freshwater phase of their lifecycle had no effects on subsequent survival, body weight, relative gonad size or various measures of iono-regulatory performance in juvenile Atlantic salmon upon transfer to seawater. The concentrations of atrazine tested exceed those likely to be experienced in the natural aquatic environment suggesting that short term exposure to atrazine does not pose a risk to Atlantic salmon during the period of smoltification.

Population-specific incidence of testicular ovarian follicles in Xenopus laevis from South Africa: a potential issue in endocrine testing.

The African clawed frog (Xenopus laevis) has been identified as an appropriate sentinel for testing endocrine activity of existing chemicals in North America and Europe. Some reports suggest that the herbicide, atrazine (CAS Number [1912-24-9]) causes ovarian follicles to form in the testes of this frog. X. laevis collected from North East (NE) sites in South Africa had testicular ovarian follicles, irrespective of exposure to atrazine, while frogs from Southwest Western (SW) Cape region sites had none. Phylogenetic analysis of mitochondrial and nuclear genes indicates that frogs from the SW Cape are evolutionarily divergent from those from NE South Africa and the rest of sub-Saharan Africa. These findings provide a possible explanation for why conflicting results have been reported concerning the impact of atrazine on amphibian sexual differentiation and highlight the importance of understanding taxonomic status of the experimental animal. Even in common laboratory animals, there is a need for their correct taxonomic characterization before their use in tests for endocrine disruption.

Does atrazine affect larval development and sexual differentiation of South African clawed frogs?

The potential impact of atrazine (ATZ) on gonadal malformations in larval Xenopus laevis has been controversially discussed, and a hypothesis has been generated that ATZ might induce the estrogen-synthesizing enzyme aromatase, leading to feminization or demasculinization. Recently, extensive long-term studies clearly indicate that no adverse effect of ATZ on larval development and sexual differentiation could be found. Therefore, to determine potential transient impacts of ATZ on sexual differentiation processes, short-term exposures were conducted using tadpoles treated for 4 days with ATZ at 25 microg/L. The expression levels of the key players for sexual differentiation in amphibians were determined in the brain, assessing aromatase, 5alpha-reductase type 1 (S1) and type 2 (S2), and the gonadotropins luteinizing hormone and follicle-stimulating hormone, and in the gonads, measuring aromatase, S1, and S2, by means of quantitative RT-PCR. No significant changes in any of these parameters have been found, implicating, in accordance with recent long-term exposures, that no aromatase induction by ATZ could be observed, and it seems likely that no further endocrine mechanism of ATZ affecting sexual differentiation in X. laevis exists.