Chantal M. Lanctôt

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frog (Lithobates sylvaticus) tadpoles. II: Agriculturally relevant exposures to Roundup WeatherMax® and Vision® under laboratory conditions

Glyphosate-based herbicides are currently the most commonly used herbicides in the world. They have been shown to affect survival, growth, development and sexual differentiation of tadpoles under chronic laboratory exposures but this has not been investigated under more environmentally realistic conditions. The purpose of this study is (1) to determine if an agriculturally relevant exposure to Roundup WeatherMax®, a relatively new and understudied formulation, influences the development of wood frog tadpoles (Lithobates sylvaticus) through effects on the mRNA levels of genes involved in the control of metamorphosis; (2) to compare results to the well-studied Vision® formulation (containing the isopropylamine salt of glyphosate [IPA] and polyethoxylated tallowamine [POEA] surfactant) and to determine which ingredient(s) in the formulations are responsible for potential effects on development; and (3) to compare results to recent field studies that used a similar experimental design. In the present laboratory study, wood frog tadpoles were exposed to an agriculturally relevant application (i.e., two pulses) of Roundup WeatherMax® and Vision® herbicides as well as the active ingredient (IPA) and the POEA surfactant of Vision®. Survival, development, growth, sex ratios and mRNA levels of genes involved in tadpole metamorphosis were measured. Results show that Roundup WeatherMax® (2.89 mg acid equivalent (a.e.)/L) caused 100% mortality after the first pulse. Tadpoles treated with a lower concentration of Roundup WeatherMax® (0.21 mg a.e./L) as well as Vision® (2.89 mg a.e./L), IPA and POEA had an increased condition factor (based on length and weight measures in the tadpoles) relative to controls at Gosner stage (Gs) 36/38. At Gs42, tadpoles treated with IPA and POEA had a decreased condition factor. Also at Gs42, the effect on condition factor was dependent on the sex of tadpoles and significant treatment effects were only detected in males. In most cases, treatment reduced the normal mRNA increase of key genes controlling development in tadpoles between Gs37 and Gs42, such as genes encoding thyroid hormone receptor beta in brain, glucocorticoid receptor in tail and deiodinase enzyme in brain and tail. We conclude that glyphosate-based herbicides have the potential to alter mRNA profiles during metamorphosis. However, studies in natural systems have yet to replicate these negative effects, which highlight the need for more ecologically relevant studies for risk assessment.

Effects of glyphosate-based herbicides on survival, development, growth and sex ratios of wood frogs (Lithobates sylvaticus) tadpoles. I: chronic laboratory exposures to VisionMax®.

The purpose of this study was to determine if chronic exposure to the glyphosate-based herbicide VisionMax(®) affects the survival, development, growth, sex ratios and expression of specific genes involved in metamorphosis of wood frog tadpoles (Lithobates sylvaticus). We hypothesized that exposure to this herbicide will affect developmental rates by disrupting hormone pathways, sex ratios and/or gonadal morphology. Tadpoles were chronically exposed in the laboratory from Gosner developmental stage 25 to 42 to four different concentrations of VisionMax(®) (ranging from 0.021 to 2.9 mg acid equivalents/L). Chronic exposures to VisionMax(®) had direct effects on the metamorphosis of L. sylvaticus tadpoles by decreasing development rates, however, there was a decrease in survival only in the group exposed to the highest dose of VisionMax(®) (2.9 mg a.e./L; from approximately 96% in the control group to 77% in the treatment group). There was a decrease in the number of tadpoles reaching metamorphic climax, from 78% in the control group to 42% in the VisionMax(®) (2.9 mg a.e./L) group, and a 7-day delay to reach metamorphic climax in the same treatment group. No effects of exposure on sex ratios or gonadal morphology were detected in tadpoles exposed to any of the concentrations of VisionMax(®) tested. Gene expression analyses in brain and tail tissues demonstrated that exposure to VisionMax(®) alters the expression of key genes involved in development. Results showed significant interaction (two-way ANOVA, P<0.05) between developmental Gosner stage and treatment in brain corticotropin-releasing factor, deiodinase type II (dio2) and glucocorticotiroid receptor (grII) and tail dio2 and grII. This demonstrates that mRNA levels may be differently affected by treatment depending on the developmental stage at which they are assessed. At the same time there was a clear dose-response effect for VisionMax(®) to increase thyroid hormone receptor β in tadpole brain (F(2,69)=3.475, P=0.037) and tail (F(2,69)=27.569, P<0.001), regardless of developmental stage. Interestingly, delays in development (or survival) were only observed in the group exposed to 2.9 mg a.e./L of VisionMax(®), suggesting that tadpoles need to be exposed to a threshold concentration of glyphosate-based herbicide to exhibit phenotypic observable effects. We suggest that the upregulation of genes that trigger metamorphosis following VisionMax(®) herbicide exposure might result from a compensatory response for the delays in development observed. Further studies are needed to determine if disruption of expression of these key genes leads to long-term effects when metamorphs reach adult stages.

Individual and Mixture Toxicity of Pharmaceuticals Naproxen, Carbamazepine, and Sulfamethoxazole to Australian Striped Marsh Frog Tadpoles (Limnodynastes peronii)

Nonsteroidal human pharmaceuticals are prevalent in domestic wastewater and may find their way into the environment at low concentrations. Since most pharmaceuticals are designed to be biologically active at low concentrations, there is a risk that these compounds may affect aquatic wildlife. Of particular concern is the occurrence of pharmaceutical mixtures, which may lead to increased adverse effects compared to individual compounds. Interactive effects were previously demonstrated for amphibians exposed to pesticide mixtures, but no such studies investigating responses of amphibians to pharmaceutical mixtures are apparently available. Results demonstrated increased toxicity (loss of tactile response) of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a mixture of naproxen, carbamazepine, and sulfamethoxazole, compared to exposures to the individual compounds. Significant timeu2009×u2009treatment interactions were observed for tadpole development following chronic exposures to 10 or 100 μg/L of each compound and the mixture; however, responses were weak and main treatment effects were not significant. Despite minor effects at low exposure concentrations, results demonstrated a potential for mixtures of nonsteroidal pharmaceuticals commonly occurring in wastewater to influence amphibian development. With the vast numbers of pharmaceuticals that exist and are found in the environment, this work highlights a need for further research into mixtures of pharmaceutically active wastewater contaminants. Further, since pharmaceuticals exert extremely varied biological actions, it is suggested that future investigations would benefit from inclusion of endpoints that are indicative of physiological or metabolic performance, as well as assessment of sensitive behavioral responses.

Effects of the glyphosate-based herbicide Roundup WeatherMax ® on metamorphosis of wood frogs (Lithobates sylvaticus) in natural wetlands

Amphibian tadpoles develop in aquatic environments where they are susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are currently the most commonly used herbicide in the world and have been shown to affect survival and development of tadpoles under laboratory and mesocosm conditions. In the present study, whole wetland manipulations were used to determine if exposure to an agriculturally relevant application of Roundup WeatherMax(®), a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the development of wood frog tadpoles (Lithobates sylvaticus) under natural conditions. Wetlands were divided in half with an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at an environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and the predicted maximum environmental concentration (PMEC, 2.89 mg a.e./L), after which abundance, growth, development, and mRNA levels of genes involved in tadpole metamorphosis were measured. Results present little evidence that exposure to this herbicide affects abundance, growth and development of wood frog tadpoles. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research demonstrates that typical agricultural use of Roundup WeatherMax(®) poses minimal risk to larval amphibian development. However, our gene expression data (mRNA levels) suggests that glyphosate-based herbicides have the potential to alter hormonal pathways during tadpole development.

Sexing frogs by real-time PCR: using aromatase (cyp19) as an early ovarian differentiation marker.

Most anurans have no identified sex-markers; therefore, alternative methods for identification of early changes in sex ratios are required. In this study, Lithobates sylvaticus and Silurana tropicalis tadpoles were sampled at different developmental stages covering the entire process of sex differentiation. Three candidate genes known to be involved in sex differentiation in other vertebrate species were selected to develop a method to identify phenotypic sex in frogs: cytochrome p450 aromatase (cyp19), forkhead box L2 (foxl2) and the cytochrome 17-alpha-hydroxylase/17,20 lyase (cyp17). Cloning of these genes revealed nucleotide identity values ranging between 75–97% when compared to other amphibian species. Gene expression of cyp17,cyp19 and foxl2 in L. sylvaticus adult gonads and gonad-mesonephros complex (GMC) of tadpoles was analyzed by real-time RT-PCR. Results showed clear sexually dimorphic patterns in the expression of the 3 genes. Our analysis reveals that GMC gene expression levels of cyp19 alone can be used as a robust predictor of phenotypic sex in L. sylvaticus tadpoles. In addition, we validated this method measuring cyp19 mRNA levels in S. tropicalis GMCs. We propose measuring cyp19 as a tool to study the effects of chemical contaminants (including endocrine disrupting compounds) on amphibian gonadal development and sex ratios in the future.

Effects of naphthenic acid exposure on development and liver metabolic processes in anuran tadpoles.

Naphthenic acids (NA) are used in a variety of commercial and industrial applications, and are primary toxic components of oil sands wastewater. We investigated developmental and metabolic responses of tadpoles exposed to sub-lethal concentrations of a commercial NA blend throughout development. We exposed Lithobates pipiens tadpoles to 1 and 2xa0mg/L NA for 75 days and monitored growth and development, condition factor, gonad and liver sizes, and levels of liver glucose, glycogen, lipids and cholesterol following exposure. NA decreased growth and development, significantly reduced glycogen stores and increased triglycerides, indicating disruption to processes associated with energy metabolism and hepatic glycolysis. Effects on liver function may explain reduced growth and delayed development observed in this and previous studies. Our data highlight the need for greater understanding of the mechanisms leading to hepatotoxicity in NA-exposed organisms, and indicate that strict guidelines may be needed for the release of NA into aquatic environments.

Behaviour, development and metal accumulation in striped marsh frog tadpoles (Limnodynastes peronii) exposed to coal mine wastewater

Coal mining generates large quantities of complex effluent, and this often contains high levels of dissolved solids, suspended solids, metals, hydrocarbons, salts and other compounds. Substantial volumes of mine wastewater are periodically discharged into the environment, through both planned and accidental releases, and this raises concerns about the potential for adverse impacts on aquatic wildlife. There have been few attempts to explore sub-lethal effects of coal mine wastewater on amphibians compared to other organisms, and this is particularly true for Australian species. To address existing knowledge gaps, we exposed striped marsh frog (Limnodynastes peronii) tadpoles to 25, 50 and 100% coal mine wastewater collected from two holding dams (CMW1 and CMW2) located at an open cut mine in Central Queensland, Australia. The exposure lasted for four weeks, after which survival, growth and development, swimming behaviour, and concentrations of metals and metalloids in tail and liver tissues were assessed. Physico-chemical parameters varied considerably between sites, with higher turbidity, nutrients, total and dissolved organic carbon, alkalinity and arsenic (As) concentrations at CMW1, and higher conductivity, salinity, dissolved solids, hardness and sulfate levels at CMW2. There was no mortality in controls and less than 5% mortality in CMW1 treatments, whereas survival was significantly decreased in tadpoles exposed to CMW2 with 40 and 55% mortality in the 50 and 100% treatments, respectively. Development was significantly delayed in 100% CMW1 wastewater, but tadpole size (growth) was not influenced by the exposure. Hepatosomatic indices were significantly increased in tadpoles exposed to 25 and 50% CMW1 but not the 100% treatment group. Exposed tadpoles (predominantly those exposed to CMW1) exhibited increased activity after very short-term exposure (24h), but this did not persist as animals approached metamorphic climax. At the end of the experiment, tadpoles exposed to both wastewaters had elevated levels of selenium (Se), cobalt (Co) and As in tail and liver tissue compared to controls. Manganese (Mn) levels were also elevated in livers and tails of CMW2 exposed tadpoles. Hepatic tissue accumulated 8-9 times higher concentrations of Co, Mn and Se compared to tail tissue, irrespective of treatments. Future research is warranted to explore possible relationships between metal bioaccumulation, morpho-physiological effects during development, and subsequent higher-level outcomes related to individual performance and population fitness.

Locomotor and behavioural responses of empire gudgeons (Hypseleotris compressa) exposed to coal mine wastewater.

Coal mining generates large quantities of complex effluent and may pose a threat to aquatic wildlife. Despite this, few studies have explored the consequences of exposure to mine wastewater on aquatic organisms, and this is particularly true for the Australian environment. We investigated sub-lethal behavioural responses in a native Australian fish exposed to wastewater from two releasing dams (CMW1 and CMW2) located at an open cut coal mine in Central Queensland. Swimming activity and movement of empire gudgeons (Hypseleotris compressa) were assessed during a two-week exposure using video-tracking software. Increased activity was observed in exposed fish after 7 and 14 days. Specifically, we found a significant increase in the mean velocity and mobility of fish exposed to CMW1 treatments. Exposed fish also spent on average 23% more time in the peripheral zone compared to controls after 14-d exposures. A similar response pattern was observed in fish exposed to CMW2, but differences between treated and control fish did not generally reach statistical significance. Alterations to normal swimming activity and movement patterns can be indicative of a stress response in fish, and could subsequently lead to negative population-level impacts by increasing the conspicuousness of exposed individuals to predators, or by altering foraging abilities. More research is warranted to explore relationships between behavioural and physiological outcomes, including endocrine disruption, and subsequent population-level outcomes in aquatic organisms at risk of exposure to coal process-affected water.

Sodium perchlorate disrupts development and affects metamorphosis- and growth-related gene expression in tadpoles of the wood frog (Lithobates sylvaticus)

Numerous endocrine disrupting chemicals can affect the growth and development of amphibians. We investigated the effects of a targeted disruption of the endocrine axes modulating development and somatic growth. Wood frog (Lithobates sylvaticus) tadpoles were exposed for 2weeks (from developmental Gosner stage (Gs) 25 to Gs30) to sodium perchlorate (SP, thyroid inhibitor, 14mg/L), estradiol (E2, known to alter growth and development, 200nM) and a reduced feeding regime (RF, to affect growth and development in a chemically-independent manner). All treatments experienced developmental delay, and animals exposed to SP or subjected to RF respectively reached metamorphic climax (Gs42) approximately 11(±3) and 17(±3) days later than controls. At Gs42, only SP-treated animals showed increased weight and snout-vent length (P<0.05) relative to controls. Tadpoles treated with SP had 10-times higher levels of liver igf1 mRNA after 4days of exposure (Gs28) compared to controls. Tadpoles in the RF treatment expressed 6-times lower levels of liver igf1 mRNA and 2-times higher liver igf1r mRNA (P<0.05) at Gs30. Tadpoles treated with E2 exhibited similar developmental and growth patterns as controls, but had increased liver igf1 mRNA levels at Gs28, and tail igf1r at Gs42. Effects on tail trβ mRNA levels were detected in SP-treated tadpoles at Gs42, 40days post-exposure, suggesting that the chemical inhibition of thyroid hormone production early in development can have long-lasting effects. The growth effects observed in the SP-exposed animals suggest a relationship between TH-dependent development and somatic growth in L. sylvaticus tadpoles.

Altered bioenergetics and developmental effects in striped marsh frog (Limnodynastes peronii) tadpoles exposed to UV treated sewage.

Effectively treating domestic wastewater so that it can be safely discharged or reused is critical for maintaining the integrity of freshwater resources, and for protecting the health of animals that rely on these systems. Amphibians are currently facing widespread population declines, so there is a particularly urgent need to investigate exposure scenarios that might result in weakened amphibian populations. Domestic sewage has received little attention as a possible factor that could influence the survival, growth and development, or general health of amphibians. However, wastewater reuse for crop irrigation and other purposes is increasing and holding ponds and constructed wetlands exist at many wastewater treatment facilities, introducing conceivable pathways that could result in the exposure of amphibians to treated wastewater. We exposed developing striped marsh frog (Limnodynastes peronii) tadpoles, to control water and 12.5, 25, 50 and 100% UV treated domestic sewage, and quantified effects on growth and development, hepatic energy reserves, and enzymatic pathways associated with detoxification and oxidative stress. Growth and development were accelerated and relative liver size was increased in exposed animals. The exposure resulted in an apparently hormetic increase in hepatic triglycerides and dose-dependent reduction in glycogen stores, as well as increased lipase and NADPH activity, indicating a general disruption to energy metabolism and/or mobilization. Contrary to expectations based on published studies with fish, we found no evidence of lipid peroxidation or induction of the detoxification enzyme Superoxide Dismutase (SOD), however, this may reflect the use of UV treatment as opposed to chlorination for disinfection. Chemical analysis and risk-based prioritization consistently identified fluoxetine, triclosan and diazinon as high-risk contaminants in the wastewater, with nonylphenol and mestranol flagged as risks during one early collection. Research is needed to explore the potential for these specific contaminants to elicit the responses identified in the present study, and to perform similar assessments using wastewater from other locations with different treatment options.