Purpose of Project Expected Results Background ...

Low Level Atrazine Exposure Effects on Crayfish Development Nathan Chandler, Mackenzie Lecher, Austin Minuto, Samuel Owens, Abbie Suttle, Caroline Williams, and Kyle J. Harris, M.S.

Research Plan

Expected Results

Eight twenty-four liter BPA-free plastic aquaria were all filled with eighteen liters of treated tap water (Figure 3). Two enclosures were assigned to each of the ATZ exposure concentrations (0.00, 0.05, 0.5, 5.0 µg/L). These concentrations were chosen to reflect values above and below the EPA safe level of 2.6 µg/L (Atrazine, 2017). Each of the eight enclosures contained four small BPA-free enclosures with holes drilled on each side and a air sponge filter to pull the surrounding water into the enclosures to facilitate movement of water within the entire enclosure and within each microenvironment. Crayfish were isolated in microenvironments to minimize predation. A layer of substrate was taken from the crayfish collection site, Opossum Creek (Campbell County, VA, 37°20’26” N, 79°08’54” W) and added to each large aquaria and each microenvironment (Figure 4). In August 2017, a kick-seine was used to collect thirty-two male and eight female juvenile crayfish (Cambarus sp.) from Opossum Creek (Figure 4). Crayfish were taken from the stream to the enclosures and were acclimated for two weeks prior to atrazine introduction. One male crayfish occupied each microenvironment, totaling four males per enclosure. Each large enclosure contained a free-swimming female. The crayfish will be feed three times a week with a mixture of crushed goldfish flake food, algae wafers, and shrimp pellets. Partial water changes (6L and 12L) will alternately be performed each week, along with water quality testing to help maintain the pH (7.6), KH (3.5 - 4), ammonia (0), and nitrate (0) using an API brand testing kit. Doses of ATZ were prepared using a serial dilution in menthol and stored at room temperature. Initial ATZ exposure (0.00, 0.05, 0.5, 5.0 µg/L) was added to treatment aquaria on September 1, 2017. The concentration and bio-degradation of the ATZ in each enclosure will be monitored using High Performance Liquid Chromatography (HPLC).

Growth and Development It is expected that low level ATZ exposure will result in suppressed crayfish growth as shown by CL, TL, and BWM (Figure 6). Growth within the groups (0.00, 0.05, 0.5, 5.0 µg/L) will be compared for the effects of low level exposure to ATZ.

Purpose of Project Herbicides designed for target organisms can have unintended consequences on nontarget organisms (Hayes & Hansen, 2017). Many non-target organisms reside within freshwater ecosystems. Crayfish are an abundant organism in Virginia streams and make an ideal model organism to study the effects of herbicides, such as Atrazine (ATZ), on non-target species. It is hypothesized that low-level ATZ exposure (0.05-5.0µg/L) will result in a reduction of overall growth and the degradation of reproductive tissue in juvenile crayfish.

Background Information Herbicides, such as ATZ, are known to be present in freshwater ecosystems and contribute to endocrine disruption in non-target organisms (Hayes & Hansen, 2017). ATZ is the second most commonly used herbicide in the United States and is observed at low-level usage in Virginia (Figure 1). Run-off introduces ATZ to stream environments where it can remain in the water for up to 200 days (Knaebel et al., 2003). Concentrations of ATZ are commonly detected at levels above the EPA standard in rural areas and some reach bursts of 50 μg/L (Wu et al., 2010).

Histology H&E staining of the testes and ovary tissues are expected to display an increased amount of degradation in relation to increasing amounts of low level ATZ exposure (Figure 7). Vimentin immunohistochemistry staining is expected to display decline in the total number of Sertoli cells in the testes with increasing amounts of low level ATZ exposure.

Figure 6. Average increase in BWM (top) and CL (bottom) after the 5-month ATZ treatment. The numbers on the x-axis represent the different concentrations of ATZ; 1 is the control, 2 is the 0.05 ppb, 3 is the 0.5 ppb, 4 is the 5.0 ppb, and 5 is 50.0 ppb (Chandler et al., 2017)

Figure 7. Effects of ATZ on the seminiferous tubules of male crayfish (Chandler et al., 2017).

References 2012 Pesticide Use Maps (2017, January 17). Retrieved October 14, 2017, from http://water.usgs.gov/nawqa/pnsp/usage/maps/show_map.php?year=2012&map=ATRAZINE&hilo=L&disp=Atrazine

Figure 1. Usage map of atrazine in the United States.

The influence of ATZ as an endocrine disrupter has been observed in a variety of invertebrates, amphibians, fish, and mammals. For example, male African clawed frogs exposed to ATZ showed feminization and chemical castration (Hayes et al., 2010). Fathead minnows and rats have shown a decrease in fertility (Abarikwu et al., 2015; Tillitt et al., 2010). The nature of ATZ’s effects on non-target organisms come in part from its biological function as a xenoestrogen, which can reduce gonad size and sperm counts and promote feminization (Figure 2). In addition, limited studies exist on the effects of ATZ on crayfish even though it is widely distributed in freshwater ecosystems (Belanger et al., 2017; Chandler et al., 2017; Mac Loughlin et al., 2016).

Atrazine – Background and Updates (2017, June 28). Retrieved October 17, 2017, from https://www.epa.gov/ingredients-usedpesticide-products/atrazine-background-and-updates#main-content

Figure 3. Aquaria set-up for trial.

Figure 4. Opossum Creek, Campbell County, VA (top) and adult crayfish (bottom) from study site.

Initial Carapace length (CL), total length (TL), and blotted wet mass (BWM), of all crayfish were recorded and will be measured monthly over the course of the trial (approximately 22 weeks). The crayfish will be grown to a size at which the reproductive tissue is large enough to perform histological staining to observe the effects of ATZ on tissue development. The gonads of the crayfish will be removed and preserved in formalin for histological staining procedures. Tissue samples taken from the crayfish will be dehydrated then embedded in paraffin wax, sectioned, and stained using Hematoxylin and Eosin (H&E). The Vimentin immunohistochemistry stain will also be used to target the Sertoli cells and further identify cell death at low dose exposure to ATZ (Figure 5).

Abarikwu, S.0., Duru, Q. C., Chinonso, O.V., & Njoku, R. (2015). Antioxidant enzymes activity, lipid peroxidation, oxidative damage in the testis and epididymis, and steroidogenesis in rats after co-exposure to atrazine and ethanol. Andrologia, 48(5), 548557. DOI:10.1111/and.12478 Belanger, R. M., Evans, K. R., Abraham, N. K., & Barawi, K. M. (2017). Diminished conspecific odor recognition in the Rusty Crayfish (Orconectes rusticus) following a 96-h exposure to Atrazine. Bulletin of Environmental Contamination and Toxicology, 1-6. Chandler, N. T., Minuto, A. M., Froese, S., Suttle, A. H., Owens, S., Grant, J., Sooklal, S., Allen, T., Blais, M., & Harris, K. J. (2017). The effects of a common herbicide (Atrazine) on juvenile crayfish growth and development. DOI:10.13140/RG.2.2.23683.20002 Hayes, T. B., Khoury, V., Narayan, A., Nazir, M., Park, A., Brown, T., Adame, L., Chan, E., Buchholz, D., Stueve, T., & Gallipeau, S. (2010). Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis). Proceedings of the National Academy of Sciences, 107(10), 4612-4617. DOI:10.1073/pnas.0909519107. Hayes, T. B., & Hansen, M. (2017). From silent spring to silent night: Agrochemicals and the Anthropocene. Elementa Science of the Anthropocene, 5, 57. DOI: https://doi.org/10.1525/elementa.246 Knaebel, D. B., Little, S. S., Plewak, D., Printup, H., & Rhoades, J. (2003). Toxicological profile for Atrazine. U.S. Department of Health and Human Services. Retrieved October 18, 2017 from https://www.atsdr.cdc.gov/toxprofiles/tp153.pdf Mac Loughlin, C. M., Canosa, I. S., Silveyra, G. R., Greco, L. S., & Rodriguez, E. M. (2016). Effects of atrazine on growth and sex differentiation, in juveniles of the freshwater crayfish Cherax quadricarinatus. Ecotoxicology and Environmental Safety, 131, 96103. DOI:10.1016/j.ecoenv.2016.05.009

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Tillitt, D. E., Papoulias, D. M., Whyte, J. J., & Richter, C. A. (2010). Atrazine reduces reproduction in fathead minnow (Pimephales promelas). Aquatic Toxicology, 99(2), 149-159. DOI:10.1016/j.aquatox.2010.04.011

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Figure 2. (A) Effects of ATZ on juvenile crayfish growth; (B) normal gonad development when exposed to ATZ(Mac Loughlin et al., 2016); (C) Evident degradation of male juvenile crayfish semiferous tubules when exposed to a range of ATZ (0.05-50 μg/L). 0.0 μg/L top and 50.0 μg/L bottom (Chandler et al., 2017).

Wu, M., Quirindongo, M., Sass, J., & Wetzler, A. (2010). Still poisoning the well. Natural Resources Defense Council. Retrieved October 18, 2017 from https://www.nrdc.org/sites/default/files/atrazine10.pdf.

Figure 5. Flow diagram of the research plan.