Abstract

Atrazine is a commonly used herbicide for the elimination of weeds that are found in agriculture plots. It is cheaply available for farmers and helps them with keeping low production costs. The chemical is found as agricultural runoff and when it is exposed to organisms it acts as an endocrine disrupting chemical, which caused an increase in estrogen and decrease in testosterone levels. In North America there has been a rapid decline of anuran amphibian species. Various scientists have linked the decline of anuran amphibians to the exposure of atrazine. The relationship between atrazine and amphibian decline is defined through abnormalities in the gonad development of tadpoles and feminized sex ratios of the amphibian population at lower concentrations than what is legally allowed in the water. Such adverse effects highlight the linked between the long-term exposure of atrazine to the population decline of anuran amphibians. This literature review will focus on how atrazine affects the reproductive system of frogs. There will be a focus on the effects of atrazine with regards to, gonads, sex ratios, and its connection to the population decline.

Keywords: Anuran Amphibians, Atrazine, Sex Ratios, Population Decline.

Introduction

Many amphibian species are at risk of extinction around the world. Nearly, 32% of the world’s amphibians are threatened with extinction (Climate Change Canada 2010). In regards to Canada of the 47 species found in the country 20% are at risk of extinction. The dwindling population of amphibians across the world has sparked an increase discussion relating to factors that may have contributed to this threat. Over the past twenty years there has been an increase in concern on what factors have possibly contributed to the population decline of amphibians. Major threats include habitat loss, invasive species, pollution, and climate change. Some areas of southern Canada have had a 90% in wetland loss with the remaining 10% containing pollutants from agricultural or urban runoff (Climate Change Canada 2010).

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  As agricultural practices become modernized chemicals such as herbicides have been applied in greater quantities. Farmers have benefited from the selective, effective and inexpensive herbicide called Atrazine. This chemical has helped farmers manage weeds and reduce production costs (Ackerman 2007). When applied to a plant, atrazine affected the plastoquinone-binding protein in photosystem II and caused oxidative damage caused the death of the plant (Appleby,Franz and Carpy 2001). Consequently, Atrazine has received attention as the main potential cause of amphibian declines particularly in frogs. Field studies have shown a correlation regarding population declines in amphibians and agricultural runoffs (Houlahan and Findlay, 2003). The chemical also acted as an endocrine disrupting chemical (EDC) at low doses when it is exposed to organisms. Atrazine interfered with the catalytic activity of aromatase. Aromatase is an enzyme that converted androgens to estrogen. An effect the disruption showed was an increased level of estrogen in an organism. Furthermore, an increase of malformed gonads in frogs living in agricultural areas where pesticides and fertilizers had been applied extensively was observed (Ouellet et al., 1997; Hayes 2010). Some studies have also observed a sex reversal in which the populations underwent increased feminization and lower instances sexual competitiveness of frogs (Hoskins and Boone 2017; Langlois et al. 2010).
 The topic of atrazine affecting the reproductive system of frogs is a western dilemma. Geographically, many of these studies were conducted in North America with no studies used in this review from the developing world. The studies are a mixture of field and lab work. This is a local issue as atrazine is heavily used in Canada and tends to be found in drinking water at low levels consisting of 5 µg/L (Health Canada 1993). There would be differences in how the studies would be treated around the world since factors such as climate, regulations, husbandry and species of frogs used vary.
 In this review I will focus on how atrazine affects the reproductive system of frogs. There will be a focus on the effects of atrazine with regards to gonads, sex ratios, and its connection to the population decline.

Amphibian Population Decline

Numerous species of Anuran amphibians (i.e frogs and toads) have experienced a population decline across a variety habitats worldwide. In the 1990s there was a high mortality rate in various amphibian populations and it caused a rapid of a population declines in wetlands ever since (Wake, 1991). An ecological decline is defined as the net loss of a population that is higher than the birth rate of the normal population (Stuart et al., 2004). Toads and frogs formed approximately 90% of all amphibian’s species and were deemed as an important connection to the health of an ecosystem (Hayes, et al., 2002). The global amphibian decline has caused a lot of concern for both researches , as amphibians are often reference point for any positive or detrimental environmental change (Wake and Vredenburg, 2008). Amphibian’s permeable skin, that is used for gas exchange and osmo-regulation, makes them sensitive to contaminants and pathogens that cause adverse effects on their overall health. Any disturbances such as habitat degradation, pollution, the increased introduction of destructive invasive species, and climate change can decrease their population size (Climate Change Canada, 2010). Particularly, popular herbicides such as atrazine received attention as frogs are exposed to the harmful effects of the environmental contaminant throughout different life-stages (Hayes, et al., 2002).

Effects of the Gonadal development of Anuran amphibians when Exposed to Atrazine

  EDCs such as atrazine have been researched heavily in frogs for numerous reasons. The time of the year when atrazine is applied is not ideal for anuran amphibians as it is mostly applied in the spring in the same time as its breeding season (Conant, 1998). The highest amount of atrazine in the water was found at the same time as the larval development of anuran amphibians (Conant, 1998). The larvae period is also the time when gonadal differentiation occurred and is regulated by hormones such as estrogen and testosterone (Hayes et al. 2002) and as the interference with the natural sexual development of the organisms caused long term gonad abnormalities that deterred their breeding capabilities.

  Various studies have examined the gonadal development of frogs when the organisms were exposed to atrazine. In the University of Berkeley, Hayes et al., 2002 had observed the full gonad development of 150 African Clawed frogs (Xenopus laevis) period over a period of 46 days. 75 frog eggs were obtained within the lab at Berkeley and 75 wild eggs were obtained from an uncontaminated pond located in Wisconsin. Each organism was exposed to different concentrations of atrazine (0,0.1,0.4,0.8,1, and 25 parts per billion).After the 46 days each frog was euthanized. A gonadal analysis of each specimen’s ovaries or testes were conducted post mortem and marked with Mallory’s trichrome stain (Hayes et al., 2002). The control group did not have any gonadal malformities (p<0.05). Consequently, Hayes et al. (2002) reported gonadal abnormalities in all the other exposure concentration; For instance, 20% of the total population had developed multiple gonads where certain frogs had developed six gonads or had developed as a hermaphrodite that contained multiple testes and ovaries at concentrations as low as 0.1 parts per billion. Frogs were classified as Hermaphrodite if they had developed testicular oocytes (figure 1)(p<0.05) (Hayes et al.,2002). Male X.laevis were more affected by the exposure to atrazine than their female counterparts. Males suffered from a 30 % underdevelopment of testicular tissue, laryngeal size and some samples were classified as hermaphrodites but were found as genetic males (p<0.05). Compared to the control the atrazine exposed male frogs had a lower amount of testosterone and obtained low mass levels (Hayes et al.,2002). A study by Mccoy et al. (2008) also observed gonadal malformities of 100 wild Bufo marinus from five different wetland sites that contained herbicides such as atrazine and round up in the South Florida area over a period of two years. Each organism was euthanized when the gonadal analysis commenced. Mccoy et al. (2008) survey results found an increasing number of organisms that had become intersex. The intersex B.marius had more estrogen than testosterone and a higher of number of ovaries than testes. The male specimen were also smaller than the control, developed oocytes, underdeveloped testes, low testosterone levels and a decreased production of sperm (p<0.001)(Mccoy et al. ,2008). Both studies theorized that the frog populations were demasculinized from chronic exposure of atrazine as the results had affected more male samples than female through underdeveloped testes, smaller laryngeal size and low testosterone levels.

Figure 1: A sample of Gonadal abnormalities found in an atrazine treated Xenopus laevis hermaphrodite. This sample wsa exposed to 1 parts per billion of atrazine. As a result, the sample produced multiple testes (labelled T) and ovaries (labelled O). The right side of the picture was a cross section of the gonads. The pairs of testes were deemed smaller than the male control group testes and lack of pigmentation in the ovaries (E). The results above was common in many hermaphrodite gonad samples in exposures between 1 -25 parts per billion (p<0.05). Credit Hayes et al. 2002.

Figure 2: Testosterone concentrations across many different groups of Bufo Marinus. The male control group had the highest amount of testosterone. The male experimental group significantly had a lower amount of testosterone. The agricultural exposed males had about the same amount of testosterone levels as the control females. The error bars are from a 95 % confidence interval and significance p value of 0.001 in all the bars except the control which the p value is 0.05.Credit to Mccoy et al 2008.

A lack of consensus existed on the point of gonadal malformities from the exposure to atrazine. Coady et al. (2004) disagreed that gonadal malformations stemmed from atrazine exposure. 2500 Rana clamitan eggs were collected from an uncontaminated rural pond in Michigan and placed the eggs in filtered water tanks until the samples hatched in which was completed in 8 days (Coady et al., 2004). After the sample organisms hatched samples were placed in 10 and 25 µg/L of atrazine in which a total of nine replicated of each sample was created. The solution was applied into the water every 72 hours for a period of 273 days. When all the samples of R.clamitan reached metamorphosis the specimens were transferred to filtered water tanks and left in the tanks for another 234 days when the sample were terminated. The gonads of each sample were analyzed in post mortem (Coady et al. ,2004). Only 5.9% of the population suffered from gonad abnormalities and none of the sample gonads were hermphodites (p<0.05) (Coady et al., 2004). Coady et al., (2004) stated, the abnormalities found such as underdeveloped testes could be connected to a normal amphibian testicular development and not to the exposure of atrazine (Coady et al., 2004). From the three studies Coady et al. (2004), presented various limitations since the experimental study presented many inconsistences. Firstly, the study’s mortality rate from all specimens was approximately 73.3-83 % which was significantly higher the other studies mortality rate of 10% (Mccoy et al.,2004; Hayes et al.,2002; Mccoy et al., 2008). Usually when there is a mortality rate of above 15 % the study is deemed as insignificant since the specimens were bad conditions throughout the experiments. Therefore, it is not possible to truly evaluate the effects of atrazine on the development of frog gonads. The samples in the Coady et al. study were also not placed in the chemically induced water for the whole study like the other two studies observed in their experiments and that could have also effected the results of the experiment. There was also a conflict of interest in the study. Syngenta the company that funded the study is a major producer of atrazine. The company is known to hire scientists to argue against any results that may present atrazine negatively (Rohr and Mccoy,2010). Although the company should defend its product if the results are unwarranted and are found to have serious flaws , the direction the company tries to discredit independent researchers is bad. By downplaying the credibility of researchers, it misleads the public to think that atrazine does not affect anuran amphibians and the environment. (Rohr and Mccoy,2010). It can then be concluded that gonad malformations negatively affected the frog populations as it acted as an environmental stressor and demasculinized the populations sampled.Effects of Sex Ratios in Atrazine Exposed Frogs

There is strong convergent evidence that the sex ratios of frogs were feminized due to the exposure of atrazine. Genetic males have ZW sex chromosomes while genetic female anuran amphibians have ZZ sex chromosomes. In Cornwall, Ontario , Canada a study by Langlois et al. 2010 reported the sex ratios of a population of 750 wild Rana pipiens tadpoles . The samples were found within five atrazine contaminated sites of the Raisin river (Ontario). The rivers contained 1.6 μg/L of atrazine and when transferred to the lab each group was exposed to 0, 0.1 and 1.8 μg/L of atrazine over a period of 78 days when the R.pipiens reached the climax of metamorphosis. Before the sex ratio count was conducted all the samples were terminated with a 1% solution of MS-222 and dry ice. The sex ratio test conducted through a dissection of the whole populations gonads. Furthermore, each gonad was classified as male (ZW) or female (ZZ) through a microscope (4x) (Langlois et al., 2010). The study stated a 1:0.6 and of male to female R.pipens in the control group and was within the normal sex ratio range for the species (1:0.5). Tadpoles exposed to 1.8 μg/L of atrazine skewed the sex ratio to 1:1.4 male to female. Low exposures to atrazine stayed with the ideal sex ratio range (1:0.8) (p<0.05) (Langois et. al ,2010). The long term result of a continuous feminized sex ratio was a low availability of male (ZW) R.pipens for breeding. Certain genetic male(ZW) R.pipens exposed to 1.8 µg/l of atrazine had the appearance of a female were deemed as sex reversed subjects and were included in the female group of sex ratios. A recent study by Boone and Hoskins 2017 also determined a connection between feminized sex ratios and the exposure to atrazine. 280 Blanchard’s cricket frogs (Acris blanchardi) were exposed to various concentrations of atrazine ( 0, 0.1, 1, and 10 μg /L) over a period of two months. The experiment was terminated with a 1% solution of MS 222. The determination of sex ratios was also conducted by a visual inspection for male testes or female ovaries from the gonads of A. blanchardi (Boone and Hoskins, 2017). Atrazine feminized the sex ratios of A.blanchardi at a concentration of 0.1 μg /L and the study theorized the skewed ratio resulted from a atrazine mediated a male(ZW) to female sex reversal (ZZ) (figure 3). Furthermore the study connected atrazine as an estrogen mimicker in which had the ability through sex reversal to lower the fertility rate of males and produced a scarcity of males available to breed with female A.blanchardi (Hoskins and Boone, 2017). Both experimental studies determined atrazine created Female-biased sex ratios and a consequence of the skewed results was the possibility of a declined amphibian population.

Figure 3: A logistical regression of the effects atrazine had on the sex ratios of Acris blanchardi. The dotted lined presented a 1:1 ratio of male to female population. Concentrations of 0.1 and 10 μg /L represent a feminized sex ratio of approximately 1:0.4 and 1:0.35 respectively. Both results were deemed different then the control as it was lower than the ideal sex ratio range of 1:1. The statistical significance is at p<0.05. Credit Boone and Hoskins 2017.

Connection of Atrazine to the Population Decline of Anuran Amphibians

 Anuran amphibian species are heavily reliant on the their habitat for the for reproduction and larval development; Therefore, the organism were vulnerable to pollutants such as atrazine. As an estrogen induced endocrine disrupting chemical (EDC) the effects of atrazine is deemed more of a concern for male frogs as it created gonadal malformations such as testicular oogenesis and sex reversal (Hayes et al.,2002). When an atrazine exposed male had a decline in testosterone morphologies such as the demasculinized larynx, low sperm count and smaller body muscle mass it is unable to attract a female for breeding as it mimics characteristics of female (Hayes et al. 2002). If it does attract a mate the low sperm count lowered the chance of breeding success. Sex reversal also feminizes the sex ratios in which there would be a lower amount of truly genetic males than females. As a result, over time the population would decline as there are not enough males to mate with females. A recent study from 2010 from North Carolina linked the low body mass of atrazine exposed Rana pipens overall impeded their overall development and survival (Koprivnikar,2010). Koprivnikar placed 384 tadpoles in different concentrations of atrazine (0,3,300 μg /L). 450 tadpoles were placed in dechlorinated tap water and after 216 hours each the tadpoles were exposed to 20 cercariae of a trematodes (Echinostoma trivolvis). The control group of each study were only exposed to dechlorinated tap water. A group of 32 tadpoles were exposed to both atrazine (3 μg /L) and trematodes (Koprivnikar,2010). The study was terminated after 46 days when the tadpoles were in mid metamorphosis. Tadpoles exposed to atrazine at 300 μg /L were annexed in the results as the mortality rate was at 100 percent. For tadpoles exposed to only 3 μg /L of atrazine had a mortality rate of 25 percent. Parasite ridden tadpoles only experienced a mortality rate of 20 percent. The mortality rate was at 35 percent when the tadpoles were exposed to both atrazine and trematodes. Koprivnikar theorized that the higher rate of mortality for tadpoles exposed to both atrazine and trematodes was due to low body mass and a slower developmental rate. When the body mass was lower many the tadpoles had become stressed, could not produce a sufficient immune response as the immune system of the organisms were underdeveloped. Consequently, the tadpoles low body mass was linked to the atrazine exposure (Koprivnikar,2010). When amphibians were exposed to various stressors the metamorphic process of frogs are hindered. Impediment to development is detrimental to the amphibian population as lower number of creatures would have reached full sexual development. Over time the population would decline due to feminization and a high susceptibility to pathogens.

 Conclusion

 Through the review of several studies it can be concluded that the exposure to atrazine can affect the gonadal development and sex ratios of anuran amphibians in North America. Numerous studies determined the feminization of the population as male frogs had underdeveloped reproductive parts, fertility problems and a smaller body mass. Some population of genetic males had become hermaphrodites as the studies observed the development of testicular oocytes (Mccoy et al.,2008; Hayes 2010). Another study determined that the exposure to atrazine did not produce gonadal malformations to exposed amphibians (Coady et al.,2004). The results of the study could not be deemed as valid due to the high mortality rate of the control group. Feminization of sex ratios were due to the sex reversal of male to female frogs. Many of genetic males were deemed as female as the organism had female characteristics. The skewed sex ratio produced a scarcity of male frogs to breed with females (Hoskins and Boone,2017). The population decline of anuran amphibian was linked to the gondal development and feminized sex ratios.

Despite, the consensus of the various experimental studies whom suggest that atrazine produced adverse effects on Anuran amphibians, the results should not be assumed in all amphibian species and other organisms. Firstly, the development of the amphibians in the studies were mainly produced in the lab and the results may differ in long term field studies. The differences may come as contaminated wetlands may have a combination of herbicides and other contaminants in the water that could also affect the metamorphic development of anuran amphibians. Many organisms have different dose-response rates may not be affected by the atrazine levels as shown in the review. Further research is needed to determine if atrazine or a mixture of herbicides had a long-term effect on several amphibian species in the field rather than just the lab. Exposure of atrazine should also be researched with other species such as fish and mammals. If more studies proved that the herbicides produced an adverse effect on amphibians that produced a decline in the population, there would be a need for governmental policy reform. Examples for policy reforms could be lowering the amount of pesticides allowed in the water and to place incentives (e.g. tax breaks, subsidies) for farmers move towards alternative management methods such as biological control.

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