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This paper provides an overview of relevant studies which mainly addresses the impacts of atrazine on amphibians. This paper assesses the strengths and limitations of the available studies to assess the extent to which atrazine may bring out developmental effects in amphibians and the degree of accordance for the entire information derived from laboratory and field studies to review the plausibility that atrazine can effect development of amphibians; and if so dose-response relationships associated with its nature and strength.
The laboratory studies reviewed different endpoints: time for metamorphosis, growth i.e body length and weight , gonadal abnormality, sex ratios, laryngeal dilator muscle area, plasma steroid concentration, and brain/gonad aromatase activity.
Some of the major limitations of these laboratory studies, that make it difficult to draw conclusions about the effects of atrazine on amphibian species, include the following:
â€¢ Studies conducted at atrazine concentrations in the range of 0.1 to 25 ug/L, the interpretation of dose-response relationships for measured endpoints was problematic as atrazine was detected in the dilution water for the control organisms at concentrations comparable to low concentration treatments.
â€¢ The extensive variability in its study design made it difficult to determine if lack of producing demasculinizing effects (decreased laryngeal dilator muscle area), and inverted dose-response relationship for other gonadal developmental endpoints, were valid results or just artifact of the design and quality of the investigations done.
â€¢ The potential gonadal developmental effects of atrazine has been proposed to be resulted from induction of aromatase activity. This increased enzyme activity may in turn elevated estrogen levels and ultimately the observed effects, i.e., ovotestes and reduced secondary sex characteristics, in males.
The field studies, which included evaluated growth (body weight and length), gonadal abnormalities, sex ratios, plasma steroid concentration and brain/blood aromatase activity in X. laevis, cricket frogs, bullfrogs, and cane toads.
The currently available field studies are limited due to the high variability in environmental conditions (e.g., photoperiod, temperature, water quality etc). In addition, the actual or possible co-occurrence of additional chemical and/or non-chemical stressors confounds attempts to attribute any observed responses to atrazine exposure.
Overall, currently available studies did not show that atrazine produces constant effects across the range of concentrations and amphibian species tested. The current knowledge has deficiencies and uncertainties that limit its effectiveness in interpreting potential atrazine effects. Although the Florida cane toads studied in the field showed both demasculinizing effects (genetic males with female coloration) and feminizing effects (oogenesis in male Bidder's organ), there were insufficient data to conclusively link atrazine exposure to the phenomena. Thus, the available data do not give a concrete answer to indicate that atrazine will or will not cause adverse developmental effects in amphibians.
The future studies should be conducted in an approach to ensure that evaluation of potential effects of atrazine are done in a systematic and efficient manner, that is by minimizing the level of resources and efforts required, while maximizing reduction in the existing uncertainties.
CHAPTER 1 INTRODUCTION
1.1 Background on Atrazine
Atrazine is the most commonly used herbicide in the world that has been used for over 40 years in more than 80 countries. It can be transported to more than 1,000 km from the area of rainfall to remote areas where it is not used (Hayes et al, 9 March 2010). Atrazine's causes inhibition of photosynthesis in plants by disturbance of the Photosystem II pathway. It is likely to persist in water as it is resistant to degradation of abiotic routes and moderately resistant to biotic degradation. So, these characteristics along with the extensive use of herbicide, contribute to its widespread. Every year in United States more than a half million pounds of atrazine is precipitated in rainfall (Hayes et al, 9 March 2010).
1.2 Chemistry and biochemistry
It is made from cyanuric chloride which is treated in sequence with ethylamine and isopropyl amine. Atrazine functions by joining to the plastoquinone-binding protein in photosystem II which is absent in animals. Death of plant is due to starvation and oxidative damage which is caused by breakdown in the electron transport chain. Oxidative damage is increases by intensity of high light (Appleby et al.2001). Atrazine was made in 1958 in the Geigy laboratories as the second of a series of 1,3,5-triazines (Wolfgang et al. 2007).
1.3 Health and environmental effects
In 2004 atrazine was banned in the European Union because of its groundwater contamination (Ackerman, Frank.2007). It is has effects on endocrine, possible carcinogenic effect and connection to low sperm levels in men caused many researchers to banning in the US (Ackerman, Frank. 2007). Atrazine was featured in the New York Times as a probable cause of defects of birth, low weight during birth and menstrual problems when taken at concentrations below federal standards (Duhigg, Charles. 2009).
1.4 Impacts of Atrazine on Different Oraganisms
Atrazine causes different rate of acute and chronic toxicity to animals especially aquatic organisms. In direct effects, atrazine is fairly toxic to fish but highly toxic to aquatic invertebrates in an acute exposure. In case of terrestrial organisms, atrazine was slightly toxic to birds and mammals on an acute exposure basis. In mammals it showed chronic sub lethal effects on the hypothalamic pituitary in rats (HED Science Chapter 2002). Oral normal Lethal Dose for atrazine is 3090 mg/kg in rats, 1750 mg/kg in mice, and 1000 mg/kg in hamsters, 750 mg/kg in rabbits. The dermal Lethal Dose in rabbits is 7500 mg/kg which is more than 3000 mg/kg in rats. (Extension Toxicology Network, June 1996).
1.5 Impacts of Atrazine on amphibians
Atrazine effects the sexual development of frogs at concentrations that are thirty times less than levels allowed by the Environmental Protection Agency. According to Hayes et al. (2002a) exposure to atrazine concentrations at as low as 0.1 Î¼g/L can cause demasculinization i.e. decreased laryngeal muscle growth and feminization i.e testicular Oogenesis in African clawed frogs (Xenopus laevis) in the laboratory studies. Single exposure of atrazine at concentration of atrazine of 21 Î¼g/L caused the production of both primary and secondary oogonia in ovaries in Xenopus laevis (Tavera-Mendoza et al. 2001a) and reduced volume of testes, nurse cells and spermatogonia in testes (Tavera-Mendoza et al. 2001b). Field studies on leopard frogs collected from atrazine exposed sites showed testicular oogenesis and hermaphroditism at a rate as high as 92% (Hayes et al. 2002b, c).
CHAPTER 2 MATERIALS AND METHODS
2.1 Gonadal Analysis.
The sexes of all individuals were found on basis of overall gonadal morphology but were vague. Histology was conducted according to Hayes 1995. The frogs are dissected and dehydrated in alcohols followed by penetration with histo clear and paraffin and later sections were cut at 8 Î¼m and stained in Mallory's trichrome stain.
2.2 Adult Treatments.
Small amount of plasma is obtained to measure hormone levels in newly metamorphosed amphibians, so studies for effects of atrazine on basis of hormone levels was done on adults. They were treated for 46 days and latter killed by decapitation and the blood is collected and plasma is extracted which is later frozen until analysis.
Plasma extracted from controls and treated animals were assayed in the same assay at three doses and the assay was repeated 3 times.
2.4 Atrazine Exposure.
ZZ larvae were grown in atrazine from hatching through metamorphosis according to Nieuwkoop and Faber (NF) stage 66 and throughout its post metamorphic life for association with control i.e ethanol treated animals.
2.5 Morphometric Analysis at Sexual Maturity. It was done on sexually mature animals i.e. two or three years after metamorphosis. The dilator larynges that extended below the thiohyral were examined. 8 Î¼m sections were cut for nuptial pads through the center of the nuptial pad and maximum cross sectional area of breeding glands was examined and later compared with the maximum cross sectional area of mucous and serous glands. The cross-section for testes were analyzed from the largest tubule of five random testicular tubules going through spermatogenesis and as well as the comparison of testicular tubules section from the largest cross sections with and without spermatozoa bundles.
2.6 Molecular Markers for Sex.
DNA is isolated from toe tips by tissue lysis and proteinase k protein digestion. The ZW genotype was determined by using multiplex PCR amplification with 37 cycles of DM-W (W specific) (Britson,Threlkeld, 2000) .
2.7 Mate Choice.
To compare the capacity of control and atrazine-exposed males to attract female frogs and to achieve amplexus, both male and female frogs were marked. ZW females were injected with hCG at 1500 hours while four control males and four atrazine-exposed males with no hCG injection were placed in a circular pool that was filled with fresh water and left overnight.At 0600 hours of the next day, the pairs and single males were removed for blood sampling (Oberdorster and Cheek,2001).RIA was used to measure plasma testosterone that was extracted from the collected blood plasma. Frequency of successful copulations was checked by a G test (Davidson et al.2001) and for testosterone analysis ANOVA was used to check the differences in testosterone levels of control and atrazine-treated males.
2.8 Fertility Analysis.
Two studies were done for fertility analysis. In the first study, control and atrazine-treated males without hCG injections were matched with ZW females who were hCG injected. Eggs were later collected, and then allowed to develop for about seventy two hours which were later fixed in Bouin fixative for forty eight hours and then preserved in 70% ethanol. Counting the number of undeveloped eggs and the number of developed embryo was done to examine fertility. In second study, control and atrazine-exposed virgin males were tested in separate rooms so that the vocalizations did not affect the results of each other.
2.9 Gross Measurements.
Complete tail reabsorption occurs during metamorphosis at Niewkwoop Faber Stage 66, the date is recorded. Later each animal is weighed to the nearest 0.002 g on a Mettler AT 261 Delta Range balance and its length is also measured to about 0.5 mm. They were anesthetized in 0.2% benzocaine i.e. Sigma and given an identification number and later fixed in Bouins fixative and preserved in 70% ethanol.
LITERATURE REVIEW OF DIRECT AND INDIRECT IMPACTS OF ATRAZINE PESTICIDES ON AMPHIBIANS
Literature review is grouped into laboratory and field studies.
3.1 LABORATORY STUDIES
One of the advantages of conducting laboratory studies is that they allow researchers to control a range of conditions that could potentially impact the outcome of a study. Environmental factors, water quality, loading rate, chemical exposure, study animals, animal husbandry and health can all be manipulated more easily to identify actual treatment effects in laboratory studies.
3.1.1 The objective of (Hayes et al. 2002a) study was to find whether atrazine interfere with metamorphosis and sex differentiation at low doses via endocrine-disrupting mechanisms. Tadpoles Xenopus laevis were exposed to atrazine concentrations ranging from 0.01 to 200 Î¼g/L. (Nieuwkoop and Faber 1994). At the end of the exposure period, animal growth (length and weight), time to metamorphosis, gonad abnormalities and size (cross-sectional diameter) of the larynx dilator muscle were recorded. It resulted in gonadal abnormalities in 16 -20% of the animals which included multiple gonads i.e multiple testes and ovaries in the same animal however these abnormalities were not observed in controls. (Hayes et al. 2002a) hypothesized that these might be due to increased endogenous estrogen concentrations. Increased estrogens level was found to be due to increased aromatase activity.
3.1.2 According to (Tavera-Mendoza et al. 2001a), male X. laevis (NF Stage 56) were exposed to concentrations of atrazine at 18 Î¼g/L for 48 hrs. Results showed that total testicular volume decreased in atrazine-treated tadpoles, thus representing a 57% decrease. The number of spermatogonial cells decreased significantly thus representing a 70% reduction. The number of nursing cells also decreased significantly. Testicular restoration was seen in 70% of the male tadpoles that were exposed to atrazine in relation to controls and there was also 10% failure of complete development of the testis.
3.1.3 To check the impacts of atrazine on gonadal differentiation during larval tadpole development of female X. laevis, (Tavera-Mendoza et al. 2001b) exposed the tadpoles to atrazine at 18 Î¼g/L for 48 hrs. The presence of primary oogonia was considerably lower in atrazine-exposed tadpoles in relation to controls , however the presence of secondary oogonia was considerably higher in atrazine-exposed tadpoles as compared to controls . According to this study atresia could decrease the reproductive capacity of the tadpole as primary germ cells supply oocytes for oogenesis in frog. This study showed that atrazine may be affecting aromatase activity.
3.1.4 (Hecker et al. 2003) studied impacts of atrazine Rana clamitans. Green frog tadpoles after five days of hatching were exposed to atrazine for 273 days. Results showed that mortality rate of all treatment groups averaged 76.5% which was due to poor water quality and overcrowding. The positive control treatments of dihydrotestosterone and 17-áµ¦ estradiol suggested that green frogs only reacted to androgenic chemicals thus changing the sex ratio to male frogs (97.6%). Based on gross morphology there was no hermaphroditism observed in any of the treatment groups. Only two concentrations of atrazine were tested as a result few frogs survived to complete their metamorphosis, gonadal histology and aromatase levels were not provided.
3.1.5 In three separate studies done by (Villeneuve et al. 2003), involving two adult males and one adult female, frogs were exposed either to atrazine or to fresh water. In the first and second study males were exposed for 26 days and 43 days respectively and in third study females were exposed for 47 days. The results showed mortality of 3, 7, and 19% respectively. After 26 days and 43 days of atrazine exposure, mean brain aromatase activity of atrazine-exposed males was not different from the controls. In the third exposure with female frogs, ovarian aromatase activity did not differ from controls. Brain homogenates, mean aromatase activity of atrazine-exposed females did not differ significantly from control females.
3.1.6 (Hecker et al. 2003 EPA MRID No. 458677-04) studied X. laevis ,its larvae were exposed to atrazine at concentrations of 0.1, 1.0, 10, and 25 Î¼g/L. Exposures were also done using dilution water, positive (0.1 Î¼g/L17-$ estradiol and 0.1 Î¼g/L dihydrotestosterone) and solvent (0.005% ethanol) controls. The results showed that atrazine treatment did not affect mortality, time to metamorphosis, sex ratio, development of gonads, aromatase activity and steroid hormone plasma concentrations in a dose-dependent relation. It was found that estradiol in positive control treatment only appeared to increase estradiol plasma concentrations and Dihydrotestosterone in positive control increased larynx dilator muscle area in females and neither of positive controls affected sex ratios.
3.1.7 (Goleman and Carr 2003) exposed 48- to 72-hr post-hatch X. laevis larvae to concentrations of 1, 10 and 25 Î¼g atrazine/L, 0.1 Î¼g/L 17-áµ¦ estradiol, 0.1 Î¼g/L dihydrotestosterone, or solvent control (0.0025% ethanol) for 78 days. Intersex in males that were treated with 25 Î¼g atrazine/L showed evident testicular and ovarian tissue while males treated with estradiol sometimes had unclear tissue structures. There was no difference in the cross-sectional area of larynx dilator muscle in atrazine treated males in relation to dilution water controls.However, Dihydrotestosterone-treated females had considerably larger cross-sectional dilator muscle areas than the solvent control females.
In this study, atrazine did not impact length, weight, time to metamorphosis or dilator muscle area relative to the controls. However, exposure to 25 Î¼g atrazine/L appeared to increase the number of intersex males. In addition, 17-áµ¦ estradiol treatment resulted in 67% females, thus suggesting that study animals might not totally respond to the positive control.
3.1.8 (Hayes et al. 2002b) conducted studies in the laboratory as well as in field; however laboratory study is discussed here and its field study in the next section. The objective was to find impacts on gonads in a native species i.e. R. pipiens. The overall morphology and histological analysis of the larval gonads showed that about 36 percent and 12 percent of the males that were treated with atrazine that is 0.1 and 25 Î¼g/L, respectively showed gonadal dysgenesis. These animals also showed different degrees of sex reversal that is some of sex reversed males had oocytes in testicular lobules and in a few cases testicular oocytes were vitellogenic .
3.2 Field Studies
Field studies give an insight of real world responses that might actually occur in a natural setting. Under natural conditions the organisms are exposed to a wide range of non-chemical and chemical stressors at the same time, which makes the explanation of cause-effect and dose-response relationships difficult.
3.2.1 In the field studies, (Hayes et al.2002b) wanted to find out the effects of atrazine on leopard frogs that were observed under controlled laboratory conditions and so could also be observed in wild R. pipiens under natural habitats having low and high atrazine. Testicular oocytes were found in males and sites with atrazine levels more than 0.2 Î¼g/L had males that showed sex reversal that was similar to the laboratory study. The highest rate (92%) of hermaphroditism were found in animals that were collected from the North Platte River i.e. Wyoming where the measured atrazine concentrations were lower than other sites and sites with no atrazine showed that the testicular oogenesis appeared to be 18 percent. This study was helpful in producing field effects similar to those observed in laboratory studies; however this study was unable to find a quantitative dose-response relationship.
3.2.2 The main objective of (DuPreez et al.2003) studies was to find the impacts of atrazine exposure on gonad abnormalities in X. laevis metamorphs. Adults were divided into four treatments i.e. 0, 1, 10 and 25 Î¼g/L atrazine. Larvae were exposed until they reached NF stage 66 and the study was terminated after 133 days of exposure. Some animals reached stage 66 by 70 days, most did not reach metamorphosis until 126 - 133 days. Xenopus laevis tadpoles took 58 days to complete metamorphosis under controlled laboratory conditions of 20 - 25oC (Nieuwkoop and Faber 1994). Based on gross morphology, the incidence of gonadal deformities in 1, 10 and 25 Î¼g/L atrazine groups was 1.3, 0.7 and 3.3% of the total frogs examined i.e.150, respectively. Discontinuous testis was the only gonadal abnormality identified in males but no abnormalities were observed in the ovaries. Unpredictable water quality in the microcosm units may have impacted the developmental rate.
3.2.3 (Reeder et al.1998) wanted to find the occurrence of gonadal abnormalities in adult and juvenile cricket frogs and to find out, if sexual development is effected by environmental contaminants. Collection of cricket frogs was over a three-year period (1993 - 1995) from various locations of the state of Illinois. Two (3.6%) had both an ovary and testis out of 55 adult and juvenile male and female frogs collected in 1993. Six (2.5%) out of 243 frogs contained both an ovary and a testis examined in 1995, only one (2.3%) had an ovotestis. Across all three sampling years the occurrence of intersex was 2.8%. Of the five sites where intersex organisms were found, four had detectable atrazine (limit of detection: 0.5 Î¼g/L).
3.2.4 The main objective of studies done by (Smith et al. 2003 (Laboratory Study ID: ECORISK Number SA-01A); Smith et al. 2003 (Laboratory Study ID ECORISK Number SA-01B); Smith et al. 2003 (Laboratory Study ID: ECORISK Number SA-01C); (Giesy et al.2003) was to find the impacts of atrazine on X. laevis in its native habitat i.e. South Africa. The study sites had abnormally high rainfall, extremes pH of 10.2 to 10.8 and variation in the mortality rate due to predation by sharp tooth catfish (Clarius gariepinus). The authors concluded that there were no differences in the lengths and weights of either males or females collected from reference site of low atrazine and experimental site of high atrazine exposure.The testes of frogs collected at high atrazine sites had more weight than testes collected from frogs at reference sites. Testicular oocytes were also found in 3 percent of the reference frogs and in 2% of the experimental frog sites. It was also found that the males collected from ponds with the highest atrazine concentrations had considerably lower plasma median testosterone concentrations than males from reference sites. On the other hand the females collected at high atrazine exposure sites had considerably higher testosterone levels than females collected at reference sites. Similarly, plasma estradiol concentrations were also lower in males and females that were collected from high atrazine sites. Ovarian aromatase activity was not significantly different between sampling sites.
3.2.8 (Jones et al.2003) sought to find the impacts of atrazine on kidney, gonad histology, plasma steroid hormone concentrations and aromatase activity of gonads in green frogs i.e R. clamitans and other ranid species that were collected from various field sites of native Michigan ranges. About a sum of four mixed or unknown sex animals were found in all of the frogs that were collected. Plasma testosterone and estradiol levels showed significant variability while the differences between "exposed" and "reference" sites could not be found. Similar amounts of estradiole was found in exposed males as that of estradiol in females while at the reference sites males showed roughly five times the plasma estradiol concentration than females.
3.2.9 The objective of this study done by (Sepulveda et al.2003) was to find that whether toads exposed to atrazine in sugarcane agricultural areas in south Florida could cause higher incidence of intersex. It was found that approximately 29% of the males collected from Belle Glade and 39% of the males collected from Canal Point were intersexes while no intersex frogs were found among the University of Miami i.e. non agricultural site samples. About hundred percent of the cane toads that were collected at Belle Glade and about fifty five percent of the male cane toads that were collected at Canal Point showed female coloration. Additionally 71% and 0% of the intersex toads collected from Canal Point and Belle Glade respectively had nuptial pads. Intersex toads collected had vitellogenin levels that were similar to the females and was roughly double that of male toads collected from the nonagricultural site and testosterone levels in intersex males also showed twice the amount of variability as similar estimate for males. The data collected over six months of sampling period showed that agricultural sites had atrazine concentrations that ranged from 0.01 to 24.45 Î¼g/L. In this same study, the southern toads (B. terrestris) were also examined and found to have an increased frequency of intersex in both agricultural i.e. at Belle Glade and Fisheater Creek and nonagricultural sites i.e. at Archibald Biological Station.
3.2.10 (Crabtree et al.2003) designed the study to select sites and to examine the effects of atrazine on kidney and gonad histology of bullfrogs (R. catesbiena) and other species that were collected from various field sites in southern Iowa. Corn and soybean-dominated agriculture areas were the experimental sites while grassland areas were the reference sites. No major differences were found for the adult body weight or snout-vent length (SVL) but mean weight and SVL for juvenile females was found to be considerably lower in reference sites than atrazine-exposure sites. N the other hand mean SVL for juvenile males was also notably lower in reference sites than in atrazine-exposure sites however the mean weight of juvenile males was not statistically diverse between sites. Gonadosomatic index i.e. GSI = weight of gonad /body weight was not different between sites for either adults or juveniles. This study shows that bullfrogs did not show a high frequency of hermaphroditism when exposed to atrazine under field conditions.
CHAPTER 4 RESULTS
The following results in amphibians are recorded from different open literature.
4.1 Demasculanisation in Amphibians
In an article of April 16 issue (Proceedings of the National Academy of Sciences, University of California) in Berkeley, the developmental endocrinologist Tyrone B. Hayes and his colleagues report that atrazine at levels often found in the environment demasculinizes tadpoles and turns them into hermaphrodites.
Atrazine-exposed animals lacked visible nuptial pads on the forearms and had protruding cloacal labia, typical of females.These atrazine-induced females lacked the DM-W thus showing that these females were indeed chromosomal males. Thus ZZ and ZW females showed gonadal aromatase but ZZ males did not.
4.3 Laryngeal Size
Atrazine exposure altered the structure but not the size of the larynx. The portion of the dilator laryngis that extended ventral to the thiohyrals was greater in control males than in atrazine-treated males.The shape of the larynx in atrazine-exposed males was morphologically similar to typical normal (ZW) females.
4.4 Morphologic evidence
The atrazine-exposed males showed reduced plasma testosterone levels comparative to control males. Atrazine-exposed males had a lower level of testosterone-dependent morphologies.
4.5 Nuptial pads and breeding glands
The nuptial pads of control males were visibly darker than the atrazine-exposed males . The size of breeding glands was decreased in atrazine-treated males .The size of mucous glands and serous glands were not affected by atrazine (Hayes et al, 9 March 2010)
Atrazine exposed frogs showed a considerable reduction in the number of testicular tubules along with mature sperm bundles.
4.7 Behavioral evidence
In several experiments control males and atrazine-treated males were competed for females, it was found that in most cases the control males out-competed atrazine males and achieved amplexus. Control males were examined to have considerably higher testosterone levels in the presence of females when compared to atrazine-treated males.
It was found that atrazine-treated males had considerably lower fertility rate i.e. proportion of eggs fertilized. Even atrazine-treated males that had relatively high sperm content showed low fertility .
4.9 Mortality, Development, and Growth.
At the all doses tested, atrazine exposure had no effects on mortality, time to metamorphosis, length, or weight at metamorphosis .
4.10 Effects on Primary and Secondary Sex Differentiation.
Males and females were sexually identified at metamorphosis based on gonadal morphology and histology. All doses tested for atrazine except 0.01 ppb resulted gonadal abnormalities. About 20 percent of the animals examined had multiple gonads or were hermaphrodites i.e. with multiple testes and ovaries.
CHAPTER 5 STRENGTHS AND LIMITATIONS OF STUDIES
5.1 Laboratory Studies
The laboratory studies were helpful in finding measurement endpoints that might be a possible hazard to amphibians. These studies also gave important insights for designing future studies that can help in further examination of these measurement endpoints. Laboratory studies are planned to provide a chance to control possible causes of variability that might affect the concerned endpoints, however none of the studies reported for environmental and husbandry factors that are capable of affecting the endpoints. Some of the major limitations of these laboratory studies, which make it difficult to draw conclusions about the effects of atrazine on amphibian species, include the following:
â€¢ For studies that were conducted at atrazine concentrations of 0.1 to 25 ug/L, the interpretation of dose-response relationships for measured endpoints e.g. gonadal abnormalities, aromatase activity, plasma steroid concentrations and laryngeal dilator muscle diameter was difficult.
Analytical measurements of atrazine were mostly incomplete, or atrazine was detected in the dilution water for the control organisms at concentrations that were comparable to low concentration treatments.
â€¢ Gonadal abnormalities and laryngeal dilator muscle diameter effects were reported at atrazine concentrations in the range of 0.1 to 200 ug/L but not been reproduced later experimentally. The extensive unpredictability in the study design has made it difficult to decide if this lack of reproducibility of demasculinizing effects and information of an inverted dose-response relationship for other gonadal developmental endpoints are valid results.
â€¢ The gonadal developmental effects of atrazine as well as details for the dose-response curve that were reported by some investigators has been projected to result from initiation of aromatase activity. This amplified enzyme activity may in turn lead to increased estrogen levels which may ultimately result in ovotestes and reduced secondary sex characteristics, in males. Aromatase induction by atrazine has not been confirmed in any anuran in laboratory investigations.
5.2 Field Studies
Field studies can supportive in evaluating the relevancy and importance of toxicological results that can be observed in laboratory based investigations. Some of the major limitations of field studies, which make it difficult to draw conclusions, include the following:
Most of the field studies did not give enough information about study sites.
In addition, the assessment of potential effects of non-chemical stressors e.g. condition of habitat, availability of prey, nutrition provided were not described or evaluated.
There is a complexity in selecting field sites that have similar morphological characteristics as a result many of the study sites had broadly differing conditions.
Field studies should be designed on basis of the changes linked with the measurement endpoints.
Main sources of variation should be identified and controlled to the degree possible. Current studies were not designed on basis of variability related within the range of measuring endpoints. In some studies animals were collected over a large period of time i.e. is up to 6 months which may increase variations due to changes in developmental stage and reproductive status of the organisms at time of collection.
CHAPTER 6 RESERVATIONS IN ASSESSING POSSIBLE IMPACTS OF ATRAZINE
Each of the studies mentioned have deficiencies and uncertainties that limit their usefulness in differentiating treatment effects.
6.1 Endpoint's Ecological Relevancy
The ecological relation for measuring endpoints in the studies is doubtful and needs further examination. In Hayes et al. (2002c), there was little difficulty in specimen collection, although 92% of hermaphrodite northern leopard frogs were found at one site. So, It's uncertain whether hermaphroditism considerably impaired population levels. According to (Gray et al.1996; DePrado et al. 2000), weed and non target plants developed resistance to atrazine. According to Hayes et al. (2002a; c) chemical exposure may result in delayed metamorphosis in order to show resistance to the feminizing effects of atrazine, however no data is currently present to find a probable compensatory mechanism.
6.2 Dose-Response interaction
Most of the studies did not experiment below 1 Î¼g/L and many of the reference sites were contaminated with atrazine at levels, there is not enough information available to conclude the potential low-dose effect.
6.3 Apparent Validity Of Atrazine Effects
In the case of atrazine, the aromatase initiation causes elevation in estrogen levels which leads to formation of ovotestes and reduced secondary sexual characteristics in males (Hayes et al. 2002a,c). Aromatase induction caused by atrazine was not confirmed in any anuran in the laboratory studies. These studies did not show any significant increase in aromatase activity after exposure to atrazine, so there is no data available that supports hypothesis that aromatase induction is produced due to atrazine exposure.
6.4 Laboratory to Field Studies
A largest challenge is interpretation of ecological importance and its impacts in the laboratory as it is comparatively better controlled and monitored to the variable conditions in the field. For example in laboratory conditions, X. laevis needs constant water temperatures and constant photoperiod which causes it to be induces to spawn. However, in the field, organisms are subjected to fluctuating temperatures and changes in photoperiod according to the season. The studies done so far did not take into consideration the developmental stages of X. laevis, nor did they consider the continuous fluctuations in atrazine exposure.
Application of Available Studies to Assess Potential Atrazine Effects
On the basis of current studies there is sufficient certainty (Tavera-Mendoza et al. 2001a,b; Hayes et al. 2002a,b,c; and Sepulveda and Gross 2003) to create the a hypothesis plausibility that atrazine could affect the development of amphibian. However, the uncertainties that were described previously thus preventing to establish a definitive depiction of atrazine's impact on amphibian development. So, additional data would be required to find relationship between atrazine exposure and development of gonads in amphibians as well as the dose-response relationship. The current studies show the degree to which field experiments can produce variability and difficulty in identifying atrazine-specific effects.
CHAPTER 7 CONCLUSION
Atrazine is typically used when the soil is tilled and its levels is highest during rainfall season. So, highest levels of atrazine coincide with the breeding season for amphibians which might impact its developmental stages. Low-dose endocrine-disrupting impacts are not described extensively in amphibians, so further studies should be conducted. Most of atrazine effects are internal and may go unnoticed by researchers unlike that of mortality and external malformations, so the exposed populations may decline and might go extinct without any detection of the developmental effects in individuals.
On basis of a review of current open literature for the impacts of atrazine on gonadal and laryngeal development in frogs, none of the studies showed that environmental and animal husbandry factors were capable of effecting endpoints that were measured.
The current evidence does not show that atrazine produces consistent effects across the different range of its exposure concentration and the amphibian species tested. In a study (Hayes et al. 2002a) has established significant reduction in laryngeal muscle area in atrazine exposed males. Hayes et al. (2002a, b) produced feminizing gonadal developmental effects in males at atrazine concentrations of as low as 0.1 Î¼g/L and similar effects is only been shown by (Goleman et al. 2003) at 25 Î¼g/L for X. laevis at approximately the same span of exposure. Another laboratory studies indicating gonadal effects (Tavera-Mendoza et al. 2001a,b) used a relatively shorter exposure to atrazine concentration of 21 Î¼g/L and resulted in different endpoints i.e. reduced volume of testes and number of spermatogonial cells in males and reduced numbers of primary and secondary oogonia in females. In another field study by Sepulveda and Gross (2003) described increased occurrence of hermaphroditism in cane toads and southern toads that were collected in Florida but its relationship to atrazine exposure was doubtful. Studies that are conducted by Hayes et al. (2002a,b), Tavera-Mendoza et al. (2001a,b) and Goleman et al. (2003) propose that exposure of atrazine at various levels may have resulted in some amount of gonadal developmental effects and thus serve up to identify a potential risk to X. laevis. However none of the current studies provides a clear knowledge of how gonadal effects vary with exposure.
The limitations on the current data have provided significant and valuable insights into the sources of variability that can aid future study designs in order to reduce uncertainties.
The current research does not provide an ultimate conclusion about dose-response relationship quantitatively between atrazine exposure and its impacts on gonad development, however it provides sufficient information to devise a hypothesis that atrazine exposure may affect the development of gonads.