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Most commonly used herbicide in the world is atrazine, which 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. 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. About half million or more pounds of atrazine is precipitated as rainfall every year in United States (Hayes et al. 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).In 1958, atrazine was made as the second series of 1, 3, 5 triazines in the Geigy laboratories (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). In the New York Times, atrazine was featured 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). Atrazine's oral normal Lethal Dose in rats is 3090 mg/kg in rats, in mice is 1750 mg/kg in mice, in hamsters is 1000 mg/kg , and in rabbits is 750 mg/kg. 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
Sexual development in frogs is disturbed by atrazine at concentrations that is thirty times less than the amount permitted by the Environmental Protection Agency. According to (Hayes et al. 2002a),the laboratory studies done on African clawed frogs (Xenopus laevis) showed that after their exposure to atrazine concentrations at as low as 0.1 Î¼g/L caused demasculinization i.e. decreased laryngeal muscle growth and feminization i.e. testicular Oogenesis. Single exposure of atrazine at concentration of atrazine of 21 Î¼g/L caused the production of oogonia (primary and secondary)in ovaries of Xenopus laevis (Tavera-Mendoza et al. 2001a) and reduced volume of testes, nurse cells and spermatogonia in testes (Tavera-Mendoza et al. 2001b). In the field studies, about 92% of leopard frogs collected from atrazine exposed sites showed testicular oogenesis and hermaphroditism (Hayes et al. 2002b, c).
MATERIALS AND METHODS
2.1 Analysis of Gonads.
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 were present beneath the thiohyral were examined. 8 Î¼m sections were cut for nuptial pads through the center of the nuptial pad and needed cross sectional area of breeding glands was examined and later compared with mucous and serous glands cross sectional area. The cross section for testes was analyzed from the biggest random testicular tubules going through the process of spermatogenesis and as well as the comparison of testicular tubules region from the largest cross sectional area containing or lacking spermatozoa bundles.
2.6 Molecular Markers for Sex.
DNA is isolated from toe tips by lysis of tissue and proteinase k protein digestion. Multiplex PCR amplification was used to determine the ZW genotype by using with 37 cycles of DM-W (Britson 2000).
2.7 Mate Choice.
To compare the capacity of control and atrazine exposed males to draw 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. Fertility examination was done by counting the amount of immature eggs and the number of completely developed embryo. In second study, males (control and atrazine exposed) were tested in separate rooms so that the vocalizations did not affect the results of each other.
2.9 Gross Measurements.
Complete tail re-absorption occurs during metamorphosis at Niewkwoop Faber Stage 66, the date is recorded. Later each animal is weighed next to 0.002 g on Delta Range balance and 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
The main importance of conducting laboratory studies is to allow researchers to control a wide range of conditions that could intentionally impact the result of a study. Quality of water, rate of loading, exposure of chemicals, animal husbandry, animal health and mainly the environmental factors can all be easily manipulated 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 every exposure period, animal's length, animal's weight, abnormalities of gonads, metamorphosis time and muscle size of the larynx dilator were recorded. Results showed that about 16-20% of animals had abnormalities in gonads i.e. multiple gonads (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), NF Stage 56 of male Xenopus laevis 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 and nursing cells 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 differentiation of gonads during development of female Xenopus laevis larval tadpole, (Tavera-Mendoza et al. 2001b) exposed these tadpoles to atrazine at 18 Î¼g/L for 48 hrs. The presence of primary oogonia were 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. Green frogs only reacted to androgenic chemicals after dihydrotestosterone and 17 beta estradiol's positive control treatment that suggested the changes in sex ratio to male frogs i.e. 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 and as a result levels of aromatase were not provided.
3.1.5 In three separate studies done by (Villeneuve et al. 2003) that involved two adult males and one adult female, the frogs were exposed to fresh water or atrazine. 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, the aromatase activity of ovaries did not differ from controls in females. 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 whose larvae were exposed to atrazine at concentrations of 0.1, 1.0, 10, and 25 Î¼g/L. Exposures were also done using positive control of 0.1 Î¼g/L 17 beta estradiol and 0.1 Î¼g/L dihydrotestosterone and solvent control of 0.005% ethanol and dilution water. In a dose dependent association, the results showed that atrazine treatment did not affect sex ratio, mortality, metamorphosis, development of gonads, aromatase activity and plasma concentrations of steroid hormone. In positive control treatment it was found that estradiol only appeared to increase estradiol plasma concentrations while Dihydrotestosterone in positive control caused enlargement of the muscle area of larynx dilator in females, however sex ratios were not effected in none of positive controls.
3.1.7 (Goleman and Carr 2003) exposed 48- to 72-hr post-hatch X. laevis larvae to atrazine concentrations of 1, 10 and 25 Î¼g per liter,17 beta estradiol concentration of 0.1 Î¼g per liter, dihydrotestosterone concentration of 0.1 Î¼g per liter or solvent control of 0.0025% ethanol for 78 days. Males that were treated with 25 Î¼g atrazine per liter showed evident tissue of ovaries and testes tissue while unclear gonadal tissue structures were found in estradiol treated males. There was no dissimilarity in the cross section muscle area of larynx dilator in atrazine treated males in relation to dilution water controls. However, dihydrotestosterone treated females had considerably larger cross sectional dilator muscle areas than the females of solvent control.
In this study, length and weight of animal, time to metamorphosis and muscle area of laryngeal dilator In comparison to the controls was not affected by atrazine. On the other hand, exposure to 25 Î¼g atrazine per liter appeared to increase the number of intersex males. In addition, 17 Beta estradiol treatments 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 of atrazine on gonads R. pipiens (a native species). 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 clarification of dose response relationship to cause-effect 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 Wyoming (North Platte River) where the atrazine concentrations were lesser than other sites and sites with no atrazine showed that the testicular oogenesis appeared to be 18 percent. This study was helpful in producing results in field studies similar to those observed in laboratory studies, yet 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. Atrazine exposure was applied to larvae until they reached NF stage 66 and the study was terminated after 133 days of exposure. Some animals after 70 days reached stage 66 but most did not attain metamorphosis until 126 - 133 days. Under controlled laboratory conditions of 20 - 25oC, Xenopus laevis tadpoles used 58 days for completing metamorphosis (Nieuwkoop and Faber 1994). On basis of gross morphology, from the total frogs examined i.e. 150, the occurrence of deformity in gonads of 1, 10 and 25 Î¼g per liter atrazine groups was 1.3, 0.7 and 3.3% respectively. Gonadal deformities identified in males were only discontinuous testis but no abnormalities were observed in the ovaries. In the microcosm units, the unpredictable water quality could have affected the rate of development.
3.2.3 (Reeder et al.1998) wanted to find the rate of abnormalities of gonads in cricket frogs (adult and juvenile) 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. In 1993, two i.e. 3.6% out of 55 male and female (adult and juvenile) frogs collected had both an ovary and testis. In 1995, six i.e.2.5% out of 243 frogs contained both an ovary and a testis, only one had an ovotestis. Across all three sampling years the occurrence of intersex was 2.8%. Intersex organisms were also found in four out of five sites where concentrations of atrazine was detectable.
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 Xenopus laevis in South Africa i.e. its native habitat. 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). According to these studies there was no difference in the lengths and weights of both males and females collected from experimental site of high atrazine and reference site of low 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 and 2 percent of the reference frogs and experimental frog sites, respectively. 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 from elevated 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. Aromatase activity of ovaries was not notably 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. From all of the frogs collected, about a total of four mixed or unidentified gender animals were found. 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 in South Florida's sugarcane agricultural areas could cause higher incidence of intersex in toads exposed to atrazine. It was found that approximately 29% and 39% of the males collected from Belle Glade and Canal Point, respectively 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, nuptial pads were found in 0% and 71% of the intersex toads collected from Belle Glade and Canal Point respectively. Intersex toads collected had vitellogenin levels that were similar to the females and was approximately double that of male toads collected from the nonagricultural sites 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 study, higher frequency of intersex was found in both agricultural i.e. at Belle Glade and Fisheater Creek and nonagricultural sites i.e. at Archibald Biological Station in southern toads (B. terrestris).
3.2.10 (Crabtree et al.2003) designed the study to select sites and to examine the impacts of atrazine on histology of gonads and kidney of bullfrogs (R. catesbiena) and other species collected from different field sites of 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 weight of body or length of snout vents i.e. SVL in adults but in juvenile females the mean weight and SVL was found to be considerably lesser in reference sites as compared to atrazine exposed sites. On the other hand, reference site's mean SVL for juvenile males was notably lower than in atrazine exposed sites, however in juvenile males the mean weight was not different between sites. For both adults and juveniles, gonadosomatic index i.e. GSI = weight of gonad /body weight was not different between sites. This study showed that high frequency of hermaphroditism was not observed in bullfrogs when exposed to atrazine under field conditions.
The following results in amphibians are recorded from different open literature.
4.1 Demasculanisation in Amphibians
In an article issued on 6th April by the University of California in the National Academy of Sciences proceedings in Berkeley, the developmental endocrinologist Tyrone B. Hayes and colleagues reported that normal amounts of atrazine present in the environment can causes gonadal abnormalities in tadpoles and can change them into hermaphrodites.
Atrazine exposed animals lacked evident nuptial pads on the forearms and had cloacal labia protruding that is 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 distorted larynx structure but not its size. In control males, the region of the dilator laryngis that extended to the thiohyrals was more as compared to atrazine treated males, however the form 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 Breeding and Nuptial pads
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 levels of testosterone in the female's presence 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 negligible impacts on time to metamorphosis, length, weight at the time of metamorphosis and mortality rate.
4.10 Impacts on Primary and Secondary Sex Development
Males and females were identified at metamorphosis on basis of morphology of gonads and their 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.
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 chief limitations of these laboratory studies which make it difficult to draw conclusions are as follows,
â€¢ It was difficult to interpret results for dose response relationships for measured endpoints e.g. abnormalities of gonads, aromatase activity, concentrations of plasma steroid and muscle diameter of laryngeal dilator at atrazine concentrations of 0.1 to 25 ug per liter.
An analytical measurement of atrazine was mostly incomplete or was found in the dilution water for the control organisms at concentrations that was similar to low concentration treatments.
â€¢ Gonadal abnormalities and laryngeal dilator muscle diameter effects were reported at atrazine concentrations of 0.1 to 200 ug/L but not been reproduced experimentally later. The extensive unpredictability in the study design has made it difficult to decide if this lack of reproducibility of demasculinizing effects and data collected for inverted dose response relationship for other endpoints of gonadal developmental are even valid.
â€¢ The effects of atrazine on development of gonad and details for the dose response curve that were reported by some investigators have been projected to result from initiation of aromatase activity. This amplified enzyme activity in males may in turn lead to increased estrogen levels which may ultimately result in reduced secondary sex characteristics and ovotestes formation. In anurans, the aromatase induction by atrazine has not been confirmed by 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 field sites selection that have alike 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 reproductive status and developmental stage of the organisms at time of collection.
RESERVATIONS IN ASSESSING POSSIBLE IMPACTS OF ATRAZINE
Each of the field studies mentioned have uncertainties that limit their importance 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 per liter and several reference sites were contaminated with atrazine , thus not enough information is available to conclude the potential low dose effect.
6.3 Apparent Validity Of Atrazine Effects
In the case of atrazine, the aromatase initiation in males causes elevation in estrogen levels which leads to formation of ovotestes and reduced secondary sexual characteristics(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 to induce spawning. on the other hand in the field, organisms faces 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.
6.5 Importance of Available Studies To Evaluate Possible Atrazine Effects
On the basis of current studies done by (Tavera-Mendoza et al. 2001a,b, Hayes et al. 2002a,b,c and Sepulveda and Gross 2003), there is sufficient certainty to create the a plausibility hypothesis 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.
Atrazine is typically used when the soil is tilled and their level 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 development of larynx and gonads, 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 causes consistent impacts 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 muscle area of laryngeal region in atrazine exposed males. (Hayes et al.2002a, b) produced feminizing gonadal developmental effects in males of xenopus laevis at atrazine concentrations of as low as 0.1 Î¼g/L and similar effects is also shown by (Goleman et al. 2003) at 25 Î¼g per liter at approximately the same span of exposure. In another laboratory studies, effects on gonads (Tavera-Mendoza et al. 2001a,b) used a relatively shorter exposure to atrazine concentration of 21 Î¼g/L and thus resulted in different endpoints i.e. reduced volume of testes and number of spermatogonial cells in males and in females number of primary and secondary oogonia reduced. 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 were conducted by (Hayes et al. 2002a,b , Tavera-Mendoza et al.2001a,b and Goleman et al. 2003) proposed 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.