develop a sound dose response relationship, and determine the developmental sensitivity of the Page  59 test species, X. laevis. If apical effects are observed in response to atrazine exposure, then three additional phases are proposed that address the mode and mechanism of atrazine action along the lines of the working hypothesis. Before proceeding through the proposed study phases, a risk management decision would be required to determine if further reduction in uncertainty is needed.

In the second phase, sex steroid ( i. e., testosterone and estrodiol) measurements would be conducted, and in the third and fourth phases aromatase activity would be measured. These mechanistic studies would be conducted for three reasons. First, understanding the mode of action is essential for extrapolating between species. Because it is impossible to test all species of concern for apical effects, this overall approach uses X. laevis as a surrogate species where doseresponse developmental sensitivity, and mechanism of action data are used as the basis to investigate the effects of atrazine on native species. This approach also allows for studies with native anurans to be focused and efficient and permits one to test the hypothesis that native species respond similarly to atrazine as X. laevis.

Second, mechanistic information is important in that it reduces the uncertainties associated with developing a causal relationship between apical effects and atrazine exposure by demonstrating a plausible, mechanistic basis for any such effects. In this context, such data also assist with the interpretation of dose response relationships and appropriate dose metrics. Finally,

mechanistic studies set the stage for the development of potential bioindicators of response that can be applied if future field work is needed. The lack of such information is a critical factor that limits the utility of the field studies conducted thus far. Page  60 Phase 5 of this proposal attempts to develop data to establish whether or not the effects of atrazine on gonadal development, if established, are ecologically relevant. This phase would require some novel approaches for evaluating fertility of males after atrazine exposure in order to develop data suitable to an analysis with population models.

There are no universally accepted standardized methods for the studies outlined above.

Therefore, the methods proposed in the following sections represent an ad hoc approach, using information and experience of several different laboratories. Although this proposal defines a series of studies to develop a comprehensive understanding of atrazine's potential effects at the organismal and sub organismal levels, this approach does not need to be fully executed as currently outlined. If the studies proceed from the initial phase to other phases, then there is an opportunity to review the outcomes and revise future study objectives accordingly and to evaluate the utility of conducting the next phase relative to the potential improvement of an eventual risk assessment. The advantage to this approach is that it minimizes the generation of unnecessary data, thus minimizing costs and delays in completing the risk assessment. This flexibility,

however, requires iterative review and communication between the risk assessors and risk managers to ensure that the Agency's data needs are met.

Phase 1. Test for Apical Gonadal Effects The ability of only one laboratory to produce ovotestes at low atrazine concentrations is problematic because this is the endpoint on which much of the concern hinges. Therefore,

additional studies on the effects of atrazine on gonadal differentiation should be conducted.

Repeatability is the hallmark of sound science and must be considered as an important step in determining the relevancy of previously conducted studies. Positive results, besides supporting Page  61 the conclusions of previous studies, would provide the rationale for conducting further studies which attempt to elucidate the pathway of potential atrazine effects. Negative results, on the other hand, that do not support the previous studies would eliminate the need to proceed to additional phases of investigation.

Species The species used for this phase should be both amenable to laboratory experimentation and sensitive to the effects in question. Xenopus laevis and Rana pipiens meet both of these requirements. X. laevis, however, should be used as the primary species because of its ease of culture, shorter developmental time, and the ability to conduct studies throughout the year. Rana pipiens, while perhaps more environmentally relevant to North America, should only be used in focused studies for additional confirmation of results obtained using X. laevis.

Stage Sensitivity These studies should first be initiated with free swimming larvae ( 96 hr post fertilization;

NF Stage 46) to make a direct comparison to the previously published studies and to include the developmental stages known to be sensitive to feminization by exogenous estrogens ( Villalpando and Merchant Larios 1990; Figure 2). Exposures should continue through metamorphosis ( NF Stage 66), at which time the study can be terminated. If atrazine is shown to induce gonadal effects in larvae, then additional studies should be conducted at the effective atrazine concentrations. These studies should initiate exposure with different developmental stages ( e. g.

NF stages 52 and 58) to determine if there are stage sensitivity differences and whether those differences are consistent with previous studies ( Villalpando and Merchant Larios 1990; Figure Page  62 2). By establishing stage sensitivity, further experimentation in subsequent phases could be more focused.

Test Conditions The tests should be conducted using flow through conditions which favor growth,

survival, and development. Static or static renewal methods should not be used. The type of water is not specified in this method because there is no known optimal water type. Instead, the type of water used must be established to promote normal survival, growth, and development under the conditions of the proposed studies. Using a flow through system, there is no need to aerate the exposure water. Feeding should be done on a daily basis using a food type and quantity that also has been demonstrated to promote normal survival, growth, and development.

Biological loading rates are a major concern of the previously conducted studies. Loading rate is known to be an important factor in maintaining adequate water quality which is essential for valid aquatic toxicity testing. Excessive loading diminishes water quality through metabolic activity and respiration and can result in inefficacious exposures. Standards have been established by the American Society for Testing and Materials ( ASTM 1998) for acceptable loading rates of fishes, amphibians, and invertebrates in both static ( or static renewal) and flow through conditions. ASTM ( 1998) standards for loading in tests conducted using static renewal conditions at 22o C are 0.5 g/ L of test solution. All of the studies reviewed in this report exceed this recommendation and suggest that the studies failed to maintain adequate conditions for a valid bioassay ( loading rates were from 1.1 to 24.4 g/ L; see Table 2). Page  63 Table 2. Summary of loading rates used in the various laboratory studies on the effects of atrazine on amphibian gonadal development.

Exposure Type Species Number of Tadpoles per Replicate Maximum g/ organism Total g Liters of solution g/ L Hayes et al. 2002a static renewal X. laevis 30 1.51 45 4 11.3 Goleman and Carr 2003 static renewal X. laevis 60 1.51 90 4 22.5 Hayes et al 2002c static renewal R. pipiens 30 2.52 75 4 18.8 Hecker et al. 2003. static renewal R. clamitans 303 205 24 24 60 40 4 16 15.0 2.5 Du Preez et al. 2003. static X. laevis 800 1.51 1,200 1,100 1.1 Goleman and Carr 2003. static renewal X. laevis 65 1.51 97.5 4 24.4 Tavera Mendoza et al.

2001a; 2001b. static X. laevis 16 1.51 24 15 1.6 Hecker et al. 2003 static renewal X. laevis 30 1.51 45 4 11.3 1 not reported; estimated based on data provided in Figure 3.

2 not reported; estimated based on Ankley et al ( 1998).

3 initial number, but high mortality confounds analysis 4 not reported; estimated based on metamorph weights 5 not reported; estimated based on approx. 30% mortality in controls at transfer to larger tanks on day 67 The ASTM recommended loading rates for studies conducted using flow through conditions is 1 g/ L for every liter dispensed in 24 hrs. Current studies conducted with X. laevis at the EPA, Office of Research and Development's Mid Continent Ecology Division ( MED) employ flow through conditions that were designed to meet the ASTM standard. Typically, the flow rates in each exposure tank are 0.025 L/ min, which is equivalent to 36 L/ day. Biological loading Page  64 is usually 20 organisms, which reach about 1.8 g maximum body weight ( Figure 3), resulting in an approximate loading rate of 1 g/ L for each liter dispensed in 24 h. Under these conditions,

minimal mortality has been observed, and metamorphosis is typically completed in about 55 days ( personal communication: J. Tietge, U. S. EPA, Mid Continent Ecology Division, 2003).

Water concentrations of atrazine and its major degradates, i. e., diaminochlorotriazine ( DACT), desethylated atrazine ( DEA), and desisopropyl atrazine ( DIA), should be measured in duplicate at specified frequencies using an appropriate analytical method to cover the concentration range of the parent compound used in the test. Water chemistries ( i. e., dissolved oxygen, pH, ammonia) should be measured periodically throughout the study and reported.

Dose Response The studies to date provide inconsistent information as to the nature of a dose response relationship for potential atrazine effects. Further studies suggest that lower concentrations of atrazine are more effective than higher concentrations in eliciting gonadal effects although no biological basis has been demonstrated for this response. However, without knowing the biological basis, the shape of a dose response curve can only be empirically established by testing multiple concentrations in the range of interest. The highest test concentration should be at or above 25

g/ L, while the lowest should be below 0.1

g/ L. If the results of the initial study using this concentration range demonstrate non monotonic dose response patterns, then subsequent tests that focus on a specific concentration range may be indicated. If atypical dose response patterns are observed, i. e., inverted U shape dose response, then additional work may be necessary to develop a biological basis for such a response. This may require dosimetry measurements to ensure that the organismal dose, i. e., whole organism residue, is correlated and Page  65 dependent upon the applied dose, i. e., exposure water concentration. Deviations from such a relationship could indicate that other mechanisms, e. g., metabolism, active uptake, active elimination, are at work to alter the expected steady state relationship, thereby potentially altering the shape of the dose response curve.

Positive Controls Many of the atrazine studies have used " positive" controls. Typically they have used both an estrogen and an androgen. The use of an estrogen is desirable because it demonstrates the sensitivity of the organisms to a potential estrogen effect that is hypothesized to be in the pathway impacted by atrazine. Estrogen applied concentrations should be measured at specified intervals to assure adequate delivery to organisms. The use of an androgen as a " positive" control is not necessary because there is no plausible relationship, based on the working hypothesis, between androgen responsiveness and the effects of concern.

Sampling Sampling of all organisms under study is essential to avoid potential bias related to differences in developmental rate. Sampling should occur at the same developmental stage to make sure that the inter organism comparisons are valid. For X. laevis, sampling when each organism reaches NF 66 is appropriate. Replication, loading, and final sampling should be determined using statistical procedures to ensure that the hypothesis of the study can be tested appropriately.

Endpoints Page  66 The primary endpoints of interest are those indicative of abnormal sexual differentiation.

These include: gross analysis of chemically prepared ( fixed) gonads to determine the morphological condition of the gonads, histological analysis of gonads from each organism to determine whether effects such as testicular oocytes and reduced spermatogonial cell nests occur in males, and alterations in the numbers of primary and secondary oogonia occur in females.

Additionally, determination of male: female sex ratios and other observations on secondary sexual characteristics should be made. Evaluation of laryngeal musculature is not necessary, as the gonads appear to be more sensitive to the effects of atrazine exposure. In addition, the typical endpoints of daily survival, growth ( as determined by wet weight at the termination of the study),

and development ( as indicated by time to complete metamorphosis) should be evaluated.

Quality Indicators Because there are currently no standardized methods available for reproductive and developmental assays with anurans, in general, nor with X. laevis specifically, the following guidelines are proposed as quality indicators of a valid study. Of utmost importance, the study should attempt to conform to the ASTM recommended standards for biological loading rates of 1.0 g/ L for flow through studies. These loading rates should be determined using the maximal mass of the organisms, not the initial or final masses, as considerable weight loss occurs ( up to 50%) during metamorphic climax. Minor deviations can be tolerated, but the ASTM guidance should be strongly considered in test design and conduct.

Survival is obviously an important indicator of study quality. And, while there is no bright line between acceptable and unacceptable survival rates, survival of 90% or more of the organisms indicates a quality study, particularly in the controls. Although the level of Page  67 unacceptable mortality is not defined, excessive mortality serves to bias the results of the study by limiting analysis to the survivors.

Like survival, there are no set criteria for acceptable growth and development. However,

taken in aggregate, growth and development should be as optimized as practical. Maximal larval weights of X. laevis should be about 1.5 to 1.8 g, with weights of NF 66 organisms approximately half of maximal weights ( Figure 3). Development in controls should proceed without great deviations from those determined by Nieuwkoop and Faber ( 1994), with metamorphic completion in approximately 7 to 9 weeks. Conditions that delay development of unexposed organisms beyond 10 weeks suggest problems with food or water quality and introduce uncertainties regarding the effects of overall delayed development on system specific development. Although there are little or no known data regarding delayed metamorphosis on organ specific development, there is a large body of literature on the effects of accelerated development demonstrating that accelerated metamorphosis, through exposure to thyroid hormone, results in abnormal system development ( Huang et al. 2001). Clearly, development is a coordinated event with temporal constraints.

Besides the biological indicators, there are several standard exposure related measurements that are indicators of a quality study. The most common are dissolved oxygen,

ammonia, and pH. ASTM guidelines suggest that dissolved oxygen should be maintained between 60 and 100% of saturation ( ASTM 1998). These guidelines recommend that pH be within an acceptable range for the test organism, which in the case of X. laevis is suggested to be within 6.5 and 9.0 for earlier developmental stages used in the FETAX assay ( ASTM 1998).

While no specific guidance is provided for ammonia concentrations, two studies have evaluated Page  68 the toxicity of ammonia to X. laevis larvae and found that concentrations of 25 50 ppm are lethal and that concentrations of about 15 ppm significantly reduced growth ( Tietge et al. 2000;

Schuytema and Nebeker 1999) in short term studies. These studies suggest that total ammonia concentrations should be well below 10 ppm because total ammonia ( recognizing that the toxic form, unionized ammonia) is dependent on the pH of the test media.

Analysis, Interpretation, and Iteration If the study or studies indicate that atrazine affects gonadal differentiation, then the experimentation could move to the next phase or phases which deal with questions regarding the mechanism of action and reproductive fitness. Although these mechanistic and higher order questions are presented sequentially, they could be evaluated simultaneously.

Negative results, however, present a much more difficult problem. At issue is whether or not a study design is, indeed, a valid attempt to repeat previous studies using the same conditions.

Strictly speaking, the conditions of any of the atrazine studies cannot be repeated because it would be highly improbable to have organisms of the same genetic makeup, which could be a contributing factor to the outcome. The conditions recommended here do not attempt to actually replicate the conditions of any of the atrazine studies conducted thus far. Those conditions appear to have been inadequate to promote normal survival, growth, and development. The underlying assumption is that a biological phenomenon, if significant, is pervasive under a broad range of conditions that are favorable to the viability of the organism being tested. If effects are only observed in the laboratory due to specific test conditions that are not likely to be found in the field, then those effects may not be relevant. If, however, the studies are negative, after Page  69 reasonable attempts are made to use well established aquatic toxicity methods in a systematic approach, then there is no plausible rationale to proceed with the next phases of the investigation.

Phase 2: Sex Steroid Measurements If positive effects are observed in Phase 1, then it is logical to hypothesize that estrogen levels are increased and result in feminization. At this point, with the stage and dose dependency of the effects documented, it should be possible to construct a refined exposure protocol which maximizes a presumptive increase in estrogen concentrations in organisms at specific developmental stages. Kloas ( 2002) has demonstrated the ability to measure estrogen in organisms at early developmental stages, including those that presumably would be present in this hypothetical study.

If estrogen levels are determined to be elevated, then it is reasonable to propose moving into Phase 3, which proposes to measure aromatase activity. If estrogen levels are not elevated,

then an alternative path should be followed, which evaluates other mechanisms associated with gonadal differentiation. Since the alternative path is not part of the working hypothesis, no further discussion of it will be presented here.

Phase 3: Aromatase Activity Measurements The test for increased aromatase activity as a result of atrazine exposure at the appropriate developmental stages assumes that estrogen levels have already been elevated by atrazine. The question is whether or not elevated estrogen levels result from enhanced aromatase activity.

Aromatase, the enzyme responsible for conversion of testosterone to estradiol, is expressed in several tissues of X. laevis. The developmental expression of this enzyme has been determined and detectable expression of aromatase mRNA in the gonad occurs at NF Stage 51. A small Page  70 transient rise of expression occurs at Stage 53, followed by a transient depression at Stage 54, and a large rise of expression after Stage 55 ( Miyashita et al. 2000; Figure 3).

Because exposure to exogenous estrogen results in feminization of the male phenotype, it is reasonable to hypothesize that enhanced aromatase activity could lead to abnormally elevated endogenous estrogens that can also affect feminization of males. Under normal developmental circumstances, the activity of aromatase is either absent or very low during the developmental stages that are most sensitive to feminization by estrogens, suggesting that this phenomenon does not occur naturally ( Figure 3). In order for aromatase activity to effect feminization of the testes,

the activity of this enzyme must be sufficiently elevated during the developmental period sensitive to estrogen induced feminization. This requires that atrazine cause aromatase expression to occur prematurely and at high enough levels to produce sufficiently high endogenous estrogen levels to affect feminization.

If, on the other hand, atrazine does not increase aromatase activity, then consideration should be given to studying its effect on other factors that are involved in the homeostasis of estrogen. A likely research avenue would include studies on hypothalamic and pituitary factors,

which have already been shown to be affected by atrazine in rodents ( Cooper et al. 2000).

Phase 4: Aromatase Inhibitor Study Assuming that aromatase activity is elevated by atrazine, a logical confirmatory study would be to expose organisms to atrazine in combination with an aromatase inhibitor. This study could be performed at all of the preceding levels and incorporate apical endpoints as well as sex steroid and aromatase measurements. Page  71 Phase 5: Ecological Relevancy Study Quantifying the likelihood of population level responses due to the potential effects of atrazine exposure to anurans is challenging. The hypothesis is that atrazine exposure adversely affects reproductive fitness of a specific anuran species to the extent that population levels decline in response. To test this hypothesis, an anuran species that permits the analysis of reproduction is required. Currently, there are no known laboratory methods to evaluate this endpoint directly with native species. And, even though reproduction in X. laevis appears to be a useful surrogate model to identify the effects of atrazine on development of the reproduction system, it probably cannot be used as a surrogate for determining ecological effects on native anuran species.

The essential elements of determining reproductive fitness can be readily identified, but collecting data to confidently assess them is relatively difficult. Because the primary effect of concern is related to males, the primary endpoint of interest is the ability of males to attract mates and fertilize eggs during mating ( amplexus). Fecundity, per se, as an indication of female reproductive capacity is of little value, unless further experimental data support the results reported by Tavera Mendoza et al. ( 2001b), which suggest that adverse gonadal effects occur in females as well.

The essential elements affecting the male's ability to effect egg fertilization can be broken down into three main components: gamete function, gamete release, and reproductive behavior.

The first, gamete function, can be assessed using in vitro fertilization methods. Briefly, this method requires that previously exposed males are either maintained under temperature and lighting conditions that induce reproductive readiness or are induced using pituitary extracts or gonadotropins. Testes would be removed and macerated in order to release the spermatocytes, Page  72 which would be applied to a fixed number of previously collected oocytes. The efficacy of fertilization could then be analyzed by determining the prevalence of developing embryos. This type of methodology is routinely used in X. laevis and X. tropicalis and could be adapted for use in native species that are amenable to the procedure. In the case of most native anurans, this would require exposing the organisms during the period of atrazine sensitivity, followed by 1.5 to 2 years of holding to allow for sexual maturation to occur. Once again, although testing the ranid species could eventually be necessary, it may be advisable to first use X. tropicalis, which reaches sexual maturity in about six months, to determine if there is a fertility hazard. If a significant reduction in fertilization is observed, then histological analysis of male gonads of native anurans may be an important consideration in extrapolating potential effects on spermiogenesis among native and non native species.

If in vitro fertility is unimpaired, then it would be advisable to determine the ability of the males to successfully release the gametes. This concern is based on the fact that the presence of ovotestes alters the structure of the gonad, which may impede gamete release. This type of study would require an in vivo approach using induced reproduction.

Because the reported effects on the gonads result in ovarian tissue occurring in males, it is possible that males with ovotestes have elevated circulating estrogen concentrations. This could impact the normal endocrinological control of reproductive behavior and secondary sexual characteristics related to reproduction, as suggested by the Florida cane toad study where males expressed female coloration patterns and lacked nuptual pads ( Sepulveda and Gross 2003). The only viable way to test for these effects is to use natural reproductive behavior in an in vivo study.

These are the most difficult studies to successfully conduct. Page  73 Finally, the data collected from fertility studies may need to eventually be translated into an estimate of reproductive output. The changes in reproductive output would in turn need to be expressed in probability terms associated with atrazine exposure. Finally, population models would need to be developed or applied to assimilate the reproductive output data. Page  74 CHAPTER 6 CONCLUSIONS A significant body of information is available to evaluate the potential effects of atrazine on amphibian development. Clearly, the body of studies reflect the commitment of an international group of research teams to independently examine whether atrazine exposure results in effects on gonadal development, and these investigations provide useful information to help resolve this complex scientific and risk assessment challenge. Any future research will certainly benefit from and build upon this existing foundation.

Based on a review of available studies in the open literature along with recently submitted registrant sponsored studies examining the effects of atrazine on gonadal and laryngeal development in frogs, the Agency has concluded that none of the studies fully account for environmental and animal husbandry factors capable of influencing endpoints that the studies were attempting to measure. Therefore, as discussed below, EPA finds the overall weight of evidence so uncertain that it does not support any definitive conclusions with regard to whether or not atrazine exposure adversely affects amphibian development.

The current weight of evidence does not show that atrazine produces consistent,

reproducible effects across the range of exposure concentrations and amphibian species tested.

Only one study ( Hayes et al. 2002a) has demonstrated statistically significant reductions in laryngeal muscle area ( demasculinization) in atrazine exposed males. Additionally, while Hayes et al. ( 2002a, b) have reproduced " feminizing" gonadal developmental effects ( hemaphroditism/ ovotestes/ intersex) in males at atrazine concentrations as low as 0.1

g/ L,

similar gonadal effects have only been demonstrated in one other laboratory ( Goleman et al.

2003) at 25

g/ L for X. laevis at roughly the same length of exposure and stage of animal Page  75 development. The only other laboratory studies demonstrating gonadal effects ( Tavera Mendoza et al. 2001a, b) followed a considerably shorter ( 48 hr) exposure to a single atrazine concentration ( 21

g/ L) and resulted in dissimilar endpoints, i. e., reduced testicular volume and numbers of spermatogonial cell nests in males and decreased numbers of primary and secondary oogonia in females. While Hayes et al. ( 2002b, c ) demonstrated feminizing effects in male leopard frogs collected in the field, the incidence of hermaphroditism varied widely relative to atrazine concentration. In another field study, Sepulveda and Gross ( 2003) demonstrated increased incidence of hermaphroditism/ intersex in cane toads and southern toads collected in Florida, but the relationship to atrazine exposure was uncertain.

Although the weight of evidence does not show that atrazine produces a consistent,

reproducible effect, both laboratory and field studies provide evidence that atrazine exposure may be associated with effects on gonadal development and secondary sexual characteristics. The Agency believes that the lack of reproducibility across studies, which may, in part, be due to an inconsistency in the methods used by the various research teams, and the absence of a doseresponse at this point do not refute the hypothesis that atrazine exposure may result in gonadal developmental effects in amphibians. Studies conducted by Hayes et al. ( 2002a, b), Tavera

Mendoza et al. ( 2001a, b) and Goleman et al. ( 2003) suggest that atrazine exposure at various levels resulted in some degree of gonadal developmental effects and thus serve to identify a potential hazard to X. laevis. However, none of these studies permit a clear understanding of how potential gonadal effects vary with exposure.

While this paper evaluated the experimental designs employed in each of the studies reviewed in some detail, it is important to note that none of the open literature studies were conducted for regulatory purposes nor were specific protocols available to assist in the design of Page  76 registrant sponsored studies. Notwithstanding the limitations on the available data, collectively,

the research has provided valuable and useful insights into sources of variability that can facilitate future study designs to reduce uncertainties.

In summary, while the current research does not support a definitive conclusion regarding a quantitative dose response relationship between atrazine exposure and effects on gonadal development, it provides sufficient information to formulate a hypothesis that atrazine exposure may affect gonadal development. If the Agency's risk management decision requires a greater degree of certainty in the ecological risk assessment for atrazine than possible from currently available data, then additional information would be necessary to evaluate the potential causal relationships between atrazine exposure and gonadal development in amphibians. Any future studies should take into account lessons learned from the currently available research. While the Agency does not discount the utility of field based studies for examining effects under "

realworld conditions, it believes that at this time laboratory based studies provide the best opportunity to control environmental and animal husbandry factors in order to demonstrate whether a quantitative relationship exists between atrazine exposure and effects on gonadal development.

In the next chapter ( Charge to the Panel) the paper solicits input from the FIFRA Scientific Advisory Panel on the Agency's interpretation of the existing data regarding the effects of atrazine on amphibian gonadal development. Additionally, the Agency is seeking input on its proposal to further test the hypothesis that atrazine exposure results in gonadal developmental effects. Page  77 CHAPTER 7 CHARGE TO THE PANEL The Agency has reviewed relevant studies in the scientific literature, as well as studies submitted by the registrant, to evaluate the potential for atrazine to elicit developmental effects in amphibians. The strengths and limitations of the individual studies were assessed, and the extent of concordance for the entire body of information derived from these laboratory and field studies was considered to assess the plausibility that atrazine can cause developmental effects, and if so,

the nature and strength of associated dose response relationships. These developmental effects included time to metamorphosis, growth, gonadal abnormalities, sex ratios, laryngeal dilator muscle area, plasma steroid concentrations and aromatase activity of brain and gonadal tissue.

The Agency has identified a number of uncertainties in the previous studies' attempts to demonstrate whether atrazine affects development in amphibians. To resolve these uncertainties,

the Agency has outlined a conceptual model for studying atrazine action on amphibian development and has proposed an approach using focused empirical studies to test the hypotheses embedded in the conceptual model.

The Agency is seeking feedback from the SAP on a number of questions surrounding the current body of evidence regarding the potential effects of atrazine on development in amphibians and the relevancy of these potential effects to ecological risk assessment. Additionally, the Agency is requesting that the SAP review and comment on the conceptual model for potential future studies. Page  78 Questions 1) In reviewing the available laboratory and field studies, the Agency used a number of criteria to evaluate individual investigations. Criteria such as experimental design, test protocols, and quality assurance information were used to ascertain the reliability of the generated data in terms of its ability to adequately assess a hypothesis that atrazine elicits developmental effects in amphibians, and if so, the nature and strength of associated doseresponse relationships.

a) Does the SAP have any comments and recommendations regarding the EPA's approach and criteria used to evaluate the studies?

b) Given the evaluation criteria employed by the Agency, please comment on EPA's overall characterization of the currently available studies.

c) Please comment on the availability, as of February 28, 2003, of additional, relevant studies in the open literature that were not addressed in the white paper.

Since February 28, 2003, is the Panel aware of any studies that would be relevant?

2) In its evaluation of existing field studies, the Agency has concluded that these investigations are of limited value. The reasons include: ( 1) the high variability in environmental conditions and uncertainties in the pre existing status and condition of fieldcollected animals, ( 2) the spatial and temporal aspects of atrazine exposure ( i. e., spatial and temporal variability over the course of the studies and the extent to which such aspects of atrazine exposure were empirically measured or otherwise accounted for), and ( 3) the possible co occurrence of additional chemical and/ or non chemical stressors. Page  79 c) To the extent that the field studies appear to indicate that atrazine may not adversely affect development, please comment on EPA's conclusion that the body of data from field studies does not provide the means to ascertain whether the lack of a relationship between atrazine exposure and developmental effects is due to the absence of a causal relationship or limitation in study methodologies.

d) To the extent that any field studies appear to indicate that atrazine may adversely affect development, please comment on EPA's conclusion that these field studies do not provide sufficient information to resolve the potential role of additional cooccurring stressors.

3) In an evaluation of the existing laboratory based studies, the Agency concluded that there was sufficient information to establish a hypothesis that atrazine could cause adverse gonadal developmental effects. However, due to different experimental designs and variability in the nature and extent of experimental conditions ( e. g., level of excessive mortality, delayed development in untreated organisms, lack of response to positive controls) it was not possible to adequately assess the hypothesis that atrazine causes developmental effects. It was further concluded that the current body of information did not provide the means to characterize the nature of any associated dose response relationships.

d) Please comment on EPA's determination that the laboratory studies provide a plausible basis for the means to establish a hypothesis concerning the potential for atrazine to cause developmental effects. Also, please comment on whether the overall body of available data is adequate to demonstrate whether or not atrazine causes developmental effects under the conditions described in these studies. Page  80 b) Please comment on EPA's conclusion that given the variability in the available dose response data across the studies ( e. g., an approximately 250 fold difference in reported thresholds for observed developmental effects as well as reports of monotonic and non monotonic dose response curves), it is not possible to ascertain the relationship, if any, of atrazine exposure to developmental effects in amphibians.

4) Many of the available studies proposed that aromatase induction results in elevated estrogen levels that lead to feminization ( ovotestes/ intersex/ hermaphroditism) in genetically male amphibians.

e) Please comment on EPA's conclusion that, to date, aromatase induction by atrazine has not been demonstrated in any anuran in controlled laboratory investigations.

f) The variability associated with plasma sex steroid concentrations and aromatase activities is high. Is this variability normal? Please comment on any readily apparent or available methodological improvements ( e. g., changes in sampling design, analytical techniques) that could efficiently address this variability in future studies.

c) Please comment on whether there are additional data, other than those summarized in the white paper, that suggest late exposure of amphibians ( i. e., juveniles or adults) to estrogens or estrogenic chemicals can induce ovotestes formation. Page  81 d) Please comment on whether there are additional data, other than those summarized in the white paper, that suggest alternative mechanisms that could explain the apparent feminization of genetically male amphibians.

5) With regard to specific endpoints, the Agency does not currently have sufficient information to quantitatively relate gonadal/ laryngeal effects to reproductive outcomes. A major underlying uncertainty is the ecological relevance of ovotestes occurrence to the maintenance of anuran populations.

g) Can the Panel provide sources of data on background rates of ovotestes occurrence in amphibian species and any associated considerations for interpreting this information in the context of the reviewed studies?

h) Can the Panel characterize any evidence that suggests that the presence of ovotestes in male anurans results in reproductive impairment via reductions in fertility?

i) The reduction of laryngeal muscle area suggests diminished testosterone in males.

If this is found to be a valid observation and if estrogen concentrations do increase as testosterone concentrations decrease, what other endpoints ( e. g., secondary sexual characteristics and reproductive behavior) would likely be affected?

6) While some of the available data indicate there may be an association between atrazine exposure and developmental effects in amphibians, the Agency's evaluation of the existing body of laboratory and field studies has determined that there is not sufficient scientific evidence to indicate that atrazine consistently produces effects across the range of amphibian species examined. However, the current body of knowledge has deficiencies Page  82 and uncertainties that limit its usefulness in assessing potential developmental atrazine effects and the extent of any associated cause effect and dose response relationships.

Consequently, the Agency has determined that there are not sufficient data to reject the hypothesis that atrazine can cause adverse developmental effects in amphibians.

f) Does the SAP concur with these conclusions? If not, what lines of evidence would lead to an alternative conclusion?

7) Assuming the Agency determined an ecological risk assessment with a greater degree of certainty concerning developmental effects of atrazine on amphibians were needed, please comment on EPA's conclusion that additional information is required to evaluate potential causal relationships between atrazine exposure and gonadal development. Please also comment on the added utility, if any, of additional information to interpret the shape of dose response curves for potential developmental endpoints and the extent to which threshold or non threshold response relationships can be quantified.

8) The Agency has developed a conceptual model from which to develop a set of study protocols for evaluating the potential effects of atrazine on gonadal development in amphibians. The Agency has proposed a research approach using focused, empirical,

laboratory studies based on initial investigations with X. laevis followed by selective,

confirmatory studies with frog species native to North America.

a) Please comment on the proposed sequence of study objectives.

b) Please comment on whether the Agency's first set of proposed studies has accounted for the major sources of uncertainty associated with the potential effects Page  83 of atrazine on anuran sexual differentiation. In addition to time to metamorphosis,

gonadal abnormalities, and sex ratios in the proposed Phase I assays, please comment on any other endpoints that should be considered in this initial phase.

c) Please also comment on the range, spacing and number of atrazine concentrations that should be employed in the proposed testing sequence to resolve uncertainties in the shape and nature of dose response relationships for any observed developmental effects.

d) Please comment on the Agency's recommendation that X. laevis be used as the primary biological model in the proposed studies and whether or not the mechanisms involved in sexual differentiation of the ranid and pipid species are sufficiently similar to predict effects and associated dose response curves for Rana and/ or to efficiently design Rana studies.

e) In this regard, are there important differences between the species to conclude that any affected developmental processes observed in X. laevis would not occur in Rana?

f) Alternatively, are there developmental pathways in Rana, but not in X. laevis, that raise concerns about using X. laevis as the primary biological model in any future atrazine studies?

g) Assuming X. laevis and Rana are sufficiently concordant from a toxicodynamic perspective with regard to potential developmental effects of atrazine, what critical toxicokinetic processes should be considered for extrapolating X. laevis doseresponse relationships to Rana and/ or for designing subsequent studies with Rana? Page  84 REFERENCES Ankely, G. T., J. E. Tietge, D. L. DeFoe, K. M . Jensen, G. W. Holcombe, E. J. Durhan, S. A.

Diamond. 1998. Effects of ultraviolet light and methoprene on survival and development of Rana pipiens. Environmental Toxicology and Chemistry 17: 2530 2542. Page  85 Birge, W. J., A. G. Westerman and J. A. Spromberg. 2000. Comparative toxicology and risk assessment of amphibians. Pages 727  791 in D. W. Sparling, G. Linder and C. A. Bishop,

editiors. Ecotoxicology of Amphibians and Reptiles. Society of Environmental Toxicology and Chemistry Press.

Calabrese, E.. J. and L. A. Baldwin. 2001. Hormesis: a generalizable and unifying hypothesis.

2001. Critical Reviews in Toxicology 31 ( 4 & 5): 353  424.

Carr, J. A., A. Gentles, E. E. Smith, W. L. Goleman, L J. Urquidi, K. Thuett, R. J. Kendall, J. P.

Giesy, T. S. Gross, K. R. Solomon, G. Van Der Kraak, 2003. Response of larval Xenopus laevis to atrazine: assessment of growth, metamorphosis, and gonadal and laryngeal morphology.

Environmental Toxicology and Chemistry 22( 2): 396  405.

40 CFR. 1997. Code of Federal Regulations 40. Parts 158 to 189 ( Protection of the Environment). Office of the Federal Register National Archives and Records Administration.

Consent Decree ( as amended) entered in Natural Resources Defense Council v. Whitman, Case Number C  99 3701 CAL, N. D. California ( 2002).

Crabtree, C.; E. E. Smith; J. A. Carr. 2003. Histology of the gonads and analysis of hormone levels in the native bull frog ( Rana catesbiena) collected from agricultural areas in southern Iowa:

pilot project. The Institute of Environmental and Human Health, Texas Technical University, Page  86 Lubbock, Texas. Sponsor: Syngenta Crop Protection, Inc. Laboratory Identification Number ECORISK Number TTU 02.

De Prado, R., N. Lopez Martinez and J. Gonzalez Gutierrez. 2000. Identification of two mechanisms of atrazine resistance in Setaria faberi and Setari viridis biotypes. Pesticide Biochemistry and Physiology 67( 2): 114  124.

Duellman, W. E. and L. Trueb. 1994. Biology of Amphibians. Johns Hopkins University Press,

Baltimore, MD.

DuPreez, L. H., A. M. Jooste and K. R. Solomon. 2003. Exposure of Xenopus laevis larvae to different concentrations of atrazine in semi natural microcosms. School of Environmental Sciences and Development, Zoology Department, Potchefstroom University for CHE,

Potchefstroom 2520, South Africa. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number SA 01 D.

Giesy, J. P., M. Hecker, and P. D. Jones. 2003. South African analytical support   hormone and aromatase analysis ( SA 01C). Aquatic Toxicology Laboratory, Michigan State University,

National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU 07*

Goleman, W. L. and J. A. Carr. 2003. Response of larval Xenopus laevis to atrazine exposure:

assessment of metamorphosis and gonadal and laryngeal morphology. The Institute of Page  87 Environmental and Human Health, Texas Tech University, Texas Tech University Health Sciences Center, Lubbock, Texas. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number TTU 01.

Gray, J. A., N. E. Balke and D. E. Stoltenberg. 1996. Increased glutathione conjugation of atrazine confers resistance in a Wisconsin velvetleaf ( Abutilon theophrasti) biotype. Pesticide Biochemistry and Physiology 55( 3): 157  171.

Hayes, T., K. Haston, M. Tsui, A. Hoang, C. Haeffele and A. Vonk. 2002b. Atrazine induced hermaphroditism at 0.1 ppb in American leopard frogs ( Rana pipiens): laboratory and field evidence. Environmental Health Perspectives http:// dx. doi. org/ [ Online 23 October 2002]

Hayes, T. B., A. Collins, M. Lee, M. Mendoza, N. Noriega, A. A. Stuart, and A. Vonk. 2002a.

Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences 99 ( 8): 5476  5480 Hayes, T., K. Haston, M. Tsui, A. Hoang, C. Haeffele and A. Vonk. 2002c. Feminization of male frogs in the wild: water borne herbicide threatens amphibian populations in parts of the United States. Nature 419: 895  896. Abridged Version of Hayes et al. 2002b and not specifically reviewed in white paper.

Hayes, T. B. 1998. Sex determination and primary sex differentiation in amphibians: genetic and developmental mechanisms. Journal of Experimental Zoology 281: 373  399. Page  88 Hecker, M., K. K. Coady, D. L. Villeneuve, M. B. Murphy, P. D. Jones and J. P. Giesy. 2003. A pilot study of response of larval Rana clamitans to atrazine exposure: assessment of metamorphosis and gonadal and laryngeal morphology and selected hormones and enzyme activities. Aquatic Toxicology Laboratory, Michigan State University, National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU 03.

Hecker, M., K. K. Coady, D. L. Villeneuve, M. B. Murphy, P. D. Jones and J. P. Giesy. 2003.

Response of Xenopus laevis to atrazine exposure: assessment of the mechanism of action of atrazine. Aquatic Toxicology Laboratory, Michigan State University, National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU 04.

HED 2002. U. S. EPA Office of Pesticide Programs Human Health Effect Division Revised Human Health Risk Assessement for the Reregiatration Eligibility Decision ( RED). April 16,

2002. OPP Docket # 2002 0026. Docket Number 0012.

Huang, H, L Cai, BF Remo, and DD Brown. 2001. Timing of metamorphosis and the onset of the negative feedback loop between the thyroid gland and the pituitary is controlled by type II iodothyronine deiodinase in Xenopus laevis. Proceeding of the National Academy of Sciences,

98, 7348 7353 Page  89 IRED Science Chapter for Atrazine: Environmental Fate and Effects Chapter, April 22, 2002 OPP Docket Number 2002 0026 Document Number 0005.

IRED. 2003. Interim Reregistration Eligibility Decision Science Chapter for Atrazine:

Environmental Fate and Effects Chapter January 28, 2003. OPP Docket ID OPP 2003 0072;

Document ID OPP 2003 0072 0010.

Jones, P. D., M. B. Murphy, M. Hecker, J. P. Giesy. 2003. Tissue pesticide residues and histology of the larynx and gonads in native green frogs ( Rana clamitans) collected from agricultural areas in Michigan: hormone analysis. Aquatic Toxicology Laboratory, Michigan State University, 218C National Food Safety and Toxicology Center, E. Lansing, MI. Sponsor:

Syngenta Crop Protection, Inc. Laboratory Study ID: ECORISK Number MSU 02.

Miyashita, K, N Shimizu, S Osanai, and S Miyata. 2000. Sequence analysis and expression of the P450 aromatase and estrogen receptor genes in the Xenopus ovary. J Ster Bioch Molec Biol, 75,

101 107.

Nieuwkoop, P. D., and J. Faber. 1994. Normal table of Xenopus laevis ( Daudin). North Holland Publishing Company, Amsterdam, Holland.

Petrini, S. F. Zaccanti. 1998. The effects of aromatase and 5   reductase inhibitors,

anitandrogens and sex steroids on Bidder's organs development and gonadal differentiation in Bufo bufo tadpoles. Journal of Experimental Zoology 280: 245  259. Page  90 Reeder, A. L., G. L. Foley, D. K. Nichols, L. G. Hansen, B. Wikoff, S. Faeh, J. Eisold, M. B.

Wheeler, R. Warner, J. E. Murphy, and V. R. Beasley. 1998. Forms and prevalence of intersexuality and effects of environmental contaminants on sexuality in cricket frogs ( Acris crepitans). Environmental Health Perspectives 106 ( 5): 261  266 Reregistration Eligibility Decision Science Chapter for Atrazine: Environmental Fate and Effects Chapter, April 22, 2002 OPP Docket Number 2002 0026 Document Number 0005 Sanderson, J. T., J. Boerma, G. W. A. Lansbergen and M. van den Berg. 2002. Induction and inhibition of aromatase ( CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicology and Applied Pharmacology 182: 44  54.

Sepulveda, M. S. and T. S. Gross. 2003. Characterization of atrazine exposures and potential effects in Florida ecosystems dominated by sugarcane agriculture: a reconnaissance survey of amphibians in south Florida for the assessment of potential atrazine effects. Department of Physiological Sciences, University of Florida, Caribbean Science Center, Gainesville, Florida.

Sponsor: Syngenta Crop Protection, Inc. Study ID: ECORISK Number UFL 02.

Sive, H. L., R. M. Granger and R. M. Harland. 1998. Early Development of Xenopus laevis, a Laboratory Manual. Cold Springs Harbor Laboratory Press. 4* Individual DERs written on study, but report collectively reviewed as one of four component studies to the South African Xenopus investigations.

Page  91 Smith, E. E., L. DuPreez, and K. Solomon. 2003. Field exposure of Xenopus laevis to atrazine and other triazines in South Africa: feasibility study for site characterization and assessment of laryngeal and gonadal responses. The Institute of Environmental and Human Health, Texas Tech University, Lubbock, Texas ( USA) and School of Environmental Sciences and Development,

Potchefstroom University for CHE, Potchefstroom, South Africa. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID: ECORISK Number SA 01A* 4.

Smith, E., L DuPreez and K. Solomon. 2003. Field exposure of Xenopus laevis to atrazine and other triazines in South Africa: exposure characterization and assessment of laryngeal and gonadal responses. The Institute of Environmental & Human Health, Texas Tech University,

Lubbock, Texas 79490 ( USA) and School of Environmental Sciences and Development,

Potchefstroom University for CHE, Private Bag X6001, Potchefstroom 2520 ( South Africa).

Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number SA 01B*

Smith, E. E., L. DuPreez, and K. Solomon. 2003. Gonadal and laryngeal responses to field exposure of Xenopus laevis to atrazine in areas of corn production in South Africa. The Institute of Environmental and Human Health, Texas Tech University, Lubbock, TX and School of Environmental Sciences and Development, Potchefstroom University for CHE, Potchefstroom,

South Africa. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID: ECORISK Number SA 01C*. Page  92 Tavera Mendoza, L., S. Ruby, P. Brousseau, M. Fournier, D. Cyr and D. Marcogliese. 2001.

Response of the amphibian tadpole Xenopus laevis to atrazine during sexual differentiation of the ovary. Environmental Toxicology and Chemistry 21 ( 6): 1264  1267.

Tavera Mendoza, L., S. Ruby, P. Brousseau, M. Fournier, D. Cyr and D. Marcogliese. 2001.

Response of the amphibian tadpole Xenopus laevis to atrazine during sexual differentiation of the testes. Environmental Toxicology and Chemistry 21 ( 3): 527  531.

Villalpando, I and H Merchant Larios. 1990. Determination of the sensitive stages for gonadal sex reversal in Xenopus laevis tadpoles. International Journal of Developmental Biology, 34,

281 285.

Villeneuve, D. L., K. Coady, M. Hecker, M. B. Murphy, P. D. Jones and J. P. Giesy. 2003.

Methods development for the study of mechanism of action of atrazine in adult and metamophosing Xenopus laevis and Rana clamitans: aromatase induction. Aquatic Toxicology Laboratory, Michigan State University, National Food Safety and Toxicology Center, E. Lansing,

MI. Sponsor: Syngenta Crop Protection, Inc., Laboratory Study ID ECORISK Number MSU

01. Page  93 Test for Apical Gonadal Effects Aromatase Activity Measurements Sex Steroid Measurements Test for Alternative Effects Relevant to Sex Steroid Homeostasis Test for Alternative Effects Relevant Gonadal Differentiation Stop YES YES NO NO NO Aromatase Inhibitor Study YES 1 2 3 4 Working Hypothesis Ecological Relevancy Study 5 Figure 1. Scheme for laboratory studies to determine the effects of atrazine on gonadal differentiation in anuran amphibians. The grey block contains the working hypothesis for atrazine action on gonadal differentiation, which is partitioned into four logical phases. See text for details. Page  94 Developmental Stage 42 44 46 48 50 52 54 56 58 Aromatase mRNA 0 100 200 300 400 500 Sex Reversal Intersex No effect Figure 2. The effect of estradiol exposure on male gonads of different stages compared to the developmental expression of aromatase mRNA. The horizontal bar indicates that there are three periods of differential sensitivity to the feminizing effects of estrogen exposure for male X. laevis larvae ( Villalpando and Merchant Larios, 1990). The line indicates the relative expression of aromatase mRNA at different developmental stages ( Miyashita et al. 2000). Page  95 Developmental Stage 46 48 50 52 54 56 58 60 62 64 66 68 Wet Weight ( mg)

0 200 400 600 800 1000 1200 1400 1600 1800 Figure 3. The relationship of weight to developmental stage of X. laevis. Each dot represents one organism ( Tietge et al., EPA Midcontinent Ecology Division, unpublished results.)