DR. STEEGER: First question. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 103 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 the hypothesis that atrazine elicits developmental effects in amphibians, and, if so, the nature and strength of associated dose response relationships.

Then part one of a two part question.

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

And secondly, given the evaluation criteria employed by the agency, please comment on EPA's overall characterization of the currently available studies.

DR. ROBERTS: Dr. Kelley, as lead discussant, I will ask you to start out on this. I will leave it to you. Do you want to take both parts together or do you want to have discussion on Part A and then discuss Part B?

DR. KELLEY: What I thought I would do is to read a response to the question that I have written after discussion with several but 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 104 not all panel members, and it includes both parts. And then ask the panel for comments on what I have written so that it can be revised to more accurately reflect the viewpoint of the panel.

Let me ask the panel to begin with whether that's an acceptable procedure to them.

DR. ROBERTS: Sure.

DR. KELLEY: And let me just tell you. Feel free to disagree violently. Not a problem.

DR. ROBERTS: I don't want them to disagree violently.

DR. KELLEY: Violently in the academic sense. That is to say how could you possibly say that. That would be violent for an academic.

DR. ROBERTS: Proceed then.

DR. KELLEY: Bear in mind we may amend this. All right?

We felt that the review was thorough and the conclusions were appropriate, given the data reviewed by the EPA. And here I'm referring to the seventeen studies.

We agreed that additional studies are warranted. There are several studies supported by the registrant or by other agencies that indicate that atrazine can cause developmental abnormalities. On the other hand, a range of abnormalities are reported and they are not 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 105 consistent from study to study ( bearing in mind that the literature includes several anuran species.)

Further, though not considered in the white paper, the findings are consistent with studies in other vertebrate, both aquatic ( e. g. fish)

and terrestrial ( some rodents.) Given the conservation of many basic pathways for endocrine regulation, these studies are relevant to this white paper focused on anurans.

Comments from the panel?

DR. ROBERTS: I have one and then I'll open it up to other commenters. A asks regarding the approach in the criteria used by the EPA to evaluate those studies.

And maybe I missed it. But did you basically agree with their criteria?

DR. KELLEY: I thought  yes. The review was thorough. I think that's implicit. So let me add a phrase saying that the criteria were appropriate.

DR. ROBERTS: Yes. If you believe so. I think we should make that explicit in the response.

DR. KELLEY: I'll add that.

DR. ROBERTS: And then Dr. Coats. You had a comment.

DR. COATS: Yes. I'm a secondary discussant on this. I would 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 106 like to read my opinion which is certainly consistent with the opinions expressed already by Dr. Kelley. The agency's approach on criteria are valid and address a number of very important points from the exposure side of the risk assessment, which is where some of my concerns are.

Number 1, the data evaluation reports could include a description of the analytical methods, that be ELISA or chromatographic methods to enhance the completeness of the reports and their interpretation.

Measured concentrations which are extremely important of the chemical in the water need to be obtained and any potentially bioactive metabolite should also be quantified as mentioned in the white paper.

In some research papers, the exposure concentrations are nominal with no measured concentrations provided or recovery confirmed given, but no values.

The importance of having measured concentrations could be addressed more directly in weighing the validity of the work.

Thirdly, the significance of the exposure method was pointed out, including some of the shortcomings on the static renewal systems. The ASTM standards for flow through systems should be 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 107 followed and the method strongly encouraged.

Fourth, body burdens must be measured in the organisms to reflect the degree of exposure and help explain the mode of action.

Once again, bioactive metabolites also should be included.

Determination of residues in specific tissues would also be very valuable, but at the very least the whole body residues could confirm the exposure.

On the hazard side of the risk assessment equation, it is critical that the dose response relationships be shown as pointed out by the agency. Even if it is an atypical inverted U shape, nonmonotonic response, a dose response curve can be generated if appropriate concentrations are tested.

This of course would help delineate safe versus unsafe concentrations, but also could assist in elucidating mechanism of action.

DR. ROBERTS: Dr. Coats, I think I heard three different kinds of comments there. One was perhaps some deficiencies in reporting among the existing studies.

Some were aspects, experimental aspects that perhaps should be addressed in other studies. But the question really asks about the EPA's characterization of currently available studies. And I think 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 108 some of your comments touched on that.

But I want to be sure that when we respond to this question that we sort of don't mix other things, other points that we want to make in with it. So I think it is going to be important that we give it as clear a response as we can to be about things that you feel and other panel members feel that any deficiencies in their characterization where there are things that they didn't describe or that they inaccurately described that perhaps could be tweaked in the document.

Dr. Kelley?

DR. KELLEY: I actually did in my written comments separate out A and B, I see here. And so I did have an additional comment,

which is that the design of the experiments reviewed by the EPA and the analysis of the data were flawed in many instances, which was the EPA's conclusion.

And then this relates to the dose response  if there is a threshold effect for atrazine, that threshold is not firmly established.

With respect to dose response, any requirement that functions be monotonic are clearly inappropriate. And as we've heard, I think the EPA would agree with that.

So I think these were in discussions of the panel members among the most important issues that came to mind, the threshold 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 109 dose for observing an effect if, in fact, an effect can be observed strikes us as a very important piece of information to be gathered.

DR. ROBERTS: So you think that perhaps the report as the EPA said should clarify that in their criteria to evaluate the studies there was not an insistence on a monotonic dose response relationship. Is that correct?

DR. KELLEY: And I think actually that it is just a wording matter. I think use of the word threshold is useful here, unless it has some technical meaning that escapes me. So threshold is the question here.

And then of course it will have a shape, and we can't predict in advance what shape that will be.

DR. ROBERTS: Dr. Green.

DR. GREEN: I'm the third discussant on that question. And following up on what Dr. Coats just read to you, I think what we were trying to convey is that we would like for the EPA to include in their approach and criteria for evaluating the study the fact that so few studies, if any, evaluated water levels and tissue levels in the animals.

And that should be emphasized in an EPA report describing their approach and criteria and noting that so few studies did that with any. That would be quite helpful. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 110 DR. ROBERTS: Thank you.

Comments from other panel members?

Dr. Matsumura?

DR. MATSUMURA: I really appreciate what Dr. Kelley said about the comparative aspect in comparing the action of atrazine to other organisms, particularly in the vertebrates, so that we formulate some idea what happens in other organisms. I think this document could have more information on the other organisms. Some people have pointed out too.

And regarding the question that Joel Coats raised, you are using the water concentration of atrazine. In some setting, it is just crazy.

As Dr. Hayes pointed out, the next day when you come a concentration may be quite different if it has rained the next day or dried up.

So criterion that one should use really is the residue levels in the frogs themselves, including metabolites.

So I know our chairman don't want to mix up with later questions, but still other criterion we should include whether residues can be found.

In the Reed study from Illinois, they were measuring the residues. And really using the water concentration is not a really 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 111 good method. We have to identify the residues, including the metabolites. Even if it is sort of ephemeral, still we have to do that.

DR. ROBERTS: Dr. Richards?

DR. RICHARDS: I believe that in a general way that the agency's review of these is pretty much in line, particularly with respect to the field studies. I think that they have done a fairly reasonably qualitative look at availability of the studies and the data and information that was provided.

DR. ROBERTS: Are there other criteria that you would like to see added or articulated?

DR. RICHARDS: I think that we will address that in other questions, specifically.

DR. ROBERTS: Okay. Dr. DeLorme?

DR. DELORME: I just wanted to concur that for the most part the criteria they used were reasonable and that they are reflecting the need for sound science and consistency in the science used to conduct ecological risk assessments. That's their job. And they want to make sure that the science that they are using and the studies that they are using are sound.

With respect to the overall characterization of the studies, I found it to be reasonable. Certainly, there were some minor points 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 112 that we might disagree with on specific studies, but the overall characterization, I think they did a good job. They have identified their concerns with the studies from a risk assessment and from a scientific perspective.

They have also identified any conclusions or information or contributions that they felt the study made. And they have also identified uncertainties that result from the studies. And I think they have done a reasonable job in doing that.

DR. ROBERTS: Dr. Skelly.

DR. SKELLY: Overall, I think the EPA did a good job of characterizing the studies. And I just want to make a comment about how studies were divided into categories.

The distinction between laboratory and field studies was made.

And in a couple places in the report, I don't know whether it is referring to all of the studies that were submitted, but they were characterized as experiments.

I just would like to point out that the field studies that are included here include something called a microcosm experiment,

which, in my experience, is actually a mesocosm experiment. That's probably just technical.

But mesocosm experiments are not field experiments. I think 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 113 that's an important distinction when we're thinking about this. And most of the field studies were observational, which isn't a criticism,

but I think the criteria for evaluating a field observational study, a field experiment, of which from what I can tell there are none here,

and a mesocosm experiment might be distinct.

DR. ROBERTS: So some of the terminology may need to be tweaked a little bit in terms of how they are described or classified, is that your recommendation?

DR. DELORME: So that the terminology might be tweaked and also the criteria for evaluating things like the distinction between a field observational study and a field experiment in terms of what sort of expectations you have for variation and environmental conditions could be different.

And I think we're going to talk about that a bit more when we get to the next question.

DR. ROBERTS: Thank you. Just a heads up for Dr. Kelley

DR. KELLEY: Would you like me to put that wording into this first thing? I'll put it in. You can check it later.

DR. ROBERTS: Just as a heads up for Dr. Kelley as the lead discussant, when we finish our discussion, I'm going to ask you to sort of give me your sense of the capsule summary of this. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 114 DR. KELLEY: Thank you.

DR. ROBERTS: Dr. Denver.

DR. DENVER: I just want to support a point that Dr. Coats made. And that goes to the criterion used to evaluate especially the endocrine data and the need to really validate the assays that are used,

especially the ELISA assays. And that will come up in another point later and we'll discuss it further. But I think that that can be raised at this point also.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: First of all, I felt that the criteria certainly was appropriate, and I was very thankful of the EPA for making our life a lot easier, I think, in reviewing this document and assisting us in that manner.

I would like to comment on a somewhat minor but I think important terminology consideration. Particularly, in light of the fact that we have had discussions relating to aqueous, measuring aqueous concentrations versus measuring tissue residues.

I certainly think tissue residues are important, certainly from a mechanistic standpoint, to understand what the burdens are and the effect that organs might be carrying in terms of atrazine loads.

Concentrations, aqueous concentrations are important because 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 115 the bottom line is that's probably how the regulatory agency is going to make decisions, based upon exposure concentrations and not true doses that the animals get.

Throughout the document, there is discussion of dose response relationships, and I would just advise that consideration be given where appropriate to use the term concentration response relationship and use dose response relationship where appropriate, but don't interuse the two.

DR. ROBERTS: I agree. Any other comments? Dr. Delorme.

DR. DELORME: I just wanted to add something. Dr.

Matsumura and I believe Dr. Kelley indicated that one of the criteria that they felt might have been addressed was inclusion of data on other vertebrates.

I think that was  especially as goes to ecological relevance, it might be  had some consideration.

DR. ROBERTS: Other comments?

I'm thinking about, trying to think about whether or not I agree.

I think their intent was to at least, I think what this statement is, to summarize studies on a particular topic.

I think at some point you have to try and perhaps put that in context, in broader context. I think at that point you would look for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 116 analogy from other species.

But I'm not sure I would criticize this summary for not going

making that broader look. I think it had a very specific purpose, not to say those other comparisons might not be important in terms of understanding potential effects, but I thought it was very well focused.

Personally speaking, I was satisfied with it.

Any other comments? Dr. Gibbs.

DR. GIBBS: Just one quick question in terms of the case by case treatment of all the studies. I felt that on the possibility for type 2 statistical errors to be committed in all of the studies wasn't very frequently considered.

I think the focus was on the statistical significance reported,

but I didn't  and clearly EPA is aware of the problems with sample sizes. But I felt many of the studies had serious issues with not being able to detect effects should they have occurred. And I just felt across studies that that consideration of type 2 errors wasn't particularly prevalent.

DR. ROBERTS: Dr. Heeringa.

DR. HEERINGA: I just add my support to that comment. I think  as I reviewed these studies and the EPA's assessment and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 117 review of them, I generally agree with the conclusions, but I also support the last comment, too, that I think if we look at these and the other contributing sources of error we have talked about, that many of these studies are underpowered to detect the type of effects that we are measuring.

In spite of some of the identified measurement, contamination or quality problems, all of these contribute potentially to biases in results, but there are other sources of variable errors that I think really would lead us to assume that even given nominal sample sizes that a lot of these studies are underpowered.

And a recommendation I'll make later on is that, if anything,

when we start into new studies, if they are conducted, that they be overpowered to start with simply so we don't find ourself in this quandary of being right on the edge of type 1 versus type 2 error problems.

DR. ROBERTS: Any other comments or responses to 1 A and 1 B?

Dr. Kelley, I know you have been taking notes. To the extent possible, can you try and summarize your discussion?

DR. KELLEY: Yes. I'm not totally up to speed on Dr. Skelly's comments. But I will try to summarize them. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 118 So in general, the panel felt that both the evaluation criteria and the thoroughness of the EPA's white paper were appropriate.

The panel will get to the hypothesis in a moment. But the panel did support the hypothesis that there was enough data out in the literature given this evaluation to proceed with evaluating the hypothesis that atrazine may contribute to developmental abnormalities in amphibia.

Some concerns were raised by the panel with regard to the evaluation, areas of emphasis that the panel felt should have been weighed in more heavily than other areas. That includes possibility of type 2 errors, which is, for the public, also my stat students always get this confused, but the inability to detect effects that are really there for whatever reason. And some of the possible reasons were outlined here. So that was one area of concern.

Another area of concern was the ability to compare across studies between nominal concentrations of application of atrazine and actual tissue concentrations of atrazine.

And the third was that the characterization of the field data in terms of what was actually obtained was felt by panel members to be,

how can I say, somewhat superficial, I think would probably be the best. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21  119 DR. GREEN: Observational.

DR. KELLEY: Observational. Thank you.

Although, there were experiments in those field data, right, so it wasn't completely observational. The microcosm experiments did have experimental and control groups.

DR. ROBERTS: Dr. Skelly can, I think, fill in on that.

DR. SKELLY: We can talk about it afterwards DR. KELLEY: Anyway, he's going to fix my terminology here.

DR. SKELLY: The point was that a mesocosm experiment is not really a field experiment. There is an important distinction.

DR. KELLEY: Yes. So that the use of the technical terms.

In a field experiment, you don't take the animals out of the field and throw them into a tank. You manipulate them in their environment in situ. You get rid of the red on the red wing black bird's wing,

right, or something of that sort. That would be a field experiment, as opposed to bringing them into a mini lab. So there was some concern about that.

But these concerns were felt by the panel to be relatively minor,

and to not abrogate the conclusion of the panel that the initial analysis by the EPA was thorough, that the criteria were appropriate and that the evaluation was a complete one. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 120 DR. ROBERTS: Would anyone like to edit or make suggestions regarding that?

Dr. Bradbury, is our response reasonably clear?

DR. BRADBURY: One question of clarification.

In interpreting or evaluating future studies, am I to understand the consensus of the panel that future studies should have measured aqueous concentrations of atrazine at a minimum and, depending upon the nature of the hypothesis being tested in a given study, perhaps tissue concentrations as well and perhaps activated metabolites?

DR. KELLEY: I think actually the panel might feel a bit more strongly than that. It might feel that measured tissue concentrations of atrazine is not a perhaps but should be a requirement going forward.

But I don't want to speak for the panel as a whole.

DR. ROBERTS: Dr. Green.

DR. GREEN: I still think in the laboratory environment that water concentrations of atrazine are important to know through the experiment.

DR. ROBERTS: Others want to weigh in on this? Dr. Coats and then Dr. LeBlanc.

DR. COATS: I want to reemphasize. I believe that the tissue 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 121 concentrations would be very helpful and water absolutely needs to be measured.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: It is my opinion that measuring aqueous concentrations is mandatory and measuring tissue levels is desirable based upon the questions being asked in a given experiment.

DR. ROBERTS: And speaking for myself, I would concur with that as well.

Anyone else want to weigh in?

Are there any other follow ups or clarifications?

Dr. Steeger.

DR. STEEGER: I would like to make one additional comment for the benefit of many of the researchers.

Dr. Heeringa is correct that type 2 errors are a critical consideration in the design of studies. Many of the researchers did attempt to design their studies with that in mind. The data evaluation records that were prepared didn't fully capture those efforts, but I think that a genuine effort was made.

Unfortunately, as things progressed in the labs and things didn't turn out as well as they had hoped, the study designs didn't support the data in terms of controlling some of the variability that really got 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 122 out of hand.

DR. ROBERTS: I believe Dr. Heeringa wanted to respond to that.

DR. HEERINGA: Thank you very much. I agree completely. I didn't intend to say that, you know, forehand that there was inappropriate planning in terms  I think across these studies that adequate planning went in.

But as you say, during the course of the studies as we learn more about some of the measurement problems and some of the other issues that came in, that clearly this caused those sort of a priori expectations to be modified and in a way that my recommendation is really that for the future that we can anticipate these things will reoccur to varying degrees, and we may have even other unmeasured sources of error in the experimental process.

And instead of sort of delaying the result of this, let's sort of design with a margin of error on these type 2 error problems so that we can accommodate them as they arise in laboratory and testing situations.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: I would just like to revisit aqueous concentrations again for a moment. Certainly, in many of the studies 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 123 that I have read, it appeared to me that concentrations of atrazine were measured only in solutions that were freshly prepared.

And certainly depending upon the delivery system that's selected and the design of the experiments, ultimately, I think consideration should be given to make sure that concentrations are measured at times at which you would expect high levels of atrazine,

but also the lower levels that might be resulting in these treatments.

DR. ROBERTS: Any other comments or points to add on 1 A and 1 B? We also had a 1 C.

DR. BRADBURY: We have a 1 C and a 1 D.

DR. ROBERTS: I don't have a D on mine.

DR. STEEGER: Actually, it's pretty much like 1 C.

1 C is, Please comment on the availability, as of February 28th,

2003, of additional relevant studies in the open literature that were not addressed in the white paper.

And 1 D is, Since February 28th, 2003, is the panel aware of any studies that would be relevant?

DR. ROBERTS: We have both of those. The second one, at least on mine, was not separately marked as D.

Let me then go back to Dr. Kelley.

DR. KELLEY: Yes. So the review of the panel, while very 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 124 thorough and complete, was narrow in its focus. So the question is what kind of guidance would the EPA like from us about what we consider to be the relevant literature.

Let me tell you what I have in mind. So omitted from this were several studies that I found in the open literature on effects of atrazine, usually at very high doses on early mortality, say, in xenopus, tadpoles and some more recent effects on slightly later animals. So those were omitted.

The EPA analysis raised issues of consistency in the data and consistency of things like the stages of sexual differentiation. Yet,

of course, there were none of the background papers on sexual differentiation in that panel. So those are two areas.

And the third is of course the issue that the panel has raised that are relevant to things like the mechanism of action of atrazine if it, in fact, has an effect that might be addressed productively by references that included work on other than amphibians.

So I'm in the midst of collecting a series of papers of that sort.

But what I would like to know  but I have to say I didn't find a paper that you guys had left out on effect on gonadal development in amphibia due to atrazine. I haven't found such a thing yet.

So I do believe that in terms of the focus of review that you 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 125 didn't omit any obvious and glaring paper. But in view of the panels's desire to provide scientific guidance to the process going forward, I think that there are probably a number of studies that we would like to bring to your attention.

DR. ROBERTS: Other panel members?

Dr. Matsumura.

DR. MATSUMURA: I would like to ask the panel to consider to add the paper by Bevan appeared in the EHS regarding action of alkylphenols. Of course it is not the atrazine, but this particular paper really shows what the estrogenic effect of the alkylphenol really look like.

It is summarizing their work on some of the early stages of development, and the curvature that develop in the tadpoles are very clear on the melanocyte site formation. You can see what real estrogenic compounds could look like.

This is the one endpoint studied in the xenopus. And it is a very clear cut effect that you expect.

And knowing that alkylphenols, particularly in nonylphenol can be found in many detergents, including the triatonix 100 and all those agents that you use in the lab washing your cages as well as in the formulation in some of the pesticides. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 126 We should really show that this is a kind of thing, if it is a direct effect on the estrogen receptor, this is what you expect. So I would like to suggest that.

I also would like to suggest that the Christin's papers that Dr.

Green mentioned too and, yes, we are looking very narrow way and there are other effects of the atrazine on the immuno competency. So that I would like to suggest.

DR. ROBERTS: Dr. Isom.

DR. ISOM: I would just like to point out for the record that the Hayes paper in EHP came out in April 2003. It has been referred to several times throughout the proceedings. And that is actually a detailed publication of the nature paper that appeared this past year.

DR. ROBERTS: Dr. Steeger.

DR. STEEGER: The requirement for the review is that studies had to be submitted by February 28th. You are correct that the published hard copy of that report came out in EHP in April.

However, the online version was indeed published or available in October. And that was the version that was reviewed for the white paper.

DR. ROBERTS: I think Dr. Isom's response is of 1 D.

DR. STEEGER: That April publication is identical to the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 127 October one.

DR. KELLEY: Could I ask my question again of the EPA?

What sorts of guidance would you like to have? Clearly, we can give guidance going forward. I still see no  within the very narrow focus of what you reviewed, I don't think you missed anything, but we have compiled a bibliography that we thought would be helpful.

Would you like that? Or do you want to sift through it?

DR. BRADBURY: This is sort of an awkward question to answer because it presumes the path the panel may or may not go down.

Hypothetical. If the panel is thinking about a pathway about gathering additional information, my sense is that some of the information you are talking about would be very instructive in thinking about the design and nature and aspects of other information that would provide greater insight either to do a risk assessment today or insights into what kind of information and pathways to gather new information would be helpful.

So my feeling is that, yes, it would be very helpful is more in the context of the proceedings

DR. KELLEY: But what about my question about the omission of acute toxic effects of atrazine on very early tadpoles from the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 128 bibliography?

DR. BRADBURY: I'll turn it over to Tom some, but in looking at gonadal developmental effects and I think a sense of what those exposure concentrations were sort of kept us focused in that context.

Putting it all into the context of full dose response and full effects endpoints, that could be helpful in the white paper now because it provides some founding in terms of effects of exposures.

DR. STEEGER: From my perspective as a scientist, I would very much appreciate to have access to that information to improve our understanding and representation of the effects of atrazine.

DR. BRADBURY: In the context of getting through

DR. ROBERTS: Right.

DR. KELLEY: I bring this up because, of course, mortality was an endpoint that was studied in all these studies.

And even though the doses in these acute studies were much higher and the mortality was much faster, still, in all, that's the compound that you are interested in and the species that you are interested in.

So their exclusion seems to me inappropriate even if they are not directly relevant to gonadal development because they die before they had any gonad. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 129 DR. ROBERTS: Perhaps the panel could recommend that they be included if for no other reason to help provide a perspective for the doses at which developmental effects may or may not occur.

For suggestions on other papers, I think we should clearly identify that if the agency is interested in studies that do not relate specifically to gonadal development but may in fact inform  by analogy help understand whether or not a potential phenomenon occurred, here are some examples that we can point out so that it's very clear that we don't necessarily consider them omissions for the focused purpose of this paper, but they may be useful for the ecological risk assessment.

DR. KELLEY: Let me tell you how I would like to proceed.

What I would like to do is to complete my list of additional sources of information that would be helpful categorizing the papers as we have just discussed under different categories, studies and other species,

acute effects on toxicity, studies that might be useful if a risk assessment goes forward and why they might be useful.

And I will compile that list over lunch, and I will get it printed up and I will distribute it, and then I will allow  of course I will allow  the panel will make me make sure that we haven't missed studies that individual panel members have discovered. And then at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 130 that point I think I would be able to sign off on both 1 C and 1 D.

At this point I would just like to revisit them afterwards.

DR. ROBERTS: Thank you. Other comments?

We're coming right up on 12 o'clock. I'm suspecting that  I'm looking at Dr. Richards as the lead discussant on Number 2. I suspect we're probably going to want a little time to discuss responses for Number 2.

So let's go ahead and break now for lunch. And we can come back replenished and restored at, say, 1 o'clock and take up Question Number 2.

Let's adjourn now, and we'll see everyone at 1 o'clock.

( Thereupon, a luncheon recess was taken.)

DR. ROBERTS: Before we take up Question Number 2, our designated federal official, Mr. Paul Lewis, has an announcement.

MR. LEWIS: Thank you, Dr. Roberts.

The panel were distributed additional comment from Syngenta in reference to clarifying some of their remarks at yesterday's meeting. It is available to the panel and will also be available in the public docket for public review and inspection. Thank you.

DR. ROBERTS: Thank you, Paul.

Dr. Bradbury, let's go ahead and proceed to Question Number 2. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 131 DR. STEEGER: In its evaluation of the existing field studies,

the agency has concluded that these investigations are of limited value.

The reasons include, one, the high variability in environmental conditions and uncertainties in the preexisting status and condition of field collected animals.

Two, 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 three, the possible cooccurence of additional chemicals and/ or non chemical stressors.

Question 2 A is, 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 the 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.

DR. ROBERTS: Dr. Richards is our lead discussant on this question. I will ask Dr. Richards to begin our response. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 132 DR. RICHARDS: Thank you. I'm going to make some general comments on a number of different aspects of this question. I think I will tend to treat A and B together, although, will certainly address both parts of that question.

And I invite the second and third readers on this question to jump in at any point.

In essence, the field studies that have been presented in the literature and the presentations over the last couple days are very good.

In one respect, they provide a great deal of information. I think one of the most important things that they seem to indicate is that the abnormalities, gonadal abnormalities that appeared in some cases in laboratory experiments are seen in the field.

These abnormalities sometimes are seen in very different geographic regions to some degree among different species.

And from that standpoint, I think that's critical, because I think all these questions ultimately relate to an ecological role and a population role. And without looking at sort of natural field type of situations, we can't really see whether some of the things that we're looking at are relevant.

Now, however, the studies do not seem to provide substance to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 133 the nature of why these gonadal abnormalities or other endpoints are related to atrazine.

And that's where it breaks down to problems in experimental design. Often times, the experiments were never really designed to ask such questions. Typically, they have very poor statistical power to address such of a question.

Most of them are essentially descriptional studies.

Descriptional studies or observational studies can be very good. They can be powerful. They can give you relationships. But they require that a number of parameters be accounted for in order to derive any use from them.

In the studies that have been set up to use a control or a reference situation, those control and reference situations have usually not been adequately described or they have some fundamental flaws. And the sample sizes used, the end size used is typically totally inadequate given the variability and some of the measures that we're looking at.

The studies that have been descriptional in attempting to use more of aggression based approach have not adequately dealt with the problems of scale.

The studies we have looked at have included scales including 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 134 looking at continental scales, looking at the size of a county,

comparing a few ponds within a small area.

All of those have very significant things that have to be accounted for in order to account for the variance associated with individual assessment endpoints.

And I will just begin to list a few of those things. But the Number 1 thing that we're dealing with here is aquatic organisms,

organisms that have a very significant part, at least one part of their last cycle tied to the aquatic medium.

In order to do a descriptional study or to set up a good field experiment, you have to adequately account for the movement of water in and out of those systems.

A small ditch is very different than a river, is very different than a wetland. It's very different than a backwater on a river.

So it comes back to both describing the environment that the animal lives in, but very much describing in a general way or a specific way the exposure that these animals actually encounter in the field.

And for small experimental studies, that needs to be accounted for and in much detail. But on the larger, more descriptive studies,

that can be accounted for in a much better way than has even been 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 135 attempted in any of these studies.

There are many tools available that allow us to look at even relatively large geographic scale studies that can quantify and allow you to partition some of that variance in a stratified experimental design.

And basically, that's relating to how does surface water interact with the bodies of water that these experiments are being conducted in or observations are being made, what's a relative contribution of groundwater, what are flow events during a period of exposure, what are the flow events that actually occur amongst seasons, in cases where experiments have been run, have there been flooding events that may influence exposure or may influence the movement of organisms in or out of those experimental situations.

And also related to hydrology is  to have an adequate depiction of hydrology, you have to know what the watershed is. You have to know what is upstream of the event that you are observing.

And there are some fairly easy, relatively powerful ways to describe watersheds both in a strictly hydrologic sense, but also in terms of what the watershed is composed of.

Things like soil characteristics and hydrology connectivity to other water bodies, these are all things that are fundamental both to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 136 the biology and movement of these creatures, as well as the chemical and physical environments that the animals live in.

Basically, some of the studies have provided that type of information perhaps in an appendix or listing of things they may or may not have measured. Some of them did not provide any of that information.

But essentially, none of the studies attempted to use that as a co correlate in any way to try to partition variance amongst characters that again provide variation in these populations that have been observed.

So in essence, I think what we're looking at is that statistical designs have not been sufficient to actually address questions specifically related to atrazine.

And I think that that falls into the B part of this question, also,

is that there are co stressors, potential co stressors. Many of those we can guess, many of those that we could potentially stratify out in an appropriate statistical design or to partition into in the analysis,

but that takes a great deal of forethought in terms of development of a design.

In many cases, had we seen as much attention given to sound experimental design as we have at least to date with some of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 137 laboratory measures, I think we could have been a little bit further forward.

I think I'll ask my co readers to jump in after that.

DR. ROBERTS: Dr. Gibbs, you are an associate discussant. Is there anything you want to add? Or do you want me to take Dr. Skelly first?

DR. SKELLY: First of all, I would like to point out that that's a single sentence up there, and so it took me a while to figure out what it means, but I think I got a handle on it.

I guess the end of the sentence is the most critical part. So do we think that the absence of causal relationship or limitation and study methodologies is the reason why the EPA's conclusion that field studies don't provide the means to ascertain whether there is something going on here.

I guess in my opinion, I'm going to agree with Dr. Richards and say that the limitation and study methodologies is probably most important here, and comment that the absence of causal relationship has never slowed down any ecologist I have ever met.

We often like to pretend that we know why things happen when we're working in the field, but that shouldn't stop us from moving forward and trying to understand what the associations are. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 138 Because after all, everything that we're doing is keeping our fingers crossed that we get the mechanism right. Even in the finest laboratory, using the best laboratory practices, we can never know for sure that we have the mechanism right.

So specifically, I think the limitations and study methodology that were most important concur largely with what Dr. Richards said.

I think the lack of power is an issue, but it is very easy to be critical of that in hindsight.

These studies that have been done can now be used moving forward to come up with better ideas with what needs to happen.

Having said that, the scale of the field studies and I say this from the standpoint of having quite a bit of experience in field sampling amphibian population, these were not large studies in terms of the number of sites that were examined.

There are many more ambitious studies and I think we can look to a lesson from how the UV light on amphibians controversy is being resolved now.

It has taken very large scale studies looking at hundreds of wetlands over very large areas dealing with many of the same issues that we have been talking about here today.

Some of the people in this room have been involved in this 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 139 work, so they know what it takes to pull inference out of these sorts of big observational studies.

That leads me to the second point, which is the issue of site selection. I think it is hard to go out and use a map ahead of time or just go out and look at a place and say, there is a cornfield here or there isn't a cornfield here, to say whether this is the right site to be an exposure site or a control site.

And some of the studies that were not considered here and are not necessarily particularly relevant to this issue but where people have taken an alternative approach, they might take a year or two years just to pick their sites.

I'm going to come back to this point when we talk about some of the questions later on. I'll leave that for now.

The final point, the limitation in the study methodologies may be this is not a limitation study methodology, but a significant limitation in the studies that I saw was to me looking at what the EPA is going for here, the assessment endpoints that are talked about are fertility reproduction recruitment.

Ultimately, what we are trying to think about  something about viability of populations and those people in this room who know about measuring population viability know what a giant task 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 140 that is.

Even if we go back to the assessment endpoints here the motivation for doing field studies should be to get at those things. I don't think most of the field studies that have been done to date address that issue. In moving forward that's something that should be tackled very concretely.

In response to B, to the extent that we do see something going on here, is it true that these studies don't provide sufficient information to resolve the potential role of additional cooccuring stressors?

I guess that is always, always going to be true to some extent with the field study, but for reasons that we'll probably talk about later on I still think it is extremely important for much of this work to be done in a context, in a field context.

To that end, as I have harped on before and I will say now,

virtually all the field work that is talked about here and if we exclude the mesacosm experiment all of it has been observational.

It is possible even in this difficult kind of context of working with pesticides to do field experiments. Especially  we've got a registered pesticide here, we can use it in the world out there and see what it does at population scales. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 141 That can help us significantly get at the issue of co occurring factors because we can choose sites that are sort of tabularasas to begin with and add our stressor that we're interested in.

I'm going to echo  in finishing, I will echo what Dr. Richards said. That is that, I think, the critical thing here that we can't ignore is we can talk about limitations of any study we want all day long,

multiple groups have gone out and looked for gonadal abnormalities in nature and they have found them and these match the morphological characters people are seeing in the lab.

At this point, I think it is fair to say we don't know why, but the fact that those abnormalities exist I think is significant. I think if a bunch of groups had gone out there and had not found them we would be thinking about this differently. I will end there and pass to Dr.

Gibbs.

DR. ROBERTS: Dr. Gibbs.

DR. GIBBS: We have spoken extensively as a group, so I don't want to reiterate too many points. I do personally agree with the statement that essentially study designs have precluded really determining whether there is or there is not an association between the occurrence of gonadal abnormalities in the field populations of amphibians in the presence or absence of levels of atrazine. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 142 We don't want to imply that observational studies cannot address these issues. I think they are actually very important for the reason that Dr. Skelly just mentioned.

The risk assessment goal here really does focus on population viability. One needs to go to the field essentially to measure many of these parameters.

I would  perhaps this is getting away from the question, but there are other modes of studying and tackling these problems which essentially fall in the realm of field experimentation.

We have been discussing some other situations that occur in the field with temporary breeding  temporary pool breeding amphibians such as the amphibians that use vernal pools.

Vernal pools occur in isolated, small, populations in great numbers in homogenous landscapes that are really quite amenable to experimental manipulations that could get at both  would yield information both from histological perspective but also from demographic perspective quite relevant to many of the issues that the risk managers  risk assessment folks have to deal with. I think that's worth exploring at some point. But to get back to the point at hand, I agree broadly with many of the statements that Dr. Richards raised, issues with inappropriate or not outlining sampling frames, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 143 low levels of replication in terms of using wetlands as actually replicates and the amphibians therein.

And really a difficulty in establishing whether there is or is not causal relationship here between what are some fairly high levels of abnormalities being seen in the field, but whether or not there is a relationship with atrazine. In my mind that can't be attributed based on the designs of many of these studies.

DR. ROBERTS: Thank you, Dr. Gibbs. Let me ask other members of the panel if they agree or disagree?

DR. MATSUMURA: I agree with Dr. Skelly and Dr. Richards.

I certainly emphasize the importance of the field studies.

I was trying to think what the precedence I could think of, and,

of course, like Gillette's work on the alligators and how long did it take, and what started that whole project, and like finding a really contaminated site like Lake Apopka was very important for that study.

And comes from field studies like eggshell thinning, it took a long,

long time to say it was DDT.

But again, field observation that all those eggs are crashing and that's a problem and the bald eagles not just thinning but by the weight of their mothers just sitting on those eggs are crashed. Those observations really helped. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 144 And finally, I would like to make sure that one aspect to emphasize, that means analysis of burden of other contaminants. I was reading this Reeder's paper reporting this intersex confined to the  correlated to atrazine. But at the same time they had the PCDD and PCDF.

And so you have to leave some questions always. Knowing what the other stressors and other chemicals are there, that could really help.

DR. ROBERTS: Dr. Green.

DR. GREEN: I agree with your comments. For my own edification, I would like to know from your experience and perspective what denotes a healthy amphibian population observationally in a field study? What implies that the population is sickly and on the decline, absolute head counts and how long does it take to get that kind of information?

DR. GIBBS: Insofar as you linked health to decline, you are talking about a temporal phenomenon. That may indicate the need for repeated sampling to actually detect a change in population size,

population structure.

With these particular organisms, some of them have  with frogs, incredibly short life spans and then generation times and hence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 145 population turnovers.

So one can fairly quickly, unlike working with many other vertebrates, see if there actually are population declines or increases occurring because you need much less time with generation times and sometimes one to two to three years to see these kinds of effects.

Sheer abundance is one good measurement. Dr. Skelly may have other ideas.

DR. SKELLY: I would agree with what Dr. Gibbs just said. I would just add that we have settled on a group here that has notoriously volatile population dynamics and going out in a given year and seeing recruitment failure for some species is actually the norm.

Having total cohort failure is  if we were talking about grizzly bears in Yellowstone people might get quite upset about that, but most of the wood frog populations I survey every year in most years crash out.

This year they are floating across the roadways and they are doing quite well with all the rain. In most years they dry up with their pond. It definitely is something you have to look at over time.

We're fortunate many of the species that we would be interested in in terms of native amphibians do have short life spans. It does 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 146 have to be looked at over time if we're interested in any sort of understanding of population robustness.

DR. ROBERTS: There's that word again. Dr. Delorme, then Dr. Richards.

DR. DELORME: Just following up on Dr. Green's question.

Could you give us any indication of some measurement endpoints or what variables you might be looking for to look for effects in populations if you are out there with your rubber boots?

DR. SKELLY: Population

DR. GREEN: I think that will be addressed in question five.

We have some estrogenic biomarkers we put together that

DR. DELORME: I was asking from a populations perspective,

like what population variables might you go out and measure.

DR. ROBERTS: Were you planning on bringing it up later in the context of five or do you want

DR. SKELLY: I actually think we will address that later.

DR. ROBERTS: Dr. Richards.

DR. RICHARDS: Another context sort of thing is amphibian population is often very variable that is the way they are. That is the nature of the waterbodies they live in, to get back to the hydrology of these situations. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 147 One of the things that we know about amphibians is that they interact over a relatively large geographic scale. You just can't look at a pond the size of the space between our tables here, you have to be cognizant of the larger network of waterbodies that they are connected to.

Because when one local population disappears they are colonized by another population that may be half a kilometer away.

Looking at a series of small ditches that are by nature connected is very important as opposed to an isolated pool.

Are you looking at a species like the South African animal that lives entirely, its whole life cycle in water versus something like the rana species that are out moving around?

These are very critical questions. Both designing the experiment and asking questions about is a population healthy in part it is related to a little bit larger scale than we frequently focus on.

DR. ROBERTS: Dr. Skelly has a follow up, then Dr. LeBlanc.

DR. SKELLY: I guess as a follow up to that, we're painting a picture that can seem to get more and more difficult.

Actually, the genetic techniques that are being developed now are getting us to the point were we can actually distinguish the boundaries of populations. Up until recently, people just kind of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 148 crossed their fingers and assumed a pond was a population. I think we're moving well beyond that. So we are actually starting to understand in a much more detailed way how much movement there is and over what scales we should be assessing population level responses in order to say whether a population is going to remain there or is it likely to go extinct.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: This question is posed to the discussants.

Based on what I just heard, my question is would it be  due to the volatile nature of the population dynamics of frogs would it be difficult to discern population level effects of environmental contaminants?

DR. RICHARDS: Yes and no. I think sometimes with some of these measures dropping down out of the sky on one occasion and listening for how many frog calls there are you are not going get a real good answer. I think because of the fluctuating dynamics of these populations in their actual environment some manner of looking at more of a prolonged view of the population is needed.

There are some things as genetics that give us a backward view of what is going on there, and that's great, but there is much room for innovation on this. The book is not completely written. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 149 DR. ROBERTS: I think Dr. Skelly wants to respond as well.

DR. SKELLY: I guess, I would say in order to make the picture a little less bleak here, there are very volatile dynamics. But if you follow a set of populations in a defined area over a long period of time, you see this volatility from year to year.

You also see very striking patterns. I will give you an example that is directly relevant to the sort of thing we're considering here.

For many, many years people have been doing experiments in mesacosms in the laboratory that were showing that amphibians undergo interspecific competition and that interspecific competition could even be possible for population extinction.

That was the way  that was the inference that was coming out of this work. There is excellent laboratory and mesacosm based evidence that there was competition going on, and if we change the words, that's exactly what we have been talking about here, the same sort of thing where people were doing these very focused studies, they were getting mechanistic, they were looking for interference compounds, they were looking for resource based competition, and so forth.

We have been able to show more recently that from everything we can tell even though this is an actor in these experimental context, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 150 it does not seem to be important for the kinds of patterns that the EPA is interested in for assessment endpoints and for their overall criteria.

Instead we have been able to discover starting with field observations, then moving to field experiments, and then into laboratory experiments, that other factors such as hydroperiod and such as the light level in these ponds that influences temperature and food gradients is much more important.

That's been a real lesson for me in how we discover what is really going on, how to parse this out from the perspective of a field scientist so that the lab scientists are working on the right stuff so they are not just going down the garden path.

DR. ROBERTS: Dr. Isom.

DR. ISOM: Since atrazine has been applied to the environment for over 40 years in North America, is there historical population data that perhaps could be looked at to give us some answers to these longer term population issues?

DR. SKELLY: There has been  because of the overall interest in amphibian declines, everything that existed and much work over the last 12 years or so has been done along those lines.

We probably for the purposes of this panel can parasitize a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 151 much larger database than you might believe. There is much known on that. The problem of course is going to be attributing cause to these historical studies where the other measurements that we are interested in were not made.

I can give you as an example, there were surveys done in the early part of this century in Iowa that describe high abundances of amphibians over many, many counties. When the resurvey was done in 1980' s, abundances and species composition over large areas had changed.

Now that's over the same time span as the green revolution.

Does it have anything to do with that, does it have anything to do with pesticides? We have absolutely no idea.

In general, the consensus seems to be now that many, many amphibian populations are declining. So that's the state of our knowledge, the state of our sciences that many populations have declined.

When people go out in their back yards  and I give lots of lectures to the public and the first question I get from the public,

which is an excellent one, is I can be deafened sitting on the back porch by the spring peepers. So are amphibians declining?

What I say to them is because this is a temporal phenomenon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 152 maybe you would have gone deaf in one week instead of two, 100 years ago. We don't know what it was  we don't have enough data to be able to say over very broad scales what amphibian abundances were like.

But Dr. Gibbs maybe can answer this a little bit better than I can. One of the things that has happened over that same time period is that wetlands have disappeared or been modified, often in an agricultural context, in ways that change their suitability for different amphibian species over the same time span.

There has been talk about multiple co occurring factors. It is there. I think the most interesting historical data that we could get are in the animals. So this issue of evolved resistance could be really important in showing or in evaluating whether there have been broad scale evolutionary responses to pesticides.

DR. ROBERTS: Dr. Gibbs, did you want to respond also?

DR. GIBBS: Just quickly, amphibian monitoring in a systematic and large scale fashion only has begun since 1990 or so with various initiatives such as the USGS and other groups have put together. There really is not a lot of data, even that effort is limping along.

Unfortunately, we are constrained and we will remain so for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 153 quite a few more decades before we can get something like the US

the breeding bird survey on line for amphibians, that's the goal. We really don't have any good solid baselines over large areas. And a decade of data on a few spots is about as good as we can do.

This underlies a lot of the controversies and debates over amphibian decline this lack of monitoring data. Unfortunately, that is the state of the science on amphibian monitoring.

DR. ROBERTS: Dr. Richards.

DR. RICHARDS: I would like to hopefully dispel a little bit of the gloom here that I myself have been raising.

At one level amphibians are no different than any other aquatic organisms that are dealt with by EPA and aquatic ecologists for a long time.

They still reproduce, they still have fecundity, they still recruit to a population, there is predation, there is density and nondensity dependent aspects to their population sizes, and that makes them particularly amenable to experimentation. That doesn't mean we can't experiment with these animals and can't derive some creative ways of examining population parameters.

They do things just like when you are chasing little fish along the margins of a stream. If you have ever done that you know you can 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 154 go there one time and they will be there, next time they won't.

That doesn't keep us from having a great body of regulatory approaches to dealing with toxic antieffects on fish.

DR. ROBERTS: Dr. Delorme, did you want to respond to that as well? I have a couple of other folks who have questions.

DR. DELORME: I wanted to make a comment on an observation. I can wait.

DR. ROBERTS: Let me go to Dr. Denver, then Dr. Heeringa,

and then you.

DR. DENVER: I just wanted to comment on something that Dr.

Skelly said. It has to do with single species experiments versus competitive interactions, and at the risk of making this even more complicated I think it is important to realize the importance of the competitive interactions. Most of the studies that we have reviewed have been studies of single species. We published a study actually last year that showed that exposure to PCBs can actually change the competitive interactions among species.

These were studying the northern leopard frog and also wood frogs. That's another layer of complexity that needs to be considered.

DR. ROBERTS: Dr. Heeringa.

DR. HEERINGA: With much more expertise, Dr. Denver 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 155 anticipated my question. Mine is much more direct and I deal a little bit with waterfowl populations.

Is there evidence, either from zoological samples in museums,

et cetera, or other studies, on sort of either a lack of increased hybridization or competition in these natural populations?

Are there species that are invading historically, turf for other species that have been a concern to ecologists, is that practice, it could happen naturally, but do you see that happening and are these populations still relatively isolated and nonhybridized?

DR. GIBBS: Certainly, and the bullfrog is a classic example of invasions, but very much associated with the activities of people and moving them around.

I'm trying to think of an analogous situation with a hybrid zone shifting or one native species, a sympatric species, one moving into the other's range. I'm failing to

DR. SKELLY: The example that Dr. Hayes talked about that was the first one that I had seen evidence for.

DR. ROBERTS: We've got Dr. Delorme, then Dr. Matsumura.

DR. DELORME: I was sitting here listening to Dr. Skelly talk in response to Gary Isom's question and it occurred to me that one of the things that EPA has asked us to look at is the assessment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 156 endpoints and they put forth an assessment endpoint of the population.

You guys can shoot me down if you want but some of the measures that you are talking about, species diversity and relative species abundance, perhaps maybe an alternate assessment endpoint is also communities, in terms of their composition.

It gets into sensitivity of different species and whether it is different or not. Just want to know if you guys think this idea has any merit as alternate assessment endpoint?

DR. ROBERTS: You three are on the spot again. Who wants to take it?

DR. RICHARDS: I'll touch a little bit of it.

There are some studies out there that have indicated over relatively large geographic areas that anuran communities can be a fairly useful way of looking at relationship to landscape parameters.

Whether they'll be useful for looking at toxic related questions, I don't think has been delineated.

Certainly, communities can be manipulated  the data from communities can be manipulated into a series of metrics much like we use fish or invertebrates or other things in a way to look at patterns over relatively large geographic scales. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 157 DR. ROBERTS: Dr. Skelly, did you want to add anything?

DR. SKELLY: I guess I would just insert a note of caution that it can be  if we are trying to link these studies, eventually, back to a mechanism and link them to laboratory studies, logistically and pragmatically, it may be challenging enough to do on a single species level.

I'm very interested in communities. I think that they can sometimes show responses that may give us more information than single species.

But on the idea that we need to walk before we run, I guess I would say that we should try to get out into the field more working with the single species before we look at multiple species simultaneously.

DR. ROBERTS: Dr. Matsumura. DR.

MATSUMURA: I'm not doing that, but I'm collaborating with the good herbatologist in the pit studies, on the study on the yellow legged mountain frogs, rana muscosa.

As far as that particular species is concerned, it is declining,

everybody agrees, at least, in California. It has a long larval life span that it spends in such a cold place for three to almost four years in the water, and they are disappearing. 158 1 When we made a survey, we found that there are still some 2 ponds and lakes teamed with the yellow legged frog but they all on 3 the eastern slope. On the western slope, there are only about three 4 sites that we found.

5 There is no question that you can talk to those rangers. They 6 say, well, I used to know that particular pond had the muscosa, lots of 7 them and they are disappearing in the last year 10 years.

8 They are species that you can really count on not just by virtue of 9 long life in that particular case. You can really see that's how they 10 are disappearing.

11 So, it is not related to atrazine.

12 But by studying those, we start finding also, hyla regilla,

13 pacific tree frogs, are also disappearing from the same western slope.

14 That's the reason why we are suspecting air pollution and particularly 15 PCBs.

16 In the lowland area also the red legged frogs also disappearing,

17 but they are being displaced by bullfrogs. So that is the same 18 example that you are citing.

19 I get the feeling that just looking at those mountain frogs 20 endangered species which we cannot study well, that there could be 21 some combination of right kind of species, very sensitive ones that 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 159 you could pick on and try to see whether those are the ones who are effected the most by this kind of pollution.

So some field biologists can really look for, they may find a good combination. I just wanted to add.

DR. ROBERTS: Thank you.

I haven't heard much in the way of disagreement in this.

Let me ask the panel, is there anything that they disagree with that has been stated or are there any other important points that have been left out of the discussion that perhaps should be included in the response?

Dr. Kelley.

DR. KELLEY: I don't disagree with the conclusion as stated based on the available data. I just simply wish to make a comment about the field studies in South Africa, which is that if the EPA decides that it wants to adopt xenopus as some sort of model system for studying a sex environmental effects, including atrazine, but there are others as well for which it's being the considered, inclusion of field studies in gathering field data in xenopus will be extremely useful.

It will enable you to take the best studied laboratory anuran and correlate those results with the field. I don't think it is possible for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 160 any other animal. We have neglected in our literature survey the data on xenopus which exists in the South African literature.

I would, if that route is taken, I think  going forward more carefully designed and better powered field studies in xenopus will be useful in trying to relate laboratory results to potential effects in the field.

Now they won't tell you anything about our North American anurans, necessarily, but they will tell you something about the limitations of the transform.

DR. ROBERTS: Thank you. Dr. Bradbury, it looks like there is strong endorsement for the value of field studies and the panel has a number of comments about what is required to really get good information from those studies.

Are there any follow up questions that you have for the panel on this topic?

DR. BRADBURY: Just for the record, we have question 2 B.

DR. ROBERTS: I sort of sense that our discussion has covered both 2 A and 2 B. I will defer to the discussants on that.

DR. BRADBURY: I feel comfortable that we got the picture between the two aspects but if there is something left in your laptop.

DR. ROBERTS: Dr. Richards, is there anything left in your 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 161 laptop?

DR. RICHARDS: My fingers are fingered out, but I think in my own comments I was implying that it was very difficult to examine the effects of costressers given the studies we have seen so far.

DR. BRADBURY: I think we're in good shape on our end.

DR. ROBERTS: Are there any other points that need to be made or anything we need to resolve before we move onto the next question? If not, let's move on to question three.

DR. STEEGER: In 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 the different experimental designs and variability in the nature and extent of experimental conditions, i. e.,

level of excessive mortality, delayed developmental and 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 prove the means to characterize the nature of any associated dose response relationships. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 162 Please comment on EPA's determination that the laboratory studies provide a plausible basis for the means to establish hypothesis concerning the potential for atrazine to cause development effects.

Also, please comment on whether the overall body of available data are adequate to demonstrate whether or not atrazine causes developmental effects under the conditions described in these studies.

DR. ROBERTS: An important question Dr. LeBlanc, what do you think?

DR. LEBLANC: I concur with the EPA, their determination that laboratory studies on the affects of atrazine on anuran gonadal development are sufficient to establish the hypothesis that atrazine interferes with normal gonad development.

Clearly, the available data are limited. However, the existing data as related specifically to the laboratory investigation, I believe,

support the hypothesis of the eight laboratory studies that we were provided with that evaluating effects of atrazine on gonad development.

Five of these studies detected such abnormalities from three different laboratories. Certainly, in my laboratory, if a student comes to me with an observation, be it induction of enzyme, suppression of protein, or a message RNA level, the first thing I tell them is go do it 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 163 again.

They come back and I tell them to go do it again and perhaps after six, seven, or eight times I will accept that data, which doesn't mean that perhaps two of those times they weren't able to reproduce the effect.

But if the preponderance of the evidence consistently shows the effect that they initially observed and that we were pursuing, I wouldn't discount the data.

There certainly were differences observed among the various studies that we examined as related to the effects that were observed and many attributes were ascribed that could have contributed to these differences, including species differences, differences in exposure design, and specific endpoints that were selected in the individual evaluations.

Significant data gaps exist in our understanding of the affects of atrazine on anuran development. These include a lack of understanding of the mechanism by which atrazine elicits its developmental toxicity, the nature of the concentration response relationship, definition of susceptible windows of exposure, and identification of a threshold concentration.

However, it is certainly my view, that the existing data does 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 164 support acceptance of the hypothesis that atrazine interferes with normal anuran gonadal development.

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: I agree a great deal with what Gerald already told you. I think one of the points why we find so many differences, and the big difference should be that we have no standardized protocols in various studies.

I think one of the goals for the future in future studies is, first,

to design more or less a standardized protocol to work on amphibians,

especially on xenopus laevis, but also if you use ranids.

So, we are comparing apples with pears if you don't go ahead with a really standardized protocol. That's one comment on it.

DR. ROBERTS: Dr. Denver, do you have something to add?

DR. DENVER: I just agree that we have sufficient data to generate a hypothesis but insufficient data to test the hypothesis.

DR. ROBERTS: Other members of the panel? Dr. Matsumura.

DR. MATSUMURA: I agree with Dr. LeBlanc's statement so long as it is limited to gonadal abnormalities. I'm not convinced about the whole scale developmental effect even including laryngeal effect.

But regarding the abnormalities, I feel that the having Dr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 165 Carr's independent experiment as well as John Giesy's, I feel that qualitative there is some clear cut independent verification of the phenomenon. But others, I would not stick my neck. So that's just my personal feeling.

That's why I asked Dr. Carr to just tell me that he can really stand behind the data. He told me that. So, I'm satisfied by independent verification. But other parameters I would not agree that there is sufficient base.

DR. ROBERTS: Dr. Matsumura raised the point  you know the question asked about developmental effects. I guess, we need to be careful how we define those in terms of our conclusions.

I would also like to point out that I want to be clear, when the question asks whether or not the overall body of available data is adequate to demonstrate whether or not atrazine causes developmental effects.

I thought I heard two different answers to that question among panel members. I want to sort of see what people think.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: I think the question is whether the data is sufficient to entertain the hypothesis that atrazine produces developmental effects. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 166 The answer was a narrowly focused one. It said that the data on gonadal development coming from two very different frog species,

one totally aquatic and one at least partially terrestrial, terrestrial during most of the lifespan, did indicate an effect on gonadal development.

So for that reason, entertaining the hypothesis was regarded as an appropriate thing to do.

DR. ROBERTS: So, not to put words in your mouth, but your opinion is that the data are sufficient to support the hypothesis or to justify the hypothesis, but not necessarily to demonstrate which I would interpret as prove the hypothesis.

DR. KELLEY: It was very well put by the EPA. You could look at the data and say the data is such that we could reject this hypothesis. I do not believe the panel would support that conclusion although we haven't heard from everybody. I do not believe that we could reject the hypothesis. So, what that means is the hypothesis is still viable. Is the hypothesis more than a hypothesis,

do we actually believe that atrazine will, at some dose that we don't know now and some range of species that we don't know now, reliably result in gonadal abnormalities?

I don't think nor do I believe the panel thinks that the data are 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 167 sufficient to accept that hypothesis. But the data are certainly sufficient not to rule it out and to continue to entertain it as a model for looking further.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: I'm going to push you a little bit more though.

I'm just going to reread the statement just to make sure we answer the question as asked. The statement  the question ends with essentially available data  EPA is questioning whether available data is adequate to demonstrate whether or not atrazine causes developmental affects. So, you're saying no.

DR. KELLEY: Read the last phrase.

DR. LEBLANC: Under conditions described in these studies.

DR. KELLEY: So we have data. We have data from a reasonably large number of studies that indicate that at least one dose there was a gonadal defect in a varying proportion of animals.

I have a where there is smoke there is fire reaction to that. I think there is something going on, that is my bottom line, but I don't know there is something going on.

I believe it is worthwhile investigating the hypothesis further,

but I wouldn't believe that it was proven at this point. It certainly raises concerns. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 168 DR. ROBERTS: Whenever we're not sure exactly what the question is asking, we sometimes ask the agency to clarify for us.

Is this question asking whether or not the data provided by the existing studies is sufficient to demonstrate that affect or is it more along the lines of what Dr. Kelley is talking about?

Is it justify pursuit of the hypothesis but not necessarily demonstrate the phenomenon which is what I, sort of, read into the white paper. If you can clarify that for us.

DR. BRADBURY: It's amazing how one can wordsmith, and work and work, and think it's clear, and then you need to work on it.

The second phrase is commenting on whether the overall body of available data is adequate to demonstrate in the describe studies.

I think that's an important phrase. Based on the body of information is there some sense, given the quality of the studies and the characteristics of the studies, is there something going on, is there some smoke?

That is sort of in the context in is there enough smoke to say, I think it's reasonable to formulate a hypothesis to look to see if there is fire associated with the smoke. That's the context of the question,

intended meaning of the question.

I apologize if you're confused. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 169 DR. ROBERTS: With that clarification, then, both of you have answered in the affirmative then?

DR. LEBLANC: I have.

DR. ROBERTS: Dr. Kelley?

DR. KELLEY: I have as well.

DR. ROBERTS: Dr. Denver?

DR. DENVER: Yes.

DR. ROBERTS: Is there anyone else that would like to weigh in on this?

Dr. Kloas you were a discussant did you want to indicate one way or the other on this? Dr. Coats?

DR. COATS: Yes, I will weigh in. I think there is enough data to pursue forward movement to test the hypothesis.

DR. DELORME: I would concur as well. I think one of the things we saw is there are a number of factors with the husbandry and whatnot that might have resulted in something going on. So, it is not really, really, clear that atrazine is the root cause. So, we need to pursue the hypothesis.

DR. ROBERTS: Would anyone else like to weigh in on this before we move on to B?

DR. SKELLY: I will concur broadly. I think the way that Dr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 170 LeBlanc and the others responded reflects my feelings as well.

DR. ROBERTS: Dr. Green?

DR. GREEN: I concur as well.

DR. ROBERTS: Good. Dr. Richards?

DR. RICHARDSON: I'll join the party.

DR. ROBERTS: Then let's go ahead and take part B?

DR. STEEGER: Please comment on EPA's conclusion that given the variability in the available dose response data across the studies, e. g., approximately 250 fold difference in reported thresholds for observed developmental effects as well as reports of monotonic and nonmonotonic dose response curves, it is not possible to ascertain the relationship, if any, of atrazine exposure to developmental effects in amphibians.

DR. ROBERTS: Dr. LeBlanc, what do you think?

DR. LEBLANC: The short answer is I agree wholeheartedly,

and I think I only need to expand on that a little bit.

There is clearly major deficiencies that exist in the data as related to describing the relationship between atrazine exposure and gonadal toxicities that have been reported.

We can extract, I think, a small amount of data as related to that relationship that presumed relationship, at this point in time, although 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 171 the conclusions are very, very limited.

For example, I think, I hope, we're all in agreement that atrazine at a concentration of 0.01 microgram per liter appears to have no effect on anuran gonadal development.

The data seems to suggest that exposure to frog larvae to atrazine concentrations greater than or equal to .1 microgram per liter can elicit developmental abnormalities, not does but can.

There is limited data available to us to make any judgments as to whether or not concentrations in the range of .1 to 10 micrograms per liter truly elicit adverse effects.

However, when we consider concentrations in the vicinity of 25 micrograms per liter, there seems to be some reasonably good concordance that this concentration does elicit gonadal effects.

Clearly, more data is required to define the concentration response relationship between atrazine and gonadal development of anuran larvae.

At this time, I would have confidence only in concluding that the threshold concentration for this material exists between .01 and 25 micrograms per liter.

DR. ROBERTS: Thoughts from other panel members? I will start with the lead discussants, Dr. Kloas, did you want to add 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 172 anything or comment on this?

DR. KLOAS: Concur.

DR. ROBERTS: Dr. Denver?

DR. DENVER: I would just concur with the statement that we put together last night.

DR. ROBERTS: Other comments from other members of the panel?

Does anyone feel that the data are adequate to describe the dose potential dose response relationships?

Dr. Matsumura?

DR. MATSUMURA: I don't think it is adequately describing,

all we're saying is that the threshold concept can be applied and that some experiments indicate that there is such a thing.

Real threshold experiments must be done rather carefully. It is not that easy. How many years it took to have any agreement on the cancer dose and the effect relationships, particularly with the hormones, there are feedbacks and  you know, Jere.

So, I would vote for the statement that the threshold is a good way to go about it and there are some indications, incipient indications.

DR. ROBERTS: In the spirit of Dr. LeBlanc's earlier comment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 173 which I concurred that these were actually concentration response relationships and not dose response relationships. So, I'm going to correct myself in agreement with that.

DR. LEBLANC: I think we need to recognize that the great majority of the studies that we have reviewed, I can think of one exception, were not designed to evaluate the concentration response relationship. Certainly, future studies need to be designed appropriately so that the response relationship can be appropriately evaluated. I made some attempts to drive some general understanding of what this relationship might be based upon all the information that was provided to us.

The best that I could come up with, and I don't have a lot of confidence in it, is that .01 micrograms per liter has no effect and all concentrations evaluated greater than that had an effect on average.

And on average, that effect regardless of the concentration seemed to have been around 20 percent incident of effect whatever the investigators may have been monitoring.

Now, I don't know what that means. It could mean that the maximum response that we should anticipate is 20 percent. And that the concentration response is going to occur between .01 and .1, the threshold will be between there. I don't know that that's the case. It 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 174 is just a suggestion.

And there are precedents for observing partial effects where perhaps we would be anticipating greater effects? What comes to mind are my own studies looking at intersex in snails as related to tributal 10. In laboratory exposures where we try and replicate what we see in the environment in 10 contaminated environments where the incidence is 100 percent intersex, the best we can generate is about 30 percent. We're not alone, other labs have had similar success in generating intersex in only about 30 percent of the animals. We really don't know why. We assume it is a deficiency in our experimental design.

We know the animals are capable of  the population is capable of totally responding, but in the lab they simply don't. I don't think we need to go into these experiments with the anticipation that we're going to see 100 percent response in a population if 20 percent is truly the maximum that we can anticipate, we should accept that and live with it.

But we should still be able to define that relationship between a threshold concentration and that maximum effect whatever it might be.

DR. ROBERTS: Okay. Are there other comments you want to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 175 express regarding this?

I'll say for myself personally, I agree that I don't think the data are adequate to describe the dose response relationship.

I have not gone through the analysis you have. I have no idea where an apparent threshold might be. Speaking personally, I have no idea what the shape of the dose response curve might be. But I do agree there is not enough data to establish that.

Anyone else? Let's go ahead and move on to the next.

DR. STEEGER: Many of the available studies proposed that aromatase induction results in elevated estrogen levels that lead to feminization as characterized by ovotestes, intersex, and hermaphroditism in genetically male amphibians.

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

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: First of all, I would like to say that it is not the only hypothesis which could raise such phenomenons, there are feminizational demasculization phenomenon.

We would have two ways how to obtain them estrogenic and anti androgenic ones, that's the first remark I want to make. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 176 Answering question 4 A, of course it is correct. There are no data showing up any aromatase induction or stimulation caused by atrazine. But however, I think we have already discussed there is no approach done which would be appropriate to demonstrate it.

So, I think that experimental designs done up to now, they don't support this hypothesis but they cannot support such a hypothesis for aromatase stimulatory effects.

Would you like to add something?

DR. ROBERTS: Thank you. So in your opinion it is still an open question?

DR. KLOAS: Yes.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: I would agree. I would like to point out that the aromatase gene or at least one of them, they probably have two, the spene laevis ( ph) has been cloned in xenopus laevis. It will be possible to study the expression of the aromatase gene in the developing gonad in the presence and the absence of agents that are thought to affect it.

While the MRNA expression is not definitive with respect to protein expression or the activity, the enzymatic activity of that protein, on the other hand, you do have to have the gene expressed to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 177 have it there. So we now have available the tools to test this hypothesis in a rigorous fashion. It should be possible to test that.

I do concur absolutely that although this is a popular hypothesis, it is not the only hypothesis that could provide a mechanism of action for effects that have been reported.

And so to focus on this hypothesis to the exclusion of other kinds of ideas like changes in hormones within the animal's body and steroid hormones changes in the hypothalamic, pituitary, gonadal axis is almost certainly a mistake. There are bound to be other mechanisms that are involved. DR. ROBERTS: Dr. Kloas.

DR. KLOAS: I would also like to add one comment. It could be also due that there is a direct interaction with the enzyme aromatase. So I think if you could invitro assay, you should add atrazine at different concentrations to show up if there is any stimulatory effect which could be done very easily because already aromatase was assessed biochemically in several labs. And it is a routine assay.

I think that's second possibility. Not only looking on gene expression, but also on direct interference with the enzyme as a second possibility to rule out if there is any interference with aromatase and the output for estrogens. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 178 DR. ROBERTS: Thank you.

Dr. Denver.

DR. DENVER: The data to at least generate the hypothesis are based on studies in human cell lines and a limited analysis in alligators. The hypothesis has not been directly tested in amphibia.

Just one point I would add, to test it, not only do we need to take advantage of the molecular tools that are available, but also do it at the appropriate developmental stage at least in one that is hypothesized to be sensitivity to atrazine.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: First, I would like to just agree with everything that has been said. But I would like to make the point strongly that in agreeing that aromatase induction has not been demonstrated, it should not be construed to mean that it's not induced from my perspective.

It simply means that the appropriate experiments haven't been done to demonstrate whether or not induction occurs.

And certainly, a point that I feel extremely strongly about in future studies looking at aromatase induction is that the experiments need to be conducted in the right life stage, not in the adult, but in the larvae that we're interested in. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 179 DR. ROBERTS: Dr. Kelley, you are nodding. Agreed?

DR. KELLEY: Yes. To demonstrate that you have an effect of an agent in an adult, say, for example, hormone levels, and then to infer that that same effect with an unknown mechanism should have happened at developmental stage is taking the hypothesis quite far down the line without evidence.

So really, you have to go back to the stage when the effects are thought to occur in order for the mechanism to be established or not established.

DR. ROBERTS: So the comments so far I think have expressed the opinion that it has not been demonstrated that aromatase is induced. However, the experiments have not necessarily been appropriate design to adequately test that.

There have been some suggestions about how the best to approach that test as well as a caution from the panel that about focusing specifically on aromatase as a possible mode of action that there are other endocrine mechanisms that need to be considered as well.

Does everybody sort of agree with that summarization? Are there any other points or is there disagreement?

Dr. Matsumura. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 180 DR. MATSUMURA: Just I want to make sure that  yes, our chairman really summarized right. We should not really lock into that. Dr. Kloas also suggested some different approaches such as the antagonist of the testosterone.

This is not the typical estrogenic response. It doesn't happen in the females. It doesn't look like any effects. So should not be locked in. I agree with Dr. Kelley's statement that there are other possibilities that we have to keep our minds open.

DR. ROBERTS: Any other thoughts or comments about A? Is the feedback clear? Great.

Let's go to B.

DR. STEEGER: 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, for example, changes in sampling design, analytical techniques that could efficiently address this variability in future studies.

DR. ROBERTS: Dr. Kloas and Dr. Kelley I believe during some earlier discussions you made some points about the sex steroid measurements. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 181 What is your response to this question?

DR. KLOAS: First of all, I think all of us being aware of this kind of determinations agree that there is always in lower vertebrates a high variability and steroid levels in general and also activities in aromatase may vary widely.

I think there should be agreement here. So the variability shown up, until yet measured, seems to be normal, what you will normally get.

Anyhow, we have to commend or we have some remarks about ELISA data, especially for estradiol measurements presented.

I think there are at least one order or two orders of magnitude higher than all the old data measured also in some of our labs that would suggest to be in this level. So they are much higher. So there should be any proof that this really is the right way to assess estradiol in xenopus.

For anything else, I think changes in sampling design  I would like to show up set series ( ph) involvement in steroid or in sexual steroid genesis. I think first of all there should be short term exposures and short term measurements for estradiol as well as for androgens.

I would also refer that there could be also some interference 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 182 with ratio of testosterone, dehydro ( ph) testosterone. And I would also refer that there should be also some improvements to include five alpha reductase measurements, because this could be also another hypothesis how it works and how it could work in anti androgenic way ( ph).

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: I'm going to read my remarks.

The variability associated, the largest variability, two orders of magnitude, in other words, 1,000 times, in the studies reviewed by the EPA reflects differences in results obtained in studies sponsored by the registrant using ELISA assays and studies in the open literature using radioimmunoassay.

The most likely explanation for this large discrepancy is the method used to measure hormone levels  so that's the analytical comment up here. Resolving these discrepancies should be straightforward.

Within the open literature, on the other hand, variability is more typical typically in the two times range, a range well within diurnal and seasonal variation.

It should be recognized that species studied extensively in the laboratory, xenopus laevis, may have originated from different 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 183 populations in their native habitat.

Populations can differ in seasonality even within the same country. For example, in South Africa, the Cape population breeds in the African winter, and the Johannesburg population in the African summer.

And we would thus expect differences between the two populations in the field and in the laboratory as this species maintains circannual rhythms even after many, many generations in the laboratory.

When wild caught animals are brought into the laboratory for study, they should be characterized genetically to identify the population of origin, this is possible now based on literature, and also to verify species because I'm sorry to tell you every xenopus looks like every other xenopus pretty much, except some are big and some are small. Although, if you listen to their songs, you can tell the difference.

And the same approach must be employed in characterizing groups of animals that are used for laboratory studies. So this is by way of saying that if we're going to standardize the animal as a test species, we have to develop standards that don't just involve the laboratory assays that we do, but that involve the biology of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 184 animal that we're looking at.

DR. ROBERTS: Dr. Denver.

DR. DENVER: I just want to reiterate the importance of validation of immunoassays, especially assays for estradiol.

Estradiol is known to be present at very low concentrations in plasma and is routinely measured at least in the laboratories that are represented here by radioimmunoassays.

And historically, radioimmunoassays have been used to measure plaza sex steroids and tend to be more sensitive than ELISA's, although some of the current generation of ELISA's may be just as sensitive.

But I think it is important to compare the two methods to make sure that they are in concordance and to validate the assay methods for each species under study.

There is a tendency these days to purchase a kit and put things into it. I'm not saying that this is what was necessarily done by the investigators here, but it is really important to evaluate that kit for the species under study and show that you're actually measuring what you think you are measuring and that you're actually recovering from your sample.

In experiments where you add known amounts of cold steroid, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 185 you can actually recover that in the assay.

So those are important things. Because the data that you generate are only as good as the methods that you are using to generate them. There has been a lot of discussion about animal husbandry, which is important, but I think there should be similar discussion of the methods used to analyze steroids, because perhaps much or perhaps some of this variability, at least, could be due to analytical methods.

The fact that one lab can use a kit and get the same result doesn't necessarily mean that result is correct. You can repeat an artifact until you are blue in the face and it doesn't necessarily mean that that is a correct number.

DR. ROBERTS: Dr. Kloas.

DR. KLOAS: I think also another possibility to overcome such a problem for measuring estradiol could be to measure an estrogenic biomarker. A lot of estrogenic biomarkers are now already available.

The methodology is well established. For instance,

vitellogenin, you can measure it in the plasma, but also as vitellogenin MRNA by RTPCR techniques. That's routine measurement.

You can also maybe in an indirect way demonstrate that there 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 186 could be any estrogenic interactions or interference caused by atrazine.

So This is another possibility to have another methodological approach to demonstrate estrogenic pathways.

DR. ROBERTS: Yes. Dr. Kelley raised that issue earlier in discussion with one of the presenters.

If she wants, we could include that as part of our response to this question.

DR. KELLEY: I also want to make the comment that snapshot measurements of hormone levels at a particular point in time do not give a valid picture of the history of exposure of animal to the hormone in question.

What you really want, what I want, are biomarkers that show you in living realtime color what the animal is seeing. We are fortunate with xenopus that those are available now, actually. We will be able to look at flashing green frogs and know what tissue is being exposed to what level of hormone, when and how, which is an unusual situation.

But even without this fancy molecular method, the biomarkers such as have just been described are really very well characterized.

And you know that if you see a male that has any vitellogenin 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 187 or further that shows rapid response to estrogen challenge, you know that male has seen a level of vitellogenin that is not present in the literature.

So these are available assays and should be used and added to the armamentarium.

DR. ROBERTS: Dr. Kelley, can we provide some specifics and citations in our minutes?

DR. KELLEY: I have them in my giganto reference list.

DR. ROBERTS: Somehow I knew you would.

Dr. Tietge I believe has a question.

DR. TIETGE: I'm a little confused on one point here. Maybe I didn't catch it quite correctly.

Dr. Kloas said that there is high variability in these measurements and that's somewhat normal.

Dr. Kelley said that the typical range of variability is twofold.

Are you in agreement, the two of you? Did I get that right?

DR. KELLEY: The typical range  would you agree that the typical range of variation is not two orders of magnitude? 1,000 times?

DR. ROBERTS: That's 100.

DR. KELLEY: Sorry. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 188 Well, you know what I mean. It's not 1,000 times?

DR. ROBERTS: I think she meant two orders of magnitude perhaps.

DR. KLOAS: Not twofold.

DR. KELLEY: I'm sorry. I meant two orders of magnitude.

DR. TIETGE: That clarifies it. Thank you.

DR. KLOAS: We agree with each other.

DR. KELLEY: We agree that twofold is normal, and 1,000 times is not normal. I actually wrote that down.

DR. ROBERTS: Two orders of magnitude, yes. Three orders of magnitude, no.

DR. KELLEY: Is twofold two orders of magnitude? No.

Twofold is within an order of magnitude. It's within 10 times.

DR. MATSUMURA: I would like to support the Werler's ( ph)

comment. Really, those steroid levels can change really by the hour.

So I would like to really recommend that the endpoint of those steroid action  actually, I have plasmid for the vitellogenin on the control by ERE. It comes from xenopus. I got it from Shapiro. And I also have a PS2, which is very sensitive.

It is well constructed. It works. I offer to Joe Giesy, if he wants, and anybody who wants I have it. Of course, I have to write to 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 189 Shapiro saying that I will transfer that.

But really, those are sensitive. I was so amazed. I compared that to mammalian construct and the xenopus is better. Much purer ERE. You can detect the PS2 very easily or PGR, progesterone receptor.

Those are well, well accepted. You can run that easily. I support that. Because that is more stable a way of measuring.

Some time ago hormone was up, but its effect is still here.

DR. ROBERTS: Any other comments on this before we move on to the next one?

Clarification?

DR. BRADBURY: I'm sure you all do it. If you can get all this written down, some of the dialogue. That will be very helpful.

Based on the discussions over the last few days, still within the realm of this question, but one of the hypotheses was it was  the increase in estradiol, that the estradiol doesn't get into the plasma, it stays within the tissue.

Are there techniques available to measure changes in estradiol concentration within the tissues?

Could you comment on that?

DR. ROBERTS: Dr. Kloas. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 190 DR. KLOAS: I think it is easy to measure also from tissue's cystorrheic ( ph) levels. You can extract them easily. But, however, I think it is a completely lipophilic compound, how to keep it  it has to be really completely resorped by receptors at the same time.

Normally, there should be any leakage to the circulation. I wouldn't expect that could be stored, of course. So should be a change.

DR. ROBERTS: Dr. LeBlanc.

DR. LEBLANC: As Dr. Kelley said also, it is really a snapshot that we're taking be it in the serum or be it in a given tissue.

And that snapshot tends to reflect very well the level of synthesis that's taking place in that tissue at that point in time.

So another alternative would be rather than looking at tissue levels at a given hormone, if we're working with gonads would be perhaps to remove the gonads and in tissue culture look at the production steroid synthesis.

And another strength to that approach too is you could be at the same time looking at production of multiple steroids, androgens and estrogens.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: Meashita ( ph) and coworkers in Japan have 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 191 routinely cultured the Stage 51 gonad. In the presence of aromatase inhibitors and also in the presence of estrogen, are able to observe sexual differentiation invitro and are able to observe effects of the agents that they add.

That assay is very available.

Also, I want to point out, I know I pointed this out before, but the liver is not that close to the gonad. Typically, if you have evidence of vitellogenin  let me explain for the public what vitellogenin is. It is the yolk proteins. Birds have it. We don't have so much yolk. But the frogs have a lot of it. A mature egg has to yolk up in order for it to be oviposited, ovulated and oviposited.

If you see vitellogenin in the liver, it had to have arrived there,

I believe there is agreement, via the circulation. So it had to have circulated at some point. That would mean that it got out of the organ.

It is worth pointing out, it has been pointed out, for example,

females use testosterone as a precursor for estrogen. Dr. Hayes was right, that he didn't get the yolk up except in rana.

But xenopus uses testosterone as a precursor for estrogen. You can measure really high levels of testosterone in females when their ovaries are activated. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 192 So circulating levels can be useful and shouldn't be denied, but there are other tools available to us.

DR. ROBERTS: Let me ask Dr. LeBlanc and Dr. Kelley a question of clarification for my edification. Not for the agency's.

The experiments in gonadal culture that you described, are those  are you envisioning an exvivo experiment where they are exposed, you are removing gonads from exposed animals and culturing them and then measuring synthesis rate?

DR. LEBLANC: Yes.

DR. ROBERTS: So the exposure would occur in the animal, but not while in consult ( ph) culture.

DR. LEBLANC: That's correct.

DR. ROBERTS: And you would still expect to see a continuing effect of the atrazine even though the gonads in culture would not be exposed to atrazine?

DR. LEBLANC: It certainly depends on the mechanism of the effect. But if it were induction of the enzyme, then in the short term,

yes, you would expect to see

DR. ROBERTS: Short term until it declines, okay.

DR. KELLEY: Just to echo a point made here about short term exposure, it is a tremendous advantage that you can get sexual 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 193 differentiation over a 48 hour period in xenopus.

Because of that, you can do it in culture. You don't have to worry about the rundown of the cells so much. You don't have to worry about the compensatory mechanisms within the entire animal.

And that's a big experimental advantage.

DR. ROBERTS: Are there any follow up questions from the agency on this? Any other comments?

Let's then move on to the next question.

DR. STEEGER: 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.

DR. KLOAS: As far as we are aware, not in xenopus, at least.

I cannot give you the citation, but there is some Japanese people talking about ranids where you could reverse probably in late stages of rana rugosa. You can induce sex reversal. But not for xenopus. It is not reported, at least.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: In ranids, it has long been known that aging animals will show gonadal changes spontaneously. Whether those are associated with environmental agents has never known. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 194 I remember reading an old report where old female ranids will shed sperm. In xenopus, this has never been reported, to my knowledge.

So I don't know of any data on this, but there may be people who know better than we do.

DR. ROBERTS: Dr. Denver.

DR. DENVER: No, I don't have any additional data to add.

DR. ROBERTS: Anybody else on the panel?

DR. ROBERTS: Let's go ahead on to the next one.

DR. STEEGER: 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.

DR. ROBERTS: I suspect Dr. Kloas has an idea or two on this.

Go ahead.

DR. KLOAS: I think we don't have really completely new data and not been included in the white paper. But from the last days and from some presentations we saw, we feel that you can create quite a lot of hypothesis more or less which could account for these findings presented by different groups.

I just would like to summarize a little bit which possibilities 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 195 could happen.

First, aromatase induction or effect  change in activity of aromatase could be one. Still one of the hypotheses which could work. The next one, we could also have anti androgenic effects. A third one would be as already mentioned yesterday, influence by the hypothalamus pituitary gonad axis. So more than ( inaudible)

endocrine pathway.

First possibility would be inhibition of steroidogenesis,

especially on sex reverse steroids.

We have shown very easily that if you expose animals to atrazine, at least testosterone levels went down and really pronounced.

And I think furthermore there is not completely evidence for me that you can exclude any interference with the thyroid system. There is still some data available also from the public comments. Dr.

Sheffield also he reminds me again to one paper on larval salamanders that shows the inhibition or delay of metamorphosis. I think still from the data we got and because of conduction arose ( ph) from the husbandry, it's not completely clear that there couldn't be any interference with the thyroid system.

Next week we're going to present some data that sometimes you 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 196 will not  you cannot see it really for developmental stages, but you will still have counter regulation if you have inhibitory effects on the thyroid system.

This could be counter regulated by more pronounced TSH production. This way, with this pathway we couldn't exclude also.

I'm making up everything. I'm sorry for that. But the data presented here still suggests that we have so many facilities and possibilities which could be one of the pathways or several pathways how it could work.

DR. ROBERTS: Dr. Kelley, do you have anything to add?

DR. KELLEY: Let me comment a little bit on the thyroid hormone question. Metamorphosis in xenopus is completely dependent upon thyroid hormone and requires as has been shown in a number of studies the expression of thyroid hormone receptor, which there are two which exist in a variety of isoforms.

I was not able to detect thyroid hormone receptor alpha in developing gonads. So I do not know if the developing gonad needs expression for formation, for what I call sex determination.

In fact, in that study in which thyroid hormone was blocked, the gonad proceeded to develop to the point of having spermatids.

Although, of course, frank spermatid gonia did not develop because  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 197 this is the other side of the equation, you cannot get effects of steroid hormones in xenopus unless the animal has first been exposed to thyroid hormone.

And some effects require thyroid hormone induction of prolactin.

So it is clear in xenopus that there is this strong interaction in terms of steroid hormone effects for many tissues between pituitary hormones, thyroid hormones, this is extending now to the axis, the pituitary hypothalamic gonadal axis now to the thyroid and the steroid hormones.

While this may sound like a nightmare like the ecological studies in point effect, it's an exquisitely regulated developmental system which provides then a very good assay system for a variety of endocrine perturbations.

So I agree that we can't at this point completely exclude thyroid hormone effects. I don't know about the gonads. But if there are any other effects of the agents that are being studied, we should take this into account.

DR. ROBERTS: Dr. Denver.

DR. DENVER: I don't know of any additional data that would support any alternative mechanisms, but I think it is important to at 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 198 least entertain the hypothesis that if there are gonadal effects, that they could be mediated by nonendocrine mechanisms, that is, that there could be direct effects on genes that are important for gonadal determination that may have nothing to do with hormones.

I don't think that should necessarily be ruled out.

DR. ROBERTS: It seems we didn't have a lot of specific data to offer, but the panel members did have suggestions for a number of alternative mechanisms and seemed to strongly think that those ought to be considered as well.

Are there any other comments?

Dr. Matsumura.

DR. MATSUMURA: I was intrigued at least by the fact that the HCG reversed some of those actions, but not all the way.

I would like to suggest at least that area should be followed up to see what it is.

It remind me of the Precosin ( ph) studies, insect studies. This really is part of capsulatum ( ph).

In that case, you can reverse it except that it doesn't come back to the same level because part of the function of the capsulatum is gone. It looks like that, but again, just one experiment. I'm quite sure Dr. Hayes is intrigued by that too. So we'll see what happens 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 199 then.

DR. ROBERTS: Any other comments?

Dr. LeBlanc.

DR. LEBLANC: I just want to make a point that in relation to all the possible effects that Dr. Kloas talked about with terminal hormones, the androgens, the estrogens, thyroid, hormone, that the effects need not be self exclusive, that there could be effects at the level of the hypothalamus, the pituitary that results in several effects on these terminal hormones resulting in decreased testosterone,

increased estradiol and some effect on thyroid hormone perhaps.

So they don't have to be self exclusive. There could be a common target upstream that is affecting many hormones.

DR. ROBERTS: Anything else?

Any follow up questions or clarifications needed?

DR. ROBERTS: I would like to go ahead and take question 5 and then do a break.

DR. STEEGER: With regard to specific endpoints, the agency does not have 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. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 200 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?

DR. ROBERTS: Our lead discussant on this is Dr. Green.

DR. GREEN: Witchie reported sporadic cases of hermaphroditism early  in the late 1950s.

And there is at least one study in which the prevalence of ovotestes in a controlled population of laboratory frogs has been recently described. And this is a paper by Dr. Kloas which is in press right now.

To our knowledge, however, the background rates of ovotestes in any amphibian population have not been reported.

The panel members believe the frequency of occurrence of ovotestes in normal healthy populations of amphibians is probably very low.

This is of course based on the rare occurrence and our observations of ovotestes in our own laboratory animal populations.

So without information on the background rates of ovotestes, it is not possible to assess the impact, if any, of the presence of ovotestes on anuran populations. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 201 DR. ROBERTS: Comments by other discussants?

Dr. LeBlanc.

DR. LEBLANC: No comments. I agree.

DR. KLOAS: I agree.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: Yes, I think this falls into the category of data that we had described by the EPA panel before, which was that in some cases you will only have anecdotal data.

None of us ever thought it was important to document how many ovotestes you saw in opening up thousands of frogs. There are no published data on those.

But if you will take the collective wisdom of people who have opened up thousands of frogs will tell you that Witschi reports only one case, actually, in the 50s by somebody else. And I actually have never seen one in the laboratory.

DR. ROBERTS: Is this an issue for which we need  is there reasonably clear understanding about what you mean by ovotestes?

DR. KELLEY: I have made a PDF file of the Witschi paper.

It's actually a chapter from a book. It is has a very nice picture of a mature gonad with clear testicular tissue and very well yolked up eggs. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 202 I submit that that was what I would call an ovotestis. And everything else I regard as kind of subpar and up for grabs. But when you have an ovary that has a testis and eggs, which are pretty unmistakable, that's what I would call it. And this is an important issue, the terminology issue is very important.

The question of whether the occasional oocyte in a testis is normal or not, Witschi himself in this  for you guys who don't know who Witschi is  was he German or Swiss? I believe he was Swiss.

He was Swiss, and he started working on xenopus. He brought them in a tea kettle to Bozzle ( ph). He performed almost all the early experiments with Chang and McComo ( ph) on sexual differentiation in xenopus and was a very gifted biologist, I believe.

He has a very nice picture of this in this chapter summarizing many, many years of work.

Anyway, I have given this to the EPA. I have the paper with me. And the book should be available in the libraries. It will be in the bibliography.

DR. ROBERTS: Perhaps it would be useful since I believe we're probably going to comment subsequently on the problems created by lack of terminology that we clarify in our response what we mean by ovotestes. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 203 DR. KELLEY: That's a very good suggestion.

DR. ROBERTS: Any other comments to add on this one?

Let's go to B then.

DR. STEEGER: Can the panel characterize any evidence that suggests that the presence of ovotestes in male anurans results in reproductive impairment via reductions in fertility?

DR. GREEN: To our knowledge, there are no studies that show that the presence of ovotestes in male anurans results in reproductive impairment. However, the panel recommends that feminized males be included in grow out studies for the purposes of using them in breeding experiments to test this hypothesis.

DR. ROBERTS: Other responses? Other panel members?

And by no evidence, you mean that there is no evidence, but is there any evidence that they do not? There is just no evidence?

DR. GREEN: There is no evidence either way.

DR. ROBERTS: Either way. We should make that clear in our response.

Dr. Skelly.

DR. SKELLY: This may be the most appropriate point to make this comment. I was glad to hear that Dr. Green mentioned reproductive behavior. Because I think fertility is only part of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 204 story.

I think it is going to be very important if we're interested in ecological relevancy to see what these genetically male and phenotypically, whatever they are, how they behave when they are given the opportunity to mate.

And I see three broad categories of possibilities at the population level. One is that there may very well be no effect. And I think someone else mentioned this earlier. If you remove a few males from a population of frogs out in nature  I mean, ecologists make jokes about how males are superficial all the time.

DR. KELLEY: We make jokes about that too.

DR. GREEN: I left those out.

DR. SKELLY: Thank you. It may not take very many many males to keep a population going.

This is a group of organisms for which minority of males in many populations may routinely do most of the matings.

However, it may be possible if these animals that have developmental abnormalities, if they actually behave as males but are not fertile and they convince females that they are fertilizing their eggs and leave a bunch of rotting unfertilized eggs around, that could be a serious population level effect. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 205 On the other hand, if these developmentally abnormal genotypic males actually function and act as females, you could increase the population size. You could increase viability of populations.

I don't know how that's going to work genetically. Maybe Dr.

Kelley can comment on that.

But I see those three broad categories. And I don't see any way of figuring out how to get towards the broader goals of ecological relevancy that the EPA set out without looking at this in a field context on native species.

DR. ROBERTS: Perhaps our response could include that caution.

Anything else, Dr. Gibbs and then Dr. Matsumura.

DR. GIBBS: Just another body of evidence that pertains to this particular question is that interspecies comparisons have shown that testes size and sperm production are positively correlated.

So it stands to reason that within the individual, any structure such as  over that reduce the size of testes would reduce the sperm production.

I don't know if that's too much of an extrapolation to make. But there is that correlation there.

DR. ROBERTS: Perhaps we should mention that, then, with 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 206 some appropriate citation in our response.

DR. MATSUMURA: I think it is pretty important to at least test in the lab that those individuals with the abnormality would indeed be reproductively successful or not.

But I was just thinking how would you do that. You can't do any invasive method to say which one was, really had discontinuous ovotestes.

How would you design, Dr. Green or Dr. Kelley?

DR. ROBERTS: Dr. Green.

DR. GREEN: I have not actually tested this myself, but I have been to amphibian workshops where I have seen this done.

Some amphibians are small enough that as you know you can see through them. If you hold them up to the light, you can see internal organs.

One method would be to ultrasound the animal's abdomen because there are ultrasound probes now that have been miniaturized for the purpose of use in mice. So that's a possibility that, if you are good enough, you might be able to detect gonadal structures with an ultrasound probe.

Another possibility would be minimally invasive endoscopic techniques. There are endoscopes now that have been miniaturized 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 207 for mice, again, that would be a small puncture. You could go in and look and suture the animal up. Very similar to harvesting oocytes.

They would probably recover just fine and go on. So those are two methods.

And then, of course, fancier methods would be MRI and that sort of thing, and which they now make coils that are small enough for mice, so I'm sure they would easily accommodate xenopus laevis,

which would not require sacrificing the animal, any of those three test methods.

DR. ROBERTS: Dr. Kelley.

DR. KELLEY: I'm going to make my pitch, although not entirely appropriate now, for using known ZZ individuals.

If you ran this experiment in animals where the offspring of sex converted females that had been phenotypically female because they have been grown up in estrogen, and males, all of their offspring are male, so then you know that you have nice sibs to compare to, and then you can actually run a very well controlled experiment under those conditions.