Comments from the 

NATURAL RESOURCES DEFENSE COUNCIL

Jennifer Sass, Ph.D.

FIFRA Scientific Advisory Panel

October 9-12, 2007

The Potential for Atrazine to Affect Amphibian Gonadal Development

More information and relevant documents can be found on the EPA website
at:    HYPERLINK
"http://www.epa.gov/pesticides/reregistration/atrazine/atrazine_update.h
tm#amphibian" 
www.epa.gov/pesticides/reregistration/atrazine/atrazine_update.htm#amphi
bian 

REGULATORY HISTORY

Atrazine was first registered for use in the United States in 1958. By
1991, repeated detection of atrazine in the drinking water supply
prompted EPA’s Office of Water to set the limit for the annual average
concentration in drinking water at 3 ppb –  more permissive than the
World Health Organization international drinking water guidance of 2
ppb. In 1994, EPA designated atrazine for additional regulatory scrutiny
through a process called “Special Review,” based on “concerns
regarding the carcinogenic potential of atrazine and possible risks”
from food and water.

In contrast to the U.S. approach of allowing pollution to occur until
there is scientific evidence of its risks, the European Union has a
uniform limit of 0.1 ppb for the residue of any pesticide in drinking-
and ground-water. While scientists representing Syngenta characterize
this standard as “neither health-based nor scientifically
supported”,  it appears that the E.U. generally adopts the position
that it is unhealthy to drink pesticide-contaminated water, arguably a
health-based and scientifically-supported position. Based on the
inability to keep water contamination below this level, European
regulators announced a ban on atrazine use in October, 2003, one week
before the U.S. EPA approved its continued use. 

Many agencies have identified atrazine as an endocrine disruptor,
including the United Kingdom’s Environmental Agency, the European
Union, the Oslo and Paris Commission Convention for the Protection of
the Marine Environment of the North-East Atlantic, and the State of
Illinois. 

SUMMARY OF NRDC COMMENTS

NRDC looks forward to a fair and complete review of the available
literature, with greatest consideration given to those data from robust
and well-designed studies published in the peer-reviewed scientific
literature. However, this SAP has been hamstrung from that task by the
unduly narrow charge questions and limited literature review provided to
the SAP experts.

NRDC asks that all scientific data relevant to the activities of
atrazine as an endocrine disruptor be evaluated, including mammalian,
aquatic, and mechanistic studies. However, this SAP has been hamstrung
from that task by the unduly narrow charge questions limited to
amphibian gonadal effects, and the arbitrary and unsupported criteria
used by EPA to disregard critical scientific data that would inform this
issue.

NRDC asks that the Scientific Advisory Panel provide its expert
scientific opinion as to the effects of atrazine as an endocrine
disruptor, and its possible risks to wildlife and human health.

CHARGE QUESTIONS

  HYPERLINK
"http://www.epa.gov/scipoly/sap/meetings/2007/october/charge_to_the_pane
l.pdf" 
www.epa.gov/scipoly/sap/meetings/2007/october/charge_to_the_panel.pdf 

1.	In reviewing the available laboratory and field studies, the Agency
used a number of criteria to evaluate individual investigations.
Criteria such as experimental design, test protocols, and quality
assurance information were used to evaluate the reliability of a
study’s ability to adequately assess a hypothesis that atrazine
elicits developmental effects in amphibians, and if so, the nature and
strength of associated dose-response relationships.  (a) Please provide
comments and recommendations regarding the EPA’s approach and criteria
used to evaluate the studies. (b) Given the evaluation criteria employed
by the Agency, please comment on EPA’s overall application of these
criteria to the currently available studies

NRDC response:

The 07 White Paper references study evaluation criteria in the USEPA
2003 and SAP 2003 reports. Although not specifically identified as
evaluation criteria, the  03 White Paper does discuss the parameters by
which it evaluates a specific set of studies, including some expression
of the Agency preferences for study design and data analysis (p. 62-68).
These are not clear criteria, they are overly vague, and they are often
specific to the study being reviewed. The section as presented leaves
doubt that the criteria were developed a priori and independently. 

Further, neither the 03 nor 07 White Papers develop a biological
rationale to support their evaluations. This is a major deficiency in
the Agency’s approach.  It is not at all clear why EPA failed to
develop a transparent science-based logical framework in which to
interpret the study results a priori. This should have included
identifying specific endpoints of interest, developing standards of
variability including baseline data, and identifying criteria for
appropriate statistical analyses a priori.

In addition, the White Paper applies random and arbitrary critiques of
the available data. For example, the 03 and 07 White Papers refer to the
ASTM (1998) standards for loading rates, husbandry, and water quality
parameters (03 White Paper at 10). These standards are acknowledged to
include acceptable loading rates for both static or static renewal and
flow-through conditions (03 White Paper at 62). Yet, the 03 and 07 White
Papers arbitrarily identify flow-through systems as superior,  even to
the extreme of eliminating from consideration all of the static renewal
system studies. This severe application of the Agency preference results
in its failure to consider the results from the majority of highly
relevant studies that have been published in the peer-reviewed
literature, presented at scientific meetings, and received accolades
from scientific expert (03 White Paper at 62; 07 White Paper at 111). 

Most curious about the Agency study evaluations is that there are many
instances in which the Agency lists as a deficiency its own failure to
gather sufficient information to evaluate the study (see as examples 07
White paper at 102-104; 107). Since the overwhelming majority of the
study authors were available to Agency staff, it is not clear why the
information was not simply requested.  For example, the 07 White Paper
states that of the open literature laboratory studies,
“none…reported analyses conducted on the commercial diets fed to
test animals” (07 White Paper at 107). Surely, since it was commercial
feed, these data could have been requested by the Agency at any time
during its review; failure to gather this information represents a
failure by the Agency to take seriously these relevant data. 

In a glaring inconsistency, the Agency’s failure to gather information
was not applied to all studies. The WLI/IGB studies received
considerable personal attention and interaction. Agency staff,
“conducted inspections of each of the study laboratories”, including
“extensive review of raw data collection sheets and data summary
tables by OECA and OPP personnel”, even going so far as to inspect the
lab that processed tissues and conducted histological analyses” (07
White Paper at 35). It seems that the relationship between the Agency
and the authors of these studies, both sponsored by Syngenta, was
unusually communicative. This may have biased the Agency’s evaluation
of these studies, by providing the registrant-sponsored authors with
endless abilities respond to Agency inquiries.

EPA has so severely applied its preferences (I hesitate to call them
criteria, since they are not clearly stated, were not developed a
priori, and are not supported with a biological rationale) that its
final conclusions are precariously balanced on one study, conducted and
sponsored by the registrant. 

Questions Concerning the Open Literature Studies 

2) The Agency has concluded that the information contained in the open
literature published since the 2003 SAP does not provide any additional
information that could be used to refute or confirm the hypothesis that
exposure to atrazine alone causes adverse developmental effects in
amphibian gonads. (a) Please comment on the comprehensiveness of the
Agency’s literature reviews relative to the potential effects of
atrazine alone on amphibian gonadal development. (b) Please comment on
the Agency’s evaluation of the open literature studies and the
Agency’s conclusion that the data derived from laboratory studies,
both individually and collectively, are not sufficient to refute or
confirm the hypothesis that atrazine exposure causes developmental
effects in amphibian gonads. (c) The Agency concluded that the field
studies are not adequate for assessing the hypothesis at hand. Please
comment on the Agency’s conclusion. If the SAP concludes one or more
of the field studies do provide the means to assess the hypothesis that
atrazine exposure results in effects on amphibian gonadal development,
please suggest interpretive and statistical methods that should be
employed to evaluate the data.

NRDC response:

The response to this question was submitted as comments to the Agency in
May, 2007, in response to a notice in the federal register
(EPA-HQ-OPP-2007-0204), and are supported by Northwest Coalition for
Alternatives to Pesticides, Pesticide Action Network North America,
Beyond Pesticides, and Defenders of Wildlife.

EPA scientific review failed to fully include scientific evidence of
neuroendocrine effects in amphibians associated with atrazine. Atrazine
has been shown by numerous independent laboratories to disrupt
neuroendocrine processes in amphibians (Rohr and Palmer, 2005; Hayes et
al, 2002, 2003; Larson et al, 1998). Such effects are likely to be a
contributing mechanism to observed adverse impacts on population
survival. Important scientific papers relevant to this issue that should
be included in the EPA and SAP review include but are not limited to:

Larval-stage exposure of salamaders to atrazine (40 and 400 ppb) was
associated with hyperactivity, fewer water-conserving behaviors, and
accelerated dessication 4-8 months after exposure, compared to controls
(Rohr and Palmer, 2005).

Larval salamanders exposed to atrazine (75 ppb, 250 ppb) had elevated
plasma thyroxine levels compared with controls. The lower dose treated
group (75 ppb) also had reduced levels of corticosterone, compared with
both the control and the high dose group. The low dose group developed
slower than controls, whereas the high dose group developed at the same
time but were smaller and had reduced weights compared with controls
(Larson et al, 1998).

Exposure of Xenopus laevis tadpoles during sexual differentiation to
atrazine (0, 21 ppb) for 48 hrs resulted in a significant increase in
the frequency of secondary oogonia in atrazine-exposed ovaries of
females (Tavera-Mendoza et al, 2002a), and a significant reduction in
spermatogonial cell nests and nursing cells in atrazine-exposed testis
of males (Tavera-Mendoza et al, 2002b), compared with controls. The
authors suggest that these effects are likely to impact the reproductive
fitness of exposed animals.

EPA scientific review failed to fully include scientific evidence of
long-term or permanent effects in amphibians associated with atrazine.
Atrazine exposure during early developmental stages has been shown to
adversely impact later-life outcomes, including susceptibility to
infection (Kiesecker, 2002), risk of dessication during adult-hood (Rohr
and Palmer, 2005), and long-term survival (Rohr et al, 2006). Such
effects are likely to be a contributing mechanism to observed adverse
impacts on population survival. Important scientific papers relevant to
this issue that should be included in the EPA and SAP review include but
are not limited to:

Relative to control animals, salamanders exposed during larval stages to
atrazine at 4 ppb and 40 ppb has statistically significantly reduced
survival even 14 months after exposure. These results were identified
only when the researchers accounted for toxic effects that persist or
appear after the exposure had ended (carryover effects), and the ability
of the population to mask harm to individuals by reduced competition
among survivors (density-mediated compensation) (Rohr et al, 2006).

Exposure of salamanders to atrazine (0, 40, 400 ppb) prior to
metamorphosis resulted in greater rates of dessication, even as much as
4 and 8 months after the exposure ended, with no sign of recovery,
compared with controls (Rohr and Palmer, 2005).

Wood frog tadpoles exposed to atrazine (0, 3, 30 ppb) for four weeks
were then challenged with naturally-occurring trematode infection. Frogs
that had been pre-exposed to atrazine at both low and high doses during
tadpole-stages had a dose-dependent weakened immune response (reduced
eosinophils) and increased infection rate, compared with controls
(Kiesecker, 2002).

Numerous scientific studies report a more severe response to atrazine
exposure at low concentrations (≤ 25 ppb), suggesting that routine
environmental exposures during critical windows of development may be of
the most relevant for predicting acute and chronic adverse impacts.
Important scientific papers relevant to this issue that should be
included in the EPA and SAP review include but are not limited to:

Tadpoles of four species of frogs--spring peepers (Pseudacris crucifer),
American toads (Bufo americanus), green frogs (Rana clamitans), and wood
frogs (Rana sylvatica)—were exposed during development to atrazine (0,
3, 30, 100 ppb). Survival was significantly lower for all animals
exposed to 3 ppb compared with either 30 or 100 ppb, except the late
stages of B. americanus and R. sylvatica (Storrs and Kiesecker, 2004).

Larval-stage exposure of salamaders to atrazine (0, 4, 40, 400 ppb) was
associated with hyperactivity, fewer water-conserving behaviors, and
accelerated dessication 4-8 months after exposure, compared to controls.
The greatest impacts on survival occurred at low exposure
concentrations. Relative to control animals, the salamanders exposed to
4 ppb atrazine during early life stages had significantly lower survival
421 days after cessation of exposure, when considering both the exposure
and carryover effects.  (Rohr et al, 2006).

Larval salamanders exposed to atrazine (75 ppb, 250 ppb) had elevated
plasma thyroxine levels compared with controls. The lower dose treated
group (75 ppb) also had reduced levels of corticosterone, compared with
both the control and the high dose group. The low dose group developed
slower than controls, whereas the high dose group developed at the same
time but were smaller and had reduced weights compared with controls
(Larson et al, 1998).

The severe limits placed on SAP review are likely to bias outcome. EPA
scientific review failed to include studies demonstrating adverse
endocrine effects of atrazine in mammals. Even before evidence of
hormone disruption activity had emerged in amphibians, EPA scientists
and others had been reporting that atrazine disrupts the normal
progression of sexual development in rats. Reported findings included:
dose-dependent decreased estrogen-induced surges of circulating
prolactin and luteinizing hormone levels (Cooper et al, 2000);
prostatitis in offspring of dams treated during nursing (Stoker et al,
1999);  delayed puberty in males (Stoker et al, 2000) and females (Laws
et al, 2000) treated with atrazine by gavage from weaning until puberty;
decreased sperm number and motility in adults (Kniewald et al, 2000),
and reduced testosterone production by testicular cells of juvenile rats
exposed prior to puberty.(Friedmann et al, 2002) The disruption of
endocrine pathways is thought to be the cause of observed mammary tumors
in one strain of female rats (Sprague-Dawley); while this mechanism may
be strain-specific, it is likely to have implications for risks in other
species (IARC, 1999; Stevens et al, 1994, 1998, 1999). Important
scientific papers relevant to this issue that should be included in the
EPA and SAP review include but are not limited to:

When nursing rats were treated with atrazine the male offspring
developed prostate gland inflammation (Stoker et al, 1999).

Treatment of Wistar male and female rats with atrazine from weaning
until puberty resulted in delayed sexual maturity.  In the female, oral
gavage of 50-200 mg/kg atrazine at postnatal day 22-41 delayed vaginal
opening (puberty), in a dose-dependent manner (Laws et al, 2000). In
male rats preputial separation was significantly delayed following
treatment with 12.5, 50, 100, 150, and 200 mg/kg atrazine administered
by gavage (PND 23-53) (Stoker et al, 2000). 

Atrazine reduced sperm motility in exposed Fischer rats. Animals were
treated with 60 and 120 mg/kg atrazine administered twice weekly by
intraperitoneal (i.p.) injection over a period of 60 days. The authors
report that testicular sperm number in atrazine-treated groups increased
with the treatment time due to reduced sperm motility (Kniewald et al,
2000).

Long Evans (LE) rats were exposed in utero to atrazine, followed by
challenge with the carcinogen dimethybenz[a]anthracene. 
Atrazine-exposed pups demonstrated delayed mammary bud outgrowth,
followed by an increase in multiplicity and volume of tumors after
exposure to the carcinogen, compared to non-atrazine treated controls.
In addition, the atrazine-exposed pups showed an increase in organ
pathology (adrenal nodules, pituitary foci, large ovarian cysts, lymph
node and spleen enlargement), compared with controls. (Birnbaum and
Fenton, 2003)

A mammary tumor response (mammary fibroadenomas and adenocarcinomas) has
been consistently observed in Sprague-Dawley (SD) female rats following
chronic oral dosing of atrazine and simazine at and above the maximum
tolerated dose (400 ppm; based on three 2-year studies] (IARC, 1999)

EPA scientific review failed to include scientific reports of adverse
endocrine effects of atrazine in humans. Relevant human data have been
published suggesting that atrazine has been linked to endocrine effects
in individuals exposed through their work or home environment. Important
scientific papers relevant to this issue that should be included in the
EPA and SAP review include but are not limited to:

A multi-center case-control study of fertile men in U.S. agrarian areas
reported a significant association between poor semen quality (reduced
sperm concentration and motility) and urinary atrazine metabolite levels
above the level of detection (0.1 (g/g creatinine), compared with men
from urban centers (OR=11.3, 95% CI=1.3-98.9).(Swan et al, 2003, 2006) 

It is alarming that far higher urinary metabolite levels have been
reported in male farmers who self-applied atrazine, ranging from 0.16 to
5.0 (g/g creatinine (95% CI=0.33-1.3) (Curwin et al, 2005), suggesting
that farmers that apply pesticide themselves (not professionally trained
applicators) may represent a population at increased risk of reduced
sperm quality from pesticide exposures. 

EPA scientific review failed to consider evidence of impacts of mixtures
and co-contaminants with atrazine. The EPA review is artificially
constrained to consider exposure to pesticides in isolation; this is
extremely likely to artificially underestimate risks from exposure to
toxic mixtures that commonly occur in the real world (Chevre et al,
2006; Christin et al, 2004). One assessment of multiple pesticide
exposures among men with NHL has reported a suggested superadditive
effect of atrazine in combination with the pesticides carbofuran,
diazinon, or alachlor (DeRoos et al, 2003). Similarly, the Hayes lab
recently examined the effects of atrazine in pesticide mixtures on frog
viability and metamorphosis, and reported increased mortality of
tadpoles exposed to multiple pesticides at levels that were non-lethal
when occurring individually (0.1 ppb) (Hayes et al, 2006). Exposure to
multiple pesticides simultaneously is routine for human and wildlife
populations; the USGS reported that more than 90% of the time, watershed
streams had detections of 2 or more pesticides or metabolites, and about
20% of the time they had detections of 10 or more pesticide contaminants
(USGS, 2006). EPA should broaden its charge to the SAP to include
consideration of mixtures and co-contaminants with atrazine.

Questions Concerning the DCI Study 

NRDC response:

NRDC has no specific response to these questions. The DCI studies were
perceived of as weak, confounded, and uninformative by the FIFRA SAP in
03, and nothing has altered this evaluation in the intervening time.
This, little time should be spent on them in this review.

Concluding Questions 

Please comment on the Agency’s recommendation that the current body of
data is sufficient to refute the hypothesis that atrazine by itself can
adversely affect amphibian gonadal development and that no additional
data are required to address this hypothesis.

NRDC response:

EPA asked the wrong question:	The Agency has intentionally and unfairly
hamstrung its FIFRA Scientific Advisory Panel by developing a series of
charge questions that clumsily avoid asking the relevant regulatory
question of whether or not atrazine poses a risk to wildlife and human
health, in particular through its activity as an endocrine disruptor.
NRDC asks the experts of this SAP to move beyond this limited set of
charge questions, and request that it be reconvened to review all
scientific data relevant to atrazine as an endocrine disruptor, and its
potential impacts on wildlife and human health.

EPA legal authority to regulate pesticides:	EPA regulates pesticides
under two statutes, the Federal Food, Drug, and Cosmetic Act (FFDCA), 21
U.S.C. § 346a and the Federal Fungicide, Insecticide, and Rodenticide
Act (FIFRA), 7 U.S.C. § 136 et seq. The Food Quality Protection Act of
1996 (“FQPA”) significantly amended both the FFDCA and FIFRA by
mandating that health-based and child-protective standards drive
decisions about acceptable levels of pesticide residues in food and the
environment.  FIFRA requires that pesticides must be registered to be
sold in the United States.  EPA may not register a pesticide unless the
chemical will perform its intended function without causing any
“unreasonable adverse effects on the environment.”  

The FFDCA, as amended by the FQPA, authorizes EPA to “establish or
leave in effect a tolerance for a pesticide chemical residue in or on a
food only if the Administrator determines that the tolerance is safe.”
 The term “safe” means that “there is a reasonable certainty that
no harm will result from aggregate exposure” to the pesticide,
“including all anticipated dietary exposures and all other exposures
for which there is reliable information.”  

Before EPA can establish a tolerance, the Agency shall “ensure that
there is a reasonable certainty that no harm will result to infants and
children from aggregate exposure” to the pesticide, and shall
“publish a specific determination regarding the safety of the
pesticide chemical residue for infants and children.”  In ensuring
that the statutory safety standard is met, EPA must consider available
information concerning “the special susceptibility of infants and
children,” including “neurological differences between infants and
children and adults, and effects of in utero exposure to pesticide
chemicals.”  EPA acknowledges that, when setting new tolerances under
the standard, it “must now focus explicitly on exposures and risks to
children and infants.” 

Furthermore, “an additional tenfold margin of safety for the pesticide
chemical residue and other sources of exposure shall be applied for
infants and children to take into account potential pre- and post-natal
toxicity and completeness of the data with respect to exposure and
toxicity to infants and children.”  EPA can depart from this
requirement and use a different margin of safety “only if, on the
basis of reliable data, such margin will be safe for infants and
children.”  

  Sass, JB, Colangelo A. European Union bans atrazine, while the United
States negotiates continued use. Int J Occup Environ Health, 2006
July;12:260-267. www.ijoeh.com/pfds/IJOEH_1203_Sass.pdf

 ibid
http://www.pesticideinfo.org/PCW/Detail_Chemical.jsp?Rec_Id=PC35042

 7 U.S.C. § 136a.  

 7 U.S.C. § 136a(c)(5)(C).

 Id. § 346a(b)(2)(A)(i).  

 Id.  § 346a(b)(2)(A)(ii).  

 Id. §§ 346a(b)(2)(C)(ii)(I) & (II).  

 Id. § 346a(b)(2)(C)(i)(II).  

 EPA, Fact Sheet: Protecting Children from Pesticides (Jan. 2002)
(www.epa.gov/pesticides/factsheets/kidpesticide.htm) (“The 1996 Food
Quality Protection Act set tougher standards to protect infants and
children from pesticide risks.”)

 21 U.S.C. § 346a(b)(2)(C).  

 Id. (emphasis added).  

NRDC comments to SAP on atrazine	October 07

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