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A qualitative meta-analysis reveals consistent effects of atrazine on freshwater fish and amphibians.

OBJECTIVE: The biological effects of the herbicide atrazine on freshwater vertebrates are highly controversial. In an effort to resolve the controversy, we conducted a qualitative meta-analysis on the effects of ecologically relevant atrazine concentrations on amphibian and fish survival, behavior, metamorphic traits, infections, and immune, endocrine, and reproductive systems.

Data SOURCES: We used published, peer-reviewed research and applied strict quality criteria for inclusion of studies in the meta-analysis.

DATA SYNTHESIS: We found little evidence that atrazine consistently caused direct mortality of fish or amphibians, but we found evidence that it can have indirect and sublethal effects. The relationship between atrazine concentration and timing of amphibian metamorphosis was regularly nonmonotonic, indicating that atrazine can both accelerate and delay metamorphosis. Atrazine reduced size at or near metamorphosis in 15 of 17 studies and 14 of 14 species. Atrazine elevated amphibian and fish activity in 12 of 13 studies, reduced antipredator behaviors in 6 of 7 studies, and reduced olfactory abilities for fish but not for amphibians. Atrazine was associated with a reduction in 33 of 43 immune function end points and with an increase in 13 of 16 infection end points. Atrazine altered at least one aspect of gonadal morphology in 7 of 10 studies and consistently affected gonadal function, altering spermatogenesis in 2 of 2 studies and sex hormone concentrations in 6 of 7 studies. Atrazine did not affect vitellogenin in 5 studies and increased aromatase in only 1 of 6 studies. Effects of atrazine on fish and amphibian reproductive success, sex ratios, gene frequencies, populations, and communities remain uncertain.

CONCLUSIONS: Although there is much left to learn about the effects of atrazine, we identified several consistent effects of atrazine that must be weighed against any of its benefits and the costs and benefits of alternatives to atrazine use.

KEY WORDS: aromatase, behavior, disease, gonads, immunity, metamorphosis, parasite, reproduction, testicular ovarian follicles, vitellogenin. Environ Health Perspect 118:20-32 (2010). doi:10.1289/ehp.0901164 available via http://dx.doi.org/ [Online 23 September 2009]

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The herbicide atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine) is the second most commonly used pesticide in the United States (Kiely et al. 2004) and perhaps the world (Solomon et al. 1996; van Dijk and Guicherit 1999). It is a photosynthesis inhibitor used to control certain annual broadleaf weeds, predominantly in corn but also in sorghum, sugarcane, and other crops and landscaping. The environmental risk posed by atrazine to aquatic systems is presently being reevaluated by the U.S. Environmental Protection Agency (U.S. EPA 2003, 2007). One of the challenges in evaluating the safety of atrazine has been that its biological effects are highly controversial, and much of the debate in the literature has been targeted at its effects on freshwater vertebrates (Hayes 2004; Renner 2004).

There have been four reviews on the biological effects of atrazine, all of which were funded by the corporation that produced or produces this chemical (Giddings et al. 2005; Huber 1993; Solomon et al. 1996, 2008). However, none of the past reviews used a meta-analytical approach to identify generalities in responses to atrazine exposure. Meta-analysis, as paraphrased from the U.S. EPA, is the systematic analysis of studies examining similar end points to draw general conclusions, develop support for hypotheses, and/or produce an estimate of overall effects (U.S. EPA 2009a). This sort of weight-of-evidence approach would provide directional hypotheses for future work on atrazine. Furthermore, it would offer invaluable information to regulatory agencies on general and expected impacts of atrazine on freshwater vertebrates that might help resolve much of the controversy surrounding atrazine. Given the lack of a meta-analytical assessment and the potential importance of any atrazine effects, we set out to conduct an objective, qualitative meta-analysis on the effects of atrazine on amphibian and fish survival, behavior, metamorphic traits, and immune, endocrine, and reproductive systems.

Atrazine Persistence, Transport, and Exposure

To place the results of this meta-analysis within an ecologic context and to evaluate the relevance of studied atrazine concentrations and exposure regimes, we briefly discuss the fate, transport, and field concentrations of atrazine. Atrazine is persistent relative to most current-use pesticides. Ciba-Giegy Corporation (1994), the company that previously produced atrazine, reported no detectable change in atrazine concentration after 30 days in hydrolysis studies conducted at pHs between 5 and 7, and an aqueous photolysis half-life of 335 days under natural light and a neutral pH. Half-lives from field and mesocosm studies are variable because degradation can depend on various environmental conditions. Nevertheless, several field and mesocosm studies report half-lives > 3 months (e.g., de Noyelles et al. 1989; Klaassen and Kadoum 1979).

Atrazine is also relatively mobile--regularly entering water bodies through runoff--and concentrations in surface waters often peak after rains. Several researchers have suggested that atrazine can be transported 1,000 km aerially (van Dijk and Guicherit 1999). Indeed, atrazine has been found regularly in surface waters and precipitation, great distances from where it is used, such as above the Arctic Circle, albeit at low concentrations (van Dijk and Guicherit 1999).

Wet deposition of atrazine might also be important in some areas. In a review on atmospheric dispersion of current-use pesticides, van Dijk and Guicherit (1999) reported more studies detecting atrazine in rain or air (from European and U.S. sites) than any other current-use pesticide. The maximum reported wet deposition of atrazine is 154 [micro]g/L from Iowa precipitation (Hatfield et al. 1996). Wet deposition > 1 [micro]g/L was reported regularly in North America and Europe between 1980 and the early 1990s (reviewed by van Dijk and Guicherit 1999). As a reference point, the maximum contaminant level for drinking water set by the U.S. EPA is 3 [micro]g/L atrazine (U.S. EPA 2002).

Surface water is likely the primary source of atrazine exposure for freshwater vertebrates. Data on atrazine concentrations in surface water, however, are more abundant for lotic (streams and rivers) than lentic (lakes, ponds, wetlands, ditches) systems (Solomon et al. 2008), primarily because of the extensive stream monitoring conducted by the U.S. Geological Survey National Water Quality Assessment project and Syngenta Crop Protection, Inc. (U.S. EPA 2007). In lentic systems, water is not replenished as it is in lotic systems, and chemicals can concentrate as lentic systems dry. Maximum reported concentrations in lentic systems are often 2.5-10 times higher than maximum concentrations in lotic systems (Baker and Laflen 1979; Edwards et al. 1997; Evans and Duseja 1973; Frank et al. 1990; Kadoum and Mock 1978; Kolpin et al. 1997). Additionally, many amphibians develop in ephemeral agricultural ponds that might receive and concentrate atrazine (Knutson et al. 2004).

Given the limited data on atrazine concentrations in lentic systems, the expected (or estimated) environmental concentration (EEC) is a reasonable alternative for estimating concentrations to which aquatic organisms are likely to be exposed. GENEEC2 software (U.S. EPA 2009b) calculates standardized EECs used by the U.S. EPA for Tier-1 chemical risk screening. EECs are important because chemical registration decisions entail comparing lowest observable effect concentrations (LOECs) to EECs to determine whether higher-level modeling is warranted. Hence, effects of a chemical near or below the EEC can affect the decision to approve its use.

For present atrazine application rates, EECs based on GENEEC2 software are typically near 100 [micro]g/L but can be higher for some crops. However, the recommended application rates (~ 2 lb active ingredient/acre) are now two to four times less than they were in the early 1990s (~ 8 lb active ingredient/acre). Hence, at the time of atrazine registration, LOECs near or below 500 [micro]g/L, a feasible EEC at the time, might have triggered Tier-2 testing and might have raised concerns about the safety of atrazine that could have compromised its registration. Given both past and present-day conditions, the lack of thorough data on atrazine concentrations in lentic systems, and the common use of agricultural ponds, ditches, and wetlands by amphibians and fish, we suggest that concentrations near or below historical EECs ([less than or equal to] 500 [micro]g/L) are ecologically relevant when considering the findings of this meta-analysis. This is arguably conservative given that atrazine concentrations > 500 [micro]g/L have been regularly recorded in agricultural ponds and ditches (Baker and Laflen 1979; Edwards et al. 1997; Evans and Duseja 1973; Frank et al. 1990; Kadoum and Mock 1978; Kolpin et al. 1997).

Methods

We selected studies for this meta-analysis beginning with those cited by Solomon et al. (2008), the most recent review of atrazine effects on amphibians and fish. We then supplemented these studies by searching Web of Science (Thomson Reuters, New York, NY) to identify studies that might have been missed by Solomon et al. (2008). The search terms were "atrazine" combined with either "amphibian*" or "fish*".

Selection criteria for inclusion of studies in meta-analyses can affect the conclusions that are drawn (Englund et al. 1999). Hence, we excluded from this meta-analysis studies that had substantial contamination in control treatments or reference sites (unless a regression approach was taken to analyze the data); no presentation of statistics and within-group variance estimates; considerable inconsistencies that could affect the biological conclusions; spatial confounders associated with atrazine treatments; pseudoreplication; or other considerable flaws in experimental design. We evaluated whether the exclusion of these studies changed the conclusion of the meta-analysis for each end point (Englund et al. 1999). For the 15 response variables, the inclusion of studies that did not meet our criteria never altered the conclusions of our meta-analyses, and in some cases including these studies actually strengthened the conclusions. Because of this and space limitations, studies that were excluded and why, as well as the directions of effects in these studies, are provided in Supplemental Material available online (doi:10.1289/ehp.0901l64.Sl via http://dx.doi.org/).

To conduct a qualitative meta-analysis, we chose to use the vote-counting method--in which we tallied the number of studies that did and did not detect effects of atrazine--for several reasons. We quantified the effects of atrazine on 15 response variables from > 125 studies, and vote counting, the simplest approach to meta-analyses, made it feasible to manage this complexity. Vote counting also facilitates identifying response variables that might warrant more sophisticated metaanalyses based on effect sizes. Finally, we chose vote counting because it is a conservative approach, biasing results toward detecting no overall effect (Gurevitch and Hedges 1993). Because most atrazine studies conducted analysis of variance to test for dose responses, despite regression analyses providing much greater statistical power (Cottingham et al. 2005), we include studies that had substantial trends for effects of atrazine (i.e., a nonsignificant increase or decrease) with studies that reported statistically significant effects ([alpha] = 0.05). Our criteria for a trend were a clear dose response, a probability value < 0.1, or authors interpreting their nonsignificant result as a trend. Never did including trends change our conclusions of the meta-analysis.

Results and Discussion

Effects of atrazine on fish and amphibian survival. Many researchers have evaluated the effects of atrazine on fish (reviewed by Giddings et al. 2005; Huber 1993; Solomon et al. 1996) and amphibian survival (e.g., Allran and Karasov 2000, 2001; Brodeur et al. 2009; Diana et al. 2000; Freeman and Rayburn 2005; Rohr et al. 2003, 2004, 2006b). Our general conclusions from these studies are consistent with the conclusions of authors from previous atrazine reviews (Giddings et al. 2005; Huber 1993; Solomon et al. 1996, 2008): There is not consistent, published evidence that ecologically relevant concentrations of atrazine are directly toxic to fish or amphibians. There are, however, some important exceptions (e.g., Alvarez and Fuiman 2005; Rohr et al. 2006b, 2008c; Storrs and Kiesecker 2004). Because our conclusions are consistent with previous reviews, we did not conduct a meta-analysis on survival.

Effects of atrazine on fish and amphibian development and growth. Background on metamorphosis. A basic understanding of four concepts about amphibian metamorphosis is necessary to interpret the effects of any chemical on time to, or size at, metamorphosis. First, amphibians must reach a minimum size before they can metamorphose (Wilbur and Collins 1973). Second, once they reach this size, they can accelerate development and metamorphose earlier if they are in a stressful environment or metamorphose later if they are in a good environment (Wilbur and Collins 1973). Last, metamorphosis is predominantly controlled by corticosterone and thyroid hormones (Larson et al. 1998); thus endocrine system disruption can lead to inappropriately timed metamorphosis.

These important facts have profound implications for understanding the effects of pollution on metamorphic traits. For example, imagine that an amphibian shunts energy away from growth to detoxify a chemical and, as a result, reaches the minimum size for metamorphosis 5 days later than amphibians not exposed to the chemical. Once this amphibian reaches the minimum size for metamorphosis, it might accelerate its developmental rate and metamorphose 5 days earlier to get out of the stressful chemical environment. In this example, there is no net effect of the chemical on time to metamorphosis despite inarguably having considerable effects on energy use, growth, and development (Larson et al. 1998). A single chemical could delay, accelerate, or have no effect on timing of metamorphosis, depending on chemical type and concentration.

This example highlights four points. First, a lack of an effect of a chemical on timing of metamorphosis does not mean there was no effect on developmental rate or hormones that drive metamorphosis, as concluded by Solomon et al. (2008). Second, nonmonotonic dose responses in the timing of metamorphosis are expected and are likely common. This is because there are several processes occurring (detoxification, growth, and modulation of developmental timing) that can be temporally offset and that likely have different (and potentially opposite) functional responses to the same chemical. Third, timing of metamorphosis in response to chemicals should be highly variable. This variation should not be interpreted as inconsistencies across studies (e.g., Solomon et al. 2008), because the complexity of metamorphosis is expected to induce extreme variability. Finally, unlike timing of metamorphosis, size at metamorphosis is expected to monotonically decrease with increasing chemical concentration across species and studies (controlling for time to metamorphosis) because energy used for detoxification is often taken away from that used for growth and development.

Effects on metamorphic traits. Our qualitative meta-analysis on the effects of atrazine on metamorphic traits is consistent with the predictions described above. Twelve of 21 studies found significant effects of atrazine on metamorphic timing, with 7 showing an increase and 7 showing a decrease in time to metamorphosis; thus, as predicted, the direction of the effect was not consistent across studies (Table 1). Seven of the 21 studies had either clear nonmonotonic dose responses or were possibly nonmonotonic (Table 1). These results are consistent with the high variability and high probability of nonmonotonicity expected for this end point.
Table 1. Summary of the results for the effects of atrazine on the
developmental rate and size at or near metamorphosis for amphibians.

 Net effect developmental rate

Taxon, species Effect Conc where Nonmonotonic Excluded
 direction effect was dose from meta
 observed response analysis?
 ([mu]g/L)

Frog

Bufo americanus ND - NA No

B. americanus [down 250, 500, Yes No
 arrow] (c) 1,000

B. americanus ND - No No

Rhinella arenarum [up arrow] 100, 1,000, Yes No
 at 100 and 5,000
 1,000,
 [down
 arrow] at
 5,000

Hyla chrysoscelis [up arrow] 192 No No

Hyla versicolor ND (e) - Possibly No

H. versicolor ND - NA No

Rana clamitans [down 10 Yes No
 arrow]

Rana pipiens Unknown - No Yes
 (g)

R. pipiens ND - NA No

R. pipiens ND - NA No

Rana ND - NA No
sphenocephala

R. sphenocephala ND - NA No

Rana sylvatica No data - No data Yes

Xenopus laevis No data - No data Yes

X. laevis ND - NA No

X. laevis [up arrow] 100, 450, No No
 800

X. laevis Unknown - Unkown Yes
 (l), (m),
 (n)

X. laevis [down No Conc No No
 arrow] differed from
 detected by controls
 regression

X. laevis No data - NA Yes

Salamander

Ambystoma [up arrow] 40, 400 No No
barbouri

Ambystoma [up arrow] 184 No No
macrodactylum

Ambystoma [up arrow] 16 vs. 1.6, Possibly; no No
tigrinum but not vs. data
 0

Ambystoma [up arrow] 250 Yes No
maculatum and [down
 arrow] (q)

A. maculatum [down 200 NA No
 arrow]

Ambystoma texanum [down 200 NA No
 arrow]

 Size at or near metamorphosis

Taxon, Effect Conc Nonmonotonic Excluded from
species direction where dose meta-analysis?
 effect response
 was
 observed
 ([mu]
 g/L)

Frog

Bufo [down arrow] 200 NA No
americanus

B. americanus [down arrow] No Conc No No
 (d) differed
 from
 controls

B. americanus No data - No data Yes

Rhinella No data - No data Yes
arenarum

Hyla No data - No data Yes
chrysoscelis

Hyla [down arrow] 200, No No
versicolor 2,000

H. versicolor No data - No data Yes

Rana [down arrow] 10 Yes No
clamitans

Rana pipiens [down arrow] Not No No
 (h) tested

R. pipiens [down arrow] 0.1 NA No

R. pipiens ND - NA No

Rana [down arrow] 200 NA No
sphenocephala

R. No data - No data Yes
sphenocephala

Rana [down arrow] Unknown; NA No
sylvatica conc in
 ponds not
 provided

Xenopus ND - No No
laevis

X. laevis No data - No data Yes

X. laevis Unknown (k) - Unknown Yes

X. laevis [down arrow] 0.01, 1, Possibly No
 (o) 100

X. laevis [down arrow] 20, 40, No No
 80, 160,
 320

X. laevis [down arrow] 400 NA No

Salamander

Ambystoma [down arrow] 400 No No
barbouri

Ambystoma [down arrow] 184 No No
macrodactylum

Ambystoma ND; trend - No data No
tigrinum toward [down
 arrow] (p)

Ambystoma [down arrow] 250 No No
maculatum

A. maculatum [down arrow] 200 NA No

Ambystoma [down arrow] 200 NA No
texanum

Taxon, Conc Atrazine Experiment Exposure Reference
species tested grade type duration
 ([mu]
 g/L)

Frog

Bufo 200 Comm; PE [less Boone and
americanus Aatrex than or James 2003
 (a) equal to] (b)
 88 days

B. americanus 250, 500, Tech SR 3 weeks Freeman et.
 1,000, al. 2005
 5,000,
 10,000

B. americanus 1,3,30 Tech SR LTM Storrs and
 Semlitsch
 2008

Rhinella 100, Tech SR LTM Brodeur et
arenarum 1,000, al. 2009
 5.000

Hyla 96, 192 Tech PE, two [less Briston and
chrysoscelis pulses than or Threlkeld
 equal to] 1998 (h)
 129 days

Hyla 20, 200, Tech PE Mean of Diana et
versicolor 2,000 13 days al. 2000 (f)

H. versicolor 1, 3, 30 Tech SR LTM Storrs and
 Semlitsch
 2008

Rana 10,25 Tech SR [less Coady et
clamitans than or al. 2004 (f)
 equal to]
 273 days

Rana pipiens 20, 200 Tech SR LTM Allran and
 Karasov 2000

R. pipiens 0.1 Tech SR LTM Hayes et
 al. 2006

R. pipiens 5 Not SR ETM, Bridges et
 provided [less al. 2004
 than or (j)
 equal to]
 45 days

Rana 200 Comm; PE [less Boone and
sphenocephala Aatrex than or James 2003
 (a) equal to] (b)
 57 days

R. 1, 3, 30 Tech SR LTM Storrs and
sphenocephala Semlitsch
 2008

Rana 3, 30 Comm FS Unknown Kiesecker
sylvatica 2002 (f)

Xenopus 1, 10, Tech SR Mean of Carr et al.
laevis 25 56 days 2003

X. laevis 1, 10, Tech SR ETM Du Preez et
 25 al. 2008

X. laevis 100,450, Tech SR 4 weeks Freeman and
 800 Rayburn
 2005

X. laevis 0.01, Tech SR [less Kloas et
 0.1, 1.0, than or al. 2009
 25, and equal to]
 100 75 days

X. laevis 20, 40, Tech SR LTM Sullivan
 80, 160, and Spence
 320 2003

X. laevis 400 Tech SR LTM Langerveld
 et al.
 2009

Salamander

Ambystoma 4, 40, Tech SR Mean of Rohr et al.
barbouri 400 52 days 2004
 exposure

Ambystoma 1.84, Tech SR 30 days Forson and
macrodactylum 18.4, Storfer
 184 2006a

Ambystoma 1.6, 16. Tech SR LTM Forson and
tigrinum 160 Storfer
 2006b

Ambystoma 75, 250 Tech SR 86 days Larson et
maculatum al. 1998

A. maculatum 200 Comm; PE [less Boone and
 Aatrex than or James 2003
 (a) equal to] (e)
 57 days

Ambystoma 200 Comm; PE [less Boone and
texanum Aatrex than or James 2003
 (a) equal to] (b), (r)
 88 days

Abbreviations: [down arrow], decreased; [up arrow], increased; Comm,
commercial; Conc, concentration; ETM, embryo to metamorphosis, or
earlier (cases where amphibians metamorphosed before atrazine exposure
ceased); FS, field survey; LTM, early larvae to metamorphosis; NA, not
applicable (used when there were too few concentrations to evaluate
nonmonotonicity); ND, not detected; PE, pulse experiment; SR, static
renewal experiment; Tech, technical, Excluded studies are listed in
Supplemental Material, Table S1 (doi:10.1289/ehp.0901164.S1).

(a) Aatrex is 59.2% inactive ingredients. (b) Community-level study.
(c) Authors show that atrazine modifies the thyroid axis for both X.
laevis and B. americanus. (d) All five atrazine concentrations tested
reduced frog size relative to controls, but no within-group variance
estimates were provided. (e) 200 ppb developed faster than 2, 000 ppb.
(f) Only a single egg mass; might not reflect general response. (g) Use
only 50% of the metamorphs in the time to metamorphosis analysis
without describing how they selected this subset of metamorphs or why
they used only 50% for time to metamorphosis but 100% of the metamorphs
for size at metamorphosis. (h) Authors report an interaction between
atrazine and time for frog length, indicating that control animals were
larger than those exposed to atrazine by the end of the experiment. (i)
Tested as a mixture of 5 [micro]/L atrazine and 5 [micro]/L carbaryl.
(j) Compared ponds with and without atrazine; effects might be due to
other factors. (k) Frogs lose weight at metamorphosis, thus mass
measurements were confounded by grouping tadpole and metamorph weights.
(l) Provide no within-group variance estimate. (m) No statistics
provided but conclude that there was no effect of atrazine. (n) Graphs
for developmental rate through time are indiscernible. (o) Detected
effects in only one of two experiments and for females only. (p) [rho]
= 0.080 for regression analysis, one-tailed test. (q) Results depended
on developmental stage; authors showed that atrazine modifies thyroxine
and corticosterone hormones. (r) Results depended on drying
conditions.


Only two studies explicitly quantified the effects of atrazine on both thyroid hormones and timing of metamorphosis, and both showed significant nonmonotonic effects (Freeman et al. 2005; Larson et al. 1998) (Table 1). Further, Larson et al. (1998) revealed delays in growth and development early in life followed by accelerated development and early metamorphosis once a critical size for metamorphosis was reached. Additional studies that quantify the impacts of atrazine on thyroid hormones, corticosteroid hormones, and changes in growth and development through time are needed.

In contrast to timing of metamorphosis, size at metamorphosis shows a clear dose-dependent response to atrazine exposure (Table 1). Fifteen of 17 studies and 14 of 14 species showed significant reductions, or considerable trends toward reductions, in amphibian size at metamorphosis associated with atrazine exposure, and all of these studies reported effects at ecologically relevant concentrations based on the above criteria (Table 1). Similar growth reductions have been observed in fish (Alvarez and Fuiman 2005; McCarthy and Fuiman 2008). Atrazine consistently reduced amphibian size, which is likely to have adverse effects on amphibian populations because smaller metamorphs generally have lower terrestrial survival, lower lifetime reproduction, and compromised immune function (Carey et al. 1999; Scott 1994; Smith 1987). However, population-level effects of atrazine have not been empirically tested for in nature and thus need to be evaluated explicitly.

Effects of atrazine on fish and amphibian behavior. Effects on locomotor activity. Twelve of 13 studies reported that atrazine exposure increased amphibian or fish locomotor activity over at least a portion of the concentration gradient tested (Table 2). Interestingly, 4 of 5 studies on fish, but none of the studies on amphibians, reported nonmonotonic dose responses. For fish, low concentrations of atrazine stimulated hyperactivity, but higher concentrations caused reductions in activity. For amphibians, hyperactivity was typically observed at the concentrations tested, but higher concentrations would likely eventually become toxic and reduce activity. All studies conducted on fish detected effects of atrazine on locomotor activity, whereas 88% of the studies on amphibians detected atrazine' effects (Table 2).
Table 2. Summary of the results for the effects of atrazine on fish and
amphibian behaviors.

Taxon, species End point Effect Conc where
 direction effect was
 observed
 ([mu]g/L)

Locomotor activity
Salamander

A. barbouri Locomotor activity [up arrow] 400
 after disturbance

A. barbouri Locomotor activity [up arrow] 400
 after disturbance

A. barbouri Locomotor activity [up arrow] 40, 400
 after disturbance

A. barbouri Locomotor activity [up arrow] 400

Frog

R. sylvatica Locomotor activity [up arrow] Two doses of
 25 separated
 by 2 weeks

B. americanus Locomotor activity ND -

X. laevis Abnormal swimming [up arrow] 25

H. chrysoscelis Burst swimming [up arrow] Positive dose
 response

Fish

Curassius auratus Burst swimming [up arrow] 0.5, 50

C. auratus Burst swimming [up arrow] 0.1, 1, 10

Oncorhynchus Locomotor activity [up arrow] 1,10
mykiss

Lepomis cyanellus Locomotor activity [up arrow]/ 400 but not
 [down arrow] 800

Larval Sciaenops Locomotor activity [up arrow] 40,80
ocellatus (b) and abnormal
 swimming

Predation-related
risk reduction

Salamander

A. barbouri Refuge use [down arrow], None
 detected with
 regression

A. barbouri Refuge use [down arrow] 400

Frog

R. sylvatica Refuge use [down arrow] Two doses of
 25 separated
 by 2 weeks

C. auratus Grouping [down arrow] 5, 50

C. auratus Sheltering in [down arrow] 5
 presence of predator
 cue

C. auratus Grouping in presence [down arrow] 5
 of predator cue

Larval S. Predation rates ND 40, 80
ocellatus (b)

Olfaction

Frog

B. americanus Chemical detection of ND -
 food, parasites, and
 predator cues

Salamander

Plethodon Chemical detection of ND -
shermani food or sex
 pheromones

P. shermani Activated olfactory ND -
 neurons

Fish

Salmo salar Olfactory response [down arrow] 2, 5, 10, 20
 (electroolfactogram)

S. salar Olfactory response [down arrow] 1
 (electroolfactogram)

S. salar Olfactory response [down arrow] 0.5, 1
 (electroolfactogram)

O. mykiss Olfactory response [down arrow] 10, 100
 (electroolfactogram)

O. mykiss Response ratio to [down arrow] 10
 L-histidine

Other behaviors

Salamander

A. barbouri Water-conserving [down arrow] 40, 400
 behaviors

Taxon, species Conc tested Nonmonotonic does Atrazine
 ([mu]g/L) respones grade

Locomotor activity
Salamander

A. barbouri 4, 40, 400 No Tech

A. barbouri 4, 40, 400 No Tech

A. barbouri 4, 40, 400 No Tech

A. barbouri 40, 400, 800 No Tech

Frog

R. sylvatica Two doses of 25 NA Tech
 separated by 2
 weeks

B. americanus 201 MA Tech

X. laevis 1, 10, 25 No Tech

H. chrysoscelis 96, 192 No Tech

Fish

Curassius auratus 0.5, 5, 50 Possibly Tech

C. auratus 0.1, 1, 10 Possibly Tech

Oncorhynchus mykiss 1, 10, 100 Yes Tech

Lepomis cyanellus 40, 400, 800 Yes, only in Tech
 presence of
 natural prey

Larval Sciaenops 40, 80 No Tech
ocellatus (b)

Predation-related risk
reduction

Salamander

A. barbouri 4, 40, 400 No Tech

A. barbouri 4, 40, 400 Mo Tech

Frog

R. sylvatica Two doses of 25 NA Tech
 separated by 2
 weeks

C. auratus 0.5, 5, 50 No Tech

C. auratus 0.5, 5, 50 Possibly Tech

C. auratus 0.5, 5,50 Possibly Tech

Larval S. ocellatus 40, 80 No Tech
(b)

Olfaction

Frog

B. americanus 201 NA Tech

Salamander

Plethodon shermani 300 NA Tech

P. shermani 700 NA Tech

Fish

Salmo salar 0.1, 1, 2, 5, 10, No Tech
 20

S. salar 0.5, 1 No Tech

S. salar 0.5, 1 No Tech

O. mykiss 1, 10, 100 No Tech

O. mykiss 1, 10, 100 Possibly Tech

Other behaviors

Salamander

A. barbouri 4, 40, 400 No Tech

Taxon, species Experiment Exposure Reference
 type duration

Locomotor activity
Salamander

A. barbouri SR 37 days Rohr et al.2003

A. barbouri SR Mean of 52 Rohr et al. 2004
 days; LTM

A. barbouri SR Mean of 47 Rohr and Palmer 2005
 days; LTM

A. barbouri PE 4 days Rohr et al.
 (unpublished data)

Frog

R. sylvatica PE 1 month Rohr and Crumrine
 2005 (a)

B. americanus PE 4 days Rohr et al. 2009

X. laevis SR Mean of 56 Carretal. 2003
 days, LTM

H. chrysoscelis PE, two [less than Briston and Threlkeld
 pulses or equal to] 1998
 129 days,
 LTM

Fish

Curassius auratus PE 1 day Sagiio and Tijasse
 1998

C. auratus PE 1 day Saglio and Tijasse
 1998

Oncorhynchus mykiss PE 30 min Tierney et al. 2007

Lepomis cyanellus PP 4 days Rohr et al.
 (unpublished data)

Larval Sciaenops PP 72 hr Alvarez and Fuiman
ocellatus (b) 2005

Predation-related risk
reduction

Salamander

A. barbouri SR 37 days Rohr et al. 2003

A. barbouri SR Mean of 52 Rohr et al. 2004
 days, LTM

Frog

R. sylvatica PE, two 1 month Rohr and Crumrine
 pulses 2005 (a)

C. auratus PE 1 day Saglio and Tijasse
 1998

C. auratus PE 1 day Saglio and Tijasse
 1998

C. auratus FP 1 day Saglio and Tijasse
 1998

Larval S. ocellatus PE 72 hr Alvarez and Fuiman
(b) 2005

Olfaction

Frog

B. americanus PE 4 days Rohr et al. 2009

Salamander

Plethodon shermani SR 28 days Lanzel 2008

P. shermani SR 28 days Lanzel 2008

Fish

Salmo salar PE 30 min Moore and Waring 1998

S. salar PE 30 min Moore and Lower 2001

S. salar PE 30 min Moore and Lower 2001
 (c)

O. mykiss PP 30 min Tierney et al. 2007

O. mykiss PE 30 min Tierney et al. 2007

Other behaviors

Salamander

A. barbouri SR Mean of 52 Rohr and Palmer 2005
 days; LTM (d)

Abbreviations: [down arrow], decreased; [up arrow], increased; Cone,
concentration; LTM, early larvae to metamorphosis; NA, not applicable
(used when there were too few concentrations to evaluate
nonmonotonicity); ND, none detected; cone, concentration; tech,
technical; PE, pulse experiment; SR, static renewal experiment; Tech,
technical. Excluded studies are listed Supplemental Material, Table S1
ldoi:10.l289/ehp.090l164.S1).

(a) Community-level study. (b)Larval red drum are often found in
freshwater, so they were included in this meta-analysis. (c) Mixture of
0.5:0.5 and 1.0: 1.0 atrazine and simazine; thus, total concentration
of triazine was 1 and 2 ppb, respectively. (d) Increased salamander
water loss and thus desiccation risk.


The effects of atrazine on amphibian and fish locomotor activity are consistent with atrazine-induced changes in locomotor activity in mammals. Atrazine seems to cause hyperactivity in mammals by competing with receptors for the inhibitory neurotransmitter gamma-aminobutyric acid, by altering monoamine turnover, and through neurotoxicity of the dopaminergic system (Das et al. 2001; Rodriguez et al. 2005). One study showed that atrazine has similar effects on the nervous system of Ranid frogs (Papaefthimiou et al. 2003), but additional studies are needed that evaluate the mechanisms responsible for atrazine-induced activity changes in fish and amphibians.

Effects on antipredator behaviors. Six of 7 studies reported that atrazine decreased amphibian and fish behaviors associated with predation-related risk reduction (Table 2). Reduced predation avoidance behaviors can increase predation risk, whereas increased hyperactivity should increase encounter rates with predators (Skelly 1994). Hence, reduced risk-reduction behaviors coupled with hyperactivity are expected to increase predation. However, there are no published studies on the effects of atrazine on predator-prey relationships of which we are aware. Given that atrazine might have effects on both predators and prey, the effects of atrazine on predator-prey interactions are difficult to predict without additional studies.

Effects on olfaction. Five of 5 studies reported that atrazine exposure reduced olfactory sensitivity of fish in a dose-dependent manner (Table 2). In contrast, 3 of 3 studies on amphibians detected no effects of atrazine on olfaction at much higher concentrations than were tested on fish (Table 2). One study on amphibians stained activated olfactory neurons with agmatine and found no difference in the stimulation of olfactory neurons between atrazine-treated and control animals (Lanzel 2008).

Effects on other behaviors. One study showed that atrazine reduced amphibian water-conserving behaviors, which increased their rate of water loss (Rohr and Palmer 2005) (Table 2). Interestingly, both the hyperactivity and the reduced water-conserving behaviors occurred hundreds of days after atrazine exposure had ceased; there was no evidence that these end points recovered from atrazine exposure, suggesting permanent effects (Rohr and Palmer 2005). Amphibians are extremely susceptible to desiccation; thus atrazine-induced changes in water conserving behaviors would be expected to increase mortality risk.

Effects of atrazine on fish and amphibian immunity and infections. Effects on immunity. Our qualitative meta-analysis revealed that atrazine exposure consistently reduced immune functioning of fish and amphibians, with 16 of 18 studies finding effects at ecologically relevant concentrations. However, many of the end points (16 of 39) were from studies where atrazine was tested as part of a mixture of pesticides, and thus the effects of atrazine were not isolated (Table 3). Nevertheless, atrazine exposure--alone (21 of 27 end points) or in a pesticide mixture (12 of 16 end points)--was associated with reduced immune functioning, resulting in an overall reduction in 77% (33 of 43) of the quantified fish and amphibian immune end points (including trends for a decrease) (Table 3). These results are somewhat conservative because in one study multiple genes associated with immunity were significantly down-regulated (Langerveld et al. 2009), but they were counted as a single end point (Table 3).
Table 3. Summary of the results for the effects of atrazine, through
water column exposure, on fish and amphibian immunity.

Taxon, species End point Effect Conc where
 direction effect was
 observed
 ([mu]g/L)

Salamander

A. tigrinum No. of peripheral [down arrow] 16, 160
 leukocytes

Frog

R pipiens Splenocyte ND -
 viability

R pipiens No. of splenocytes [down arrow] if 210
 using
 appropriate
 one-tailed
 test

R pipiens No. of phagocytic [down arrow] 210
 splenocytes postinfection

R pipiens T cell [down arrow] in 2.1, 21, 210
 proliferation presence of
 mitogens

R pipiens T cell [down arrow] in 2.1, 21, 210
 proliferation absence of
 mitogens

R pipiens Absolute no. of [down arrow] 2.1, 21, 210
 phagocytic cells in
 spleen

R pipiens No. of thymic [up arrow] 0.1
 plaques indicating
 reduced immune
 capacity

R pipiens No. of hemolytic [down arrow] 1, 10
 plaques
 representing
 antibody secreting
 B cells

R. pipiens No. of lymphocyte ND -
 from spleen

R pipiens No. of white blood [down arrow] 0.01 to 10
 cells

R pipiens No. of highly [down arrow] 0.01 to 10
 phagocytic cells

X. laevis Splenocyte ND -
 viability

X. laevis Splenocyte [down arrow] 210, 2100
 cellularity

X. laevis Relative no. of [up arrow] 21, 210, 2, 100
 phagocytic cells

X. laevis in spleen Absolute [down arrow] 210, 2, 100
 no. of phagocytic
 cells

X. laevis in spleen Tcell ND -
 proliferation

X. laevis Downregulation of [down arrow] 400
 several genes
 involved in skin
 peptide defense

X. laevis Downregulation of [down arrow] 400
 several genes
 involved in blood
 cell function

R. sylvatica No. of eosinophil [down arrow] 3, 30
 from circulating
 blood

R pipiens No. of [down arrow] < 1 Do not
 melano-macrophages know maximum
 from liver

Rana paulustris No. of [down arrow] concentration
 melano-macrophages 117
 from liver

R paulustris No. of eosinophil ND, trend 117
 from liver toward
 decrease;
 p=0.10

R clamitans No. of eosinophil [down arrow] 117
 from liver

R clamitans No. of ND 117
 melano-macrophages
 from liver

Fish

C. auratus No. of superoxide [up arrow] 4 42
 radical from and 8 weeks;
 macrophages of indicator of
 spleen and kidney oxidative
 stress

C. auratus Plasma lysozyme [up arrow] at 8 42
 activity and 12 weeks,
 argued as a
 reduction in
 resistance to
 infection

C. auratus Antibody titers [down arrow] 42
 against Aeromonas
 hydrophila

C. auratus Antioxidant enzyme [down arrow] at 42
 in spleen 4, 8, and 12
 (superoxide weeks
 dismutase)

Galaxias Leucocrit [down arrow] 3, 50
macuiatus

O. mykiss Proliferative [down arrow] > 5,000
 ability of
 circulating T
 lymphocytes (ConA)

O. mykiss Proliferative [down arrow] > 5,000
 ability of
 circulating B
 lymphocytes (LPS)

O. mykiss Respiratory burst [down arrow] > 2,500
 activity of
 circulating
 phagocytes

Liza ramada and Macrophage quality [down arrow] 25-280
Liza aurata (cells
 degenerated)

L ramada and L Melanomacrophage [up arrow] 25-280
aurata centers in liver

Salmonidae White blood cells [down arrow] 100-1,000
(species not
specified)

Salmonidae Lymphoid organ [down arrow] 100-1,000
(species not quality (evidence of
specified) atrophy)

Salvelinus Spleen weight [down arrow]/ 1,500-13,500
namaycush, no effect
Oncorhynchus
kisutch

S. namaycush, O. No. of lymphocytes [down arrow]/ 1,500-13,500
kisutch no effect

Taxon, species Conc tested ([mu]g/L) Nonmonotonic Atrazine grade
 does respones
 (a)

Salamander

A. tigrinum 1.6, 16, 160 No Tech

Frog

R pipiens 2.1, 21, 210 No Tech

R pipiens 2.1, 21, 210 No Tech

R pipiens 2.1, 21, 210 No Tech

R pipiens 2.1, 21, 210 No Tech

R pipiens 2.1, 21, 210 No Tech

R pipiens 2.1, 21, 210 No Tech

R pipiens 0.1 NA Tech

R pipiens 1, 10 No Not provided

R. pipiens 1, 10 Possibly Not provided

R pipiens 0.01, 0.1 No Tech

R pipiens 0.01, 0.1, 1, 10 No Tech

X. laevis 2.1, 21, 210, 2100 No Tech

X. laevis 2,100 2.1,21,210, No Tech

X. laevis 2, 100 2.1, 21.210, No Tech

X. laevis 2, 100 2.1, 21, 210 No Tech

X. laevis 2, 100 2.1, 21, 210 No data Tech

X. laevis 2, 100 400 NA Tech

X. laevis 400 NA Tech

R. sylvatica 3, 30 No Tech

R pipiens Unknown No Comm

Rana paulustris 117 NA Tech

R paulustris 117 NA Tech

R clamitans 117 NA Tech

R clamitans 117 NA Tech

Fish

C. auratus 42 NA Tech

C. auratus 42 NA Tech

C. auratus 42 NA Tech

C. auratus 42 NA Tech

Galaxias 0.9, 3, 10.50 Possibly Tech
macuiatus

O. mykiss 1,000-10,000 Possibly Tech

O. mykiss 1,000-10,000 Possibly Tech

O. mykiss 1,000-10,000 Possibly Tech

Liza ramada and Unknown Unknown Unknown
Liza aurata

L ramada and L Unknown Unknown Unknown
aurata

Salmonidae Unknown Unknown Unknown
(species not
specified)

Salmonidae Unknown Unknown Unknown
(species not
specified)

Salvelinus Unknown Unknown Unknown
namaycush,
Oncorhynchus
kisutch

S. namaycush, O. Unknown Unknown Unknown
kisutch

Taxon, species Experiment type Exposure Reference
 (b) duration

Salamander

A. tigrinum SR Until Forson and Storfer
 metamorphosis 2006b

Frog

R pipiens SR 21 days Christin et al.
 2003, 2004 (a)

R pipiens SR 21 days Christin et al.
 2003, 2004 (a)

R pipiens SR 21 days Christin et al.
 2003 (a)

R pipiens SR 21 days Christin et al.
 2003, 2004

R pipiens SR 21 days Christin et al.
 2003, 2004 (a)

R pipiens SR 21 days Christin et al.
 2004 (a)

R pipiens SR Until Hayes et al. 2006
 metamorphosis

R pipiens SR 4 weeks Houck and Sessions
 2006

R. pipiens SR 8 weeks Houck and Sessions
 2006

R pipiens SR 8 days Brodkin et al.
 2007 (c)

R pipiens SR 8 days Brodkin et al.
 2007 (a)

X. laevis SR 21 days Christin et al.
 2004 (a)

X. laevis SR 21 days Christin et al.
 2004 (a)

X. laevis SR 21 days Christin et al.
 2004 (a)

X. laevis SR 21 days Christin et al.
 2004 (a)

X. laevis SR 21 days Christin et al.
 2003 (a)

X. laevis SR Until Langerveld et al.
 metamorphosis 2009

X. laevis SR Until Langerveld et al.
 metamorphosis 2009

R. sylvatica SR 4 weeks Kiesecker 2002

R pipiens FS Unknown Rohr et al. 2008c
 (d)

Rana paulustris PE 4 weeks Rohr et al. 2008c

R paulustris PE 4 weeks Rohr et al. 2008c

R clamitans PE 4 weeks Rohr et al. 2008c

R clamitans PE 4 weeks Rohr et al. 2008c

Fish

C. auratus SR 12 weeks Fatima et al. 2007
 (a)

C. auratus SR 12 weeks Fatima et al. 2007
 (a)

C. auratus SR 12 weeks Fatima et al. 2007
 (a)

C. auratus SR 12 weeks Fatima et al. 2007
 (a)

Galaxias macuiatus SR 10 days Davies et al. 1994

O. mykiss PE 2 days Rymuszka et al.
 2007

O. mykiss PE 2 days Rymuszka et al.
 2007

O. mykiss PE 2 days Rymuszka etal.
 2007

Liza ramada and Unknown Unknown Biagianti-Risbourg
Liza aurata 1990 (e)

L ramada and L Unknown Unknown Biagianti-Risbourg
aurata 1990 (e)

Salmonidae (species Unknown Unknown Walsh and Ribelin
not specified) 1975 (e)

Salmonidae (species Unknown Unknown Walsh and Ribelin
not specified) 1975 (e)

Salvelinus Unknown Unknown Zeeman and
namaycush, Brindley 1981
Oncorhynchus
kisutch

S. namaycush, O. Unknown Unknown Zeeman and
kisutch Brindley 1981

Abbreviations: [down arrow], decreased; [up arrow], increased; Comm,
commercial; Cone, concentration; FS, field survey; NA, not applicable
(used when there were too few concentrations to evaluate
nonmonotonicity); ND, not detected; PE, pulse experiment; SR, static
renewal experiment, Tech, technical. Excluded studies are listed in
Supplemental Material, Table S1 (doi:10.1289/ehp.0901164.S1).

(a) Atrazine was a component of a mixture of pesticides tested, and
thus the experiment did not isolate the effects of atrazine. (b)
Atrazine alone and every mixture containing atrazine increased thymic
plaques. (c) Immune response stimulated by thioglycollate. (d) No
quantified factors correlated with atrazine could parsimoniously
explain patterns in infection. (e) As reported by Dunier and Swicki
1993; could not obtain original works.


Effects on infections. Similar to the effects of atrazine on amphibian and fish immunity, atrazine exposure was consistently associated with an increase in infection end points in fish and amphibians at ecologically relevant concentrations (Table 4). Atrazine elevated trema-tode, nematode, viral, and bacterial infections (Table 4). Of the studies with sufficient statistical power and without obvious confounders, 12 of 14 of the infection end points increased or showed a strong trend toward increasing, indicating either more infected individuals, more infections per individual, faster maturation, or greater reproduction of the parasite within the host, or greater parasite-induced host mortality (Table 4). As with immunity, these patterns should be considered with caution because many of these end points (6 of 16) came from studies where atrazine was part of a mixture of pesticides tested. Nevertheless, atrazine exposure, alone (4 of 7 end points) or in a pesticide mixture or field study (9 of 9 end points), was associated with an increase in infection end points (Table 4). In general, high concentrations of atrazine seem to be directly toxic to trematodes and viruses, possibly reducing infection risk for amphibians (Forson and Storfer 2006a; Koprivnikar et al. 2006; Rohr et al. 2008b), whereas more ecologically common concentrations seem to increase amphibian susceptibility, elevating infection risk (Forson and Storfer 2006b; Gendron et al. 2003; Kiesecker 2002; Rohr et al. 2008c).
Table 4. Summary of the results for the effects of atrazine, through
water column exposure, on fish and amphibian parasite infections.

Taxon, species End point Effect Conc where
 direction effect was
 observed
 ([mu]g/L)

Salamander

A. macrodactylum Infectivity of ATV [down arrow] Not provided

A. tigrinum Percentage infected [up arrow] at 16
 with ATV 16 but not 1.6
 or 160

A. tigrinum Viral load ND; p = 0.14 -

A. tigrinum Mortality due to ATV [up arrow] Not provided

Frog

R. pipiens Rhabdias ranae ND; trend -
 nematode prevalence toward [up
 arrow]

R. pipiens No. of adult R. ranae [up arrow], 21 + 210 >
 nematode clear dose controls, 210
 response > water
 control

R. pipiens Chryseobacterium [up arrow] 0.1
 (Flavobacterium)
 menigosepticum
 infections

R. pipiens R. ranae nematode Faster 21, 210
 within host migration

R. pipiens R. ranae nematode Earlier 21, 210
 maturation and
 reproduction

R. sylvatica No. of Ribieoriasp. [up arrow] 3, 30
 and Telorchis sp.

R. sylvatica Limb deformities [up arrow] in Ponds with
 caused by Ribieorla ponds with atrazine
 sp. atrazine

R. clamitans No. of Echinostoma [up arrow] 201
 trivolvis cercariae

R. pipiens No. of larval [up arrow] < 1 Do not
 trematodes know maximum
 Cone

R. clamitans No. of larval [up arrow] 117
 Plagiorchid
 trematodes

R. clamitans No. of Echinostoma [down arrow], 20, 200
 trivolvis cercariae but amphibians
 not exposed
 toatrazine

Fish

C. auratus Mortality due to [up arrow] 42
 Aeromonas hydrophila
 challenge

Taxon, species Cone tested ([mu]g/L) Nonmonotonic Atrazine grade
 dose
 response

Salamander

A. macrodactylum 1.84, 18.4, 184 Dose response Tech
 not provided

A. tigrinum 1.6, 16, 160 Yes Tech

A. tigrinum 20, 200 No Tech

A. tigrinum 20, 200 No Tech

Frog

R. pipiens 2.1, 21, 210 No Tech

R. pipiens 2.1, 21, 210 No Tech

R. pipiens 0.1 NA Tech

R. pipiens 2.1, 21, 210 No Tech

R. pipiens 2.1, 21.210 No Tech

R. sylvatica 3,30 No Tech

R. sylvatica Unknown NA Comm

R. clamitans 201 NA Tech

R. pipiens Unknown No Comm

R. clamitans 117 NA Tech

R. clamitans 20, 200 No Comm; Aatrex
 (g)

Fish

C. auratus 42 NA Tech

Taxon, species Experiment Exposure duration Reference
 type

Salamander

A. macrodactylum SR 30 days Forson and Storfer
 2006 (a)

A. tigrinum SR Until Forson and Storfer
 metamorphosis 2006 (b)

A. tigrinum SR 2 weeks Kerby and Storfer
 2009

A. tigrinum SR 2 weeks Kerby and Storfer
 2009

Frog

R. pipiens SR 21 days Christin et al.
 2003 (c)

R. pipiens SR 21 days Gendron et al. 2003
 (c)

R. pipiens SR Until Hayes et al. 2006 (c),
 metamorphosis (d)

R. pipiens SR 21 days Gendron et al. 2003
 (c)

R. pipiens SR 21 days Gendron et al. 2003
 (c)

R. sylvatica SR 4 weeks Kiesecker 2002

R. sylvatica FS Unknown Kiesecker 2002

R. clamitans SR 2 weeks Rohr et al. 2008b
 (e)

R. pipiens FS Unknown Rohr et al. 2008c
 (l)

R. clamitans PE 4 weeks Rohr et al. 2008c

R. clamitans PE Cercariae exposed Koprivnikar et al.
 for 2 hr 2006 (h), (i), (j)

Fish

C. auratus SR 12 weeks Fatima et al. 2007
 (c)

Abbreviations: [down arrow], decreased; [up arrow], increased; ATV.
Ambystoma tigrinum virus; Comm, commercial; Conc, concentration; FS,
field survey; NA, not applicable (used when there were too few
concentrations to evaluate nonmonotonicity); ND, not detected; PE,
pulse experiment; SR, static renewal experiment. Tech, technical.
Excluded studies are listed in Supplement Material, Table S1
(doi:10.1289/ehp.0901164.S1).

(a) Effect was observed when combining of 1.84, 18.4, and 184
treatments and comparing with controls; effect might be predominantly
due to 184. (b) 160 ppb was thought to reduce ATV infectivity
explaining nonmonotonicity. (c) Atrazine was a component of a mixture
of pesticides tested, and thus the experiment did not isolate the
effects of atrazine. (d) Saw .this effect only when atrazine was
mixed with eight other pesticides. (e) Effect was found pooling
pesticides and comparing them with control treatments. (f) No
quantified factors correlated with atrazine could parsimoniously
explain patterns in infection. (g) Aatrex is 59.2% inactive
ingredients. (h) Effects could be due to inactive ingredients. (j)
Effects could be due to chemicals other than atrazine that might be in
the pond water used to make the stock solutions. (j) All [C.sub.50]s
were a calculated incorrectly.


Several atrazine studies collected immunologic data only from animals that were also exposed to parasites, thus confounding immune parameters with parasite exposure and loads (Christin et al. 2003; Forson and Storfer 2006b; Gendron et al. 2003; Hayes et al. 2006; Kiesecker 2002; Rohr et al. 2008c). However, in each of these studies, atrazine was associated with both reduced immune parameters and elevated parasite loads. The elevated infections associated with atrazine cannot be explained by parasites reducing immune responses. Hence, the parsimonious explanation for both of these findings is that atrazine reduced immune responses, which elevated infections, especially given that it is often beneficial for vertebrates to up-regulate immunity upon infection (Raffel et al. 2006).

Despite the apparent consistency in the effects of atrazine on immunity and infections (Table 3), much remains to be learned about the effects of atrazine and other chemicals on parasite-host interactions (Raffel et al. 2008; Rohr et al. 2006a). For instance, we know little about how atrazine-induced changes affect population or community dynamics or most human diseases.

Effects of atrazine on fish and amphibian gonadal morphology. General morphologic end points. Sex differentiation is the process by which gonads develop into either testes or ovaries from an undifferentiated or bipotential gonad (Hayes 1998). This process is distinct from reproductive maturation where the differentiated gonad becomes reproductively functional (e.g., undergoes spermatogenesis in males). Determining if atrazine induces changes in gonadal morphology is an important step in evaluating whether it can influence sexual differentiation.

Atrazine consistently affected male gonadal morphology in fish and amphibians (Table 5). Seven of the 10 studies including results on males and females reported strong trends or statistically significant alterations (6 studies) in at least one aspect of general gonadal morphology associated with atrazine exposure. Alterations included discontinuous and multiple testes, sexually ambiguous gonadal tissue, testicular ovarian follicles (TOFs), altered gonadal somatic index (GSI; ratio of gonad weight to body weight), expanded testicular lobules, and spermatogenic tubule diameter (Table 5).
Table 5. Summary of the effects of atrazine on general gonadal
morphology

Taxon, species End point Effect direction Conc where
 effect was
 observed
 ([mu]g/L)

Testes

Fish

Pimephales Testis size ND 5, 50
promelas corrected for body
 size

P. promelas Spermatogenic [down arrow] 250
 tubule diameter

Frog

X. laevis Discontinuous [up arrow] 25
 gonads (abnormal
 segmentation)

X. laevis Ambiguous gonads [up arrow] 25
 (not obviously male
 or female)

X. laevis Testis size [up arrow] 10
 corrected for body
 size

X. laevis Sperm/area ND -

X. laevis Testis size ND -
 corrected for body
 size

R. clamitans Testis size [down arrow] in ND-3.13
 corrected for body juvenile males
 size

R. pipiens TOFs (testicular [up arrow] where ND-3.14
 oocytes) atrazine was
 detected in 2003
 (c)

Various spp., Discontinuous ND -
mostly R. testes (abnormal
clamitans segmentation)

Various spp.. Intersex (having ND -
mostly R. testicular and
clamitans ovarian tissues)

Various spp., TOFs (testicular [up arrow] in 1 ND-0.73
mostly R. oocytes) of 2 years in
clamitans juveniles,
 positively
 correlated with
 max atrazine Cone
 in that year

R. clamitans Testis size [up arrow] in ND-250
 corrected for body adult males at
 size agricultural
 sites in 1 of 2
 years

X. laevis Hermaphroditism ND
 (testicular
 oocytes, intersex,
 mixed sex)

Acris crepitans Intersex or Trend for [up Atrazine
Ovaries testicular oocytes arrow] p = 0.07 detections

Fish

P. promelas Ovary size Trend for i 50
 corrected for body
 size

P. promelas Proportion of ND -
 oocytes undergoing
 atresia

Frog

H. versicolor, R. Ovarian - -
sphenocephala developmental
 stage

B. americanus Ovarian ND -
 developmental rate

Taxon, species Conc tested Atrazine Experiment
 ([mu]g/L) grade type

Testes

Fish

Pimephales promelas 5, 50 Tech SR

P. promelas 25, 250 Tech FT

Frog

X. laevis 1.0, 10, 25 Tech SR

X. laevis 1.0, 10, 25 Tech SR

X. laevis 10, 100 Tech SR

X. laevis 10, 100 Tech SR

X. laevis 1, 25, 250 Tech SR

R. clamitans ND-3.13 (c) Comm FS

R. pipiens ND-3.13 (c) Comm FS

Various spp., mostly R. ND-2(e) Comm FS
clamitans

Various spp.. mostly R. ND-2 (e) Comm FS
clamitans

Various spp., mostly R. ND-2 (e) Comm FS
clamitans

R. clamitans ND-2 (e) Comm FS

X. laevis 0.1, 1, 10, 100 Tech SR

Acris crepitans Ovaries ND-70 Comm FS

Fish

P. promelas 5, 50 Tech SR

P. promelas 25, 250 Tech FT

Frog

H. versicolor, R. 1, 3, 30 (b) Tech SR
sphenocephala

B. americanus 1, 3, 30 (b) Tech SR

Taxon, species Exposure duration Reference

Testes

Fish

Pimephales promelas 21 days Bringolf et al. 2004
 (a)

P. promelas 21 days U.S. EPA 2005

Frog

X. laevis ~78 days during larval Carr et al. 2003
 period

X. laevis ~78 days during larval Carretal. 2003 (b)
 period

X. laevis 48 days Hecker et al.
 2005a(a)

X. laevis 48 days Heckeretal. 2005a (a)

X. laevis 36 days Hecker et al. 2005a
 (a)

R. clamitans Unknown McDaniel et al. 2008
 (c)

R. pipiens Unknown McDaniel et al. 2008
 (c), (d)

Various spp., mostly R. Unknown Murphy et al. 2006a
clamitans

Various spp.. mostly R. Unknown Murphy et aI. 2006a
clamitans

Various spp., mostly R. Unknown Murphy et al. 2006a
clamitans

R. clamitans Unknown Murphy et al. 2006b
 (f)

X. laevis ~65 days during Oka et al, 2008
 larval period

Acris crepitans Unknown Reeder et al. 1998(g)
Ovaries

Fish

P. promelas 21 days Bringolf et al. 2004
 (a)

P. promelas 21 days U.S. EPA 2005

Frog

H. versicolor, R. Through metamorphosis Storrs and Semlitsch
sphenocephala 2008

B. americanus Through metamorphosis Storrs and Semlitsch
 2008

Abbreviations: [down arrow], decreased; [up arrow]. increased:; Comm,
commercial; Conc, concentration; FS, field survey; FT, flow-through
experiment; ND, not detected; SR, static renewal experiment, Tech,
technical. Excluded studies are listed in Supplemental Material, Table
SI (doi:10.1289/ehp.0901164.S1).

(a) No test statistics or degrees of freedom are presented; however,
means and variances were presented either in the text or in a figure of
the article. (b) Xenopus are typically sexually differentiated at the
gross morphologic level at metamorphosis; individuals in this study
exposed to 25 [micro]g/L were so sexually ambiguous they were initially
considered intersex (having both testicular and ovarian issues). (c)
Atrazine concentration for the nonagricultural reference site during
2003 was reported incorrectly; repeated attempts to contact the authors
for clarification have not been forthcoming. (d) When atrazine
concentrations were highest (2003), TOFs per individual occurred in
higher numbers; TOFs were positively associated with atrazine, nitrate,
and quantity of pesticides in a multivariate comparison, suggesting
that atrazine is contributing to TOFs. (e) Concentrations were between
ND and 2 except on two occasions at one site, when levels were 65 and
250 [micro] g/L (f) Authors argued that differences in GSI between
agricultural and nonagricultural sites cannot be due to atrazine
because GSI does not correlate with atrazine concentration; however,
they presented no statistics to support this claim. (g) The
relationship between detection of atrazine and the presence of one or
more intersex cricket frogs approached significance (p = 0.07). (h) The
actual concentration of the 30-[micro] g/L treatment was 125 [micro]
g/L.


Effects on ovarian morphology are generally less obvious than those on testicular morphology and are typically dismissed without quantification. None of the three studies on fish or amphibians included in our metaanalysis found significant effects of atrazine on ovarian morphology, suggesting that atrazine induces fewer gonadal abnormalities in females than males. However, additional studies are necessary to fully evaluate the effects of atrazine on female gonadal morphology.

TOFs as a natural phenomenon. Jooste et al. (2005) and Solomon et al. (2008) argued that experiments with high numbers of TOFs in control Xenopus laevis support the hypothesis that TOFs are normal in some X. laevis populations. Although it was argued long ago that some anurans in some environments transition through a hermaphroditic phase during development (Witschi 1929), the literature we reviewed does not argue that adult amphibians commonly have oocytes within testicular tissue or are naturally hermaphroditic (Eggert 2004; Hayes 1998). Indeed, X. laevis sexually differentiates (without a transitional/hermaphroditic stage) during the larval period prior to sexual maturation (Iwasawa and Yamaguchi 1984). Thus, cases of gonadal abnormalities in healthy adult X. laevis populations should be rare. Given that simultaneous hermaphroditism has not been previously reported in X. laevis despite decades of research on their reproductive biology, an equally or more plausible explanation for high numbers of TOFs in control animals (e.g., Jooste et al. 2005; Orton et al. 2006) is exposure to some type of unmeasured endocrine-disrupting contaminant.

Effects of atrazine on fish and amphibian sex ratios. Given that atrazine exposure has been proposed to feminize gonadal development (Hayes et al. 2002, 2003), it might lead to female-biased sex ratios. Many studies, however, have severe methodologic errors, such as contaminated controls or inadequate data reporting [see Supplemental Material, Table SI (doi: 10.1289/ehp.0901164.S1)], preventing a conclusive synthesis of the effects of atrazine on sex ratios. None of the sex-ratio studies used the most accepted and powerful approaches for testing for changes in sex ratios (e.g., Wilson and Hardy 2002). Only four studies, all on X. laevis, were of sufficient quality to be included in our meta-analysis, and only one found that atrazine induced a female-biased sex ratio (see Supplemental Material, Table S2 (doi:10.1289/ehp.0901164.S1)].

Effects of atrazine on fish and amphibian gonadal function. Chemicals that alter gonadal development can affect gonadal function, such as germ cell (e.g., spermatogenesis in males) and steroid hormone production (McCoy et al. 2008; McCoy and Guillette, in press), and thus can lead to altered reproductive success.

Effects on testicular cell types. Spermatogenesis is the process through which mature male gametes (spermatozoa) are produced from precursor cells (spermatogenk cells). The relative ratios of different spermatogenic cell types, rather than abundance of spermatozoa alone, is the most sensitive metric of altered spermatogenesis. Unfortunately, few studies on effects of atrazine on spermatogenesis met our inclusion criteria. Two of two studies demonstrated that atrazine was associated with altered spermatogenesis and that several cell types were affected (Table 6). Thus, atrazine appears capable of altering spermatogenesis, but the contexts and generality of these effects cannot be firmly established. Our analysis once again highlights a need for more rigorous investigations.
Table 6. Summary of the effects of atrazine on gonadal function.

Taxon, species End point Effect direction

Testicular cell
types

Frog

R. clamitans Proportion of juvenile Lower at agricultural site
 males with > 50% tubules with highest atrazine
 containing spermatids and concentrations
 spermatozoa

R. pipiens Proportion of juvenile Higher at agricultural
 males with > 50% tubules site with highest
 atrazine
 containing spermatids and concentrations
 spermatozoa

Fish

P. promelas Proportion of primary [up arrow]
 spermatogonia

P. promelas Proportion of secondary Reduced
 spermatogonia

Sex hormone
concentrations

Frog

X. laevis Testosterone in adult [down arrow]
 males

X. laevis Testosterone in adult ND
 males

X. laevis Estradiol in adult males ND

X. laevis Estradiol in adult males ND

X. laevis Testosterone in adult [down arrow]
 males

X. laevis Testosterone in females [down arrow] at
 agricultural sites,
 negatively correlated with
 concentration of atrazine
 and breakdown product

X. laevis Testosterone in males Negatively correlated with
 diamino-chlorotriazine
 concentration (a product
 of atrazine breakdown)

X. laevis Estradiol in females [down arrow] at
 agricultural sites,
 negatively correlated with
 cone of atrazine and
 breakdown product

R. pipiens Testosterone in juvenile [down arrow] at
 males (2003) agricultural sites

R. pipiens Testosterone in juvenile Negatively correlated with
 males (2003) atrazine concentration

R. pipiens 11-Ketotestosterone in Negatively correlated with
 juvenile males (2003) atrazine concentration

R. pipiens Testosterone in adult Negatively correlated with
 females (2003) atrazine concentration

R. clamitans 11-Ketotestosterone to [up arrow] at agricultural
 testosterone ratio in sites
 adult females (late
 summer Aug-Sep 2002)

R. clamitans 11-Ketotestosterone to [up arrow] at agricultural
 testosterone ratio in sites
 adult males (late summer
 Aug-Sep 2002)

R. clamitans 11-Ketotestosterone to [up arrow] at agricultural
 testosterone ratio in sites
 adult males (early summer
 May 2003)

R. clamitans Estradiol to testosterone [up arrow] at agricultural
 ratio in adult females sites
 (late summer Aug-Sep
 2002)

R. clamitans Estradiol to testosterone [up arrow] at agricultural
 ratio in adult males sites
 (Late summer Aug-Sep
 2002)

R. clamitans Estradiol to testosterone [down arrow] at
 ratio in adult males agricultural sites
 (early summer May 2003)

R. clamitans Estradiol to testosterone [up arrow] at agricultural
 ratio in juvenile males sites
 (Jul 2003)

R. clamitans Testosterone in adult [up arrow] at agricultural
 males (early summer May sites
 2003)

R. clamitans Testosterone in juvenile [up arrow] at agricultural
 females (Jul 2003) sites

R. clamitans Testosterone in juvenile [up arrow] at agricultural
 males (Jul 2003) sites (d)

Fish

P. promelas Testosterone female ND

P. promelas Estradiol female Trend (up to a 44% [down
 arrow])

P. promelas Testosterone male Trend (up to a 31% [down
 arrow])

P. promelas 11-Ketotestosterone male Trend (up to a 47% [down
 arrow])

Reproductive
success

Salamander

A. barbouri Proportion hatched and ND
 timing of hatching

A. barbouri Proportion hatched and [down arrow] and delayed
 timing of hatching hatching

Frog

R. pipiens Proportion hatched ND

R. clamitans Proportion hatched ND

B. americanus Proportion hatched ND

Fish

P. promelas Eggs per spawning of Trend for a [down arrow]
 exposed adults

P. promelas Number of spawnings of Trend for a [down arrow]
 exposed adults

P. promelas Fertilization success of Trend for a [down arrow]
 exposed adults

P. promelas Proportion hatched and ND
 larval development of
 offspring form exposed
 adults

P. promelas Egg production of exposed ND
 adults

P. promelas Fertilization success of ND
 exposed adults

P. promelas Proportion hatched and ND
 larval development of
 offspring from exposed
 adults

Taxon, species Cone where effect was Cone tested Atrazine grade
 observed ([mu]g/L) ([mu]g/L)

Testicular cell
types

Frog

R. clamitans Range of medians, ND-3.13 (a) Comm
 0.068-0.78

R. pipiens 0.342 (mean of median ND-3.13 (a) Comm
 concentrations)

Fish

P. promelas 25,250 25, 250 Test

P. promelas 25, 250 25, 250 Test

Sex hormone
concentrations

Frog

X. laevis 25 25 Tech

X. laevis - 10,100 Tech

X. laevis - 10,100 Tech

X. laevis - 1,25,250 Tech

X. laevis 250 1,25,250 Tech

X. laevis < 0.1-4.14 < 0.1-4.14 Comm

X. laevis < 0.1-4.14 < 0.1-4.14 Comm

X. laevis < 0.1-4.14 < 0.1-4.14 Comm

R. pipiens Range of medians, ND-3.13 (a) Comm
 0.380-0.780

R. pipiens ND-3.13 ND-3.13 (a) Comm

R. pipiens ND-3.13 ND-3.13 (a) Comm

R. pipiens ND-3.13 ND-3.13 (a) Comm

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to 250

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to 250

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to 250

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to 250

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

R. clamitans Agricultural sites ND-250 Comm
 ranged from ND to
 0.73

Fish

P. promelas - 25, 250 Tech

P. promelas 25, 250 25, 250 Tech

P. promelas 25, 250 25, 250 Tech

P. promelas 25, 250 25, 250 Tech

Reproductive
success

Salamander

A. barbouri - 4, 40,400 Tech

A. barbouri 400 4, 40,400 Tech

Frog

R. pipiens - 2,590-20,000 Tech

R. clamitans - 2,590-20,001 Tech

B. americanus - 2,590-20,002 Tech

Fish

P. promelas 5 5, 50 Tech

P. promelas 50 5, 50 Tech

P. promelas 50 5, 50 Tech

P. promelas - 5, 50 Tech

P. promelas - 25, 250 Tech

P. promelas - 25, 250 Tech

P. promelas - 25,250 Tech

Taxon, species Experiment Exposure duration Reference
 type

Testicular cell
types

Frog

R. clamitans FS Unknown McDaniel et al. 2008
 (a)

R. pipiens FS Unknown McDaniel et al. 2008
 (a)

Fish

P. promelas FT 21 days U.S. EPA 2005

P. promelas FT 21 days U.S. EPA 2005

Sex hormone
concentrations

Frog

X. laevis SR 46 days Hayes et al. 2002 (b)

X. laevis SR 48 days Hecker et al. 2005a

X. laevis SR 48 days Hecker et al. 2005a

X. laevis SR 36 days Hecker et al. 2005b

X. laevis SR 36 days Hecker et al. 2005b

X. laevis FS Unknown Hecker et al. 2004

X. laevis FS Unknown Hecker et al. 2004

X. laevis FS Unknown Hecker et al. 2004

R. pipiens FS Unknown McDaniel et al. 2008
 (a)

R. pipiens FS Unknown McDaniel et al. 2008
 (a), (c)

R. pipiens FS Unknown McDaniel et al. 2008
 (a), (c)

R. pipiens FS Unknown McDaniel et al. 2008
 (a), (c)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

R. clamitans FS Unknown Murphy et al. 2006b
 (d)

Fish

P. promelas FT 21 days U.S. EPA 2005

P. promelas FT 21 days U.S. EPA 2005 (e)

P. promelas FT 21 days U.S. EPA 2005 (e)

P. promelas FT 21 days U.S. EPA 2005 (e)

Reproductive
success

Salamander

A. barbouri SR 37 days Rohr et al. 2003

A. barbouri SR Mean of 52 days Rohr et al. 2004

Frog

R. pipiens SR 10 days Allran and Karasov 2001

R. clamitans SR 10 days Allran and Karasov 2001

B. americanus SR 10 days Allran and Karasov 2001

Fish

P. promelas SR 21 days Bringolf et al. 2004
 (b)

P. promelas SR 21 days Bringolf et al. 2004
 (b)

P. promelas SR 21 days Bringolf et al. 2004
 (b)

P. promelas SR 21 days Bringolf et al. 2004
 (b)

P. promelas FT 21 days U.S. EPA 2005

P. promelas FT 21 days U.S. EPA 2005

P. promelas FT 21 days U.S. EPA 2005

Abbreviations: [down arrow], decreased; [up arrow], increased; Comm,
commercial; Conc, concentration; FS, field survey; FT, flow-through
experiment; ND, not detected; SR, static renewal experiment, Tech,
technical. Excluded studies are listed in Supplemental Material, Table
S1 (doi:10. 1289/ehp.0901164.S1).

(a) Atrazine concentration for the nonagricultural reference site
during 2003 was reported incorrectly; repeated attempts to contact the
authors for clarification have not been forthcoming. (b) No test
statistics or degrees of freedom were presented; however, means and
variances were presented either in the text or in a figure of the
article. (c) Authors reported no significant correlation between
atrazine and sex hormones in their abstract when, in fact, these end
points were negatively correlated; contrary to the authors' conclusion,
the negative correlations across sexes and age groups reported in their
study are unlikely to occur because of a low sample size or sampling
error. (d) Authors argued that differences in hormone levels between
agricultural and nonagricultural sites cannot be due to atrazine
because hormone concentrations do not correlate with atrazine
concentration; however, they presented no statistics to support this
claim. (e) Low samples sizes (7-8 fish) likely precluded detecting
these considerable effects.


Effects on sex hormone concentrations. Sex hormone production is an important function of gonads that can be altered by gonadal abnormalities (McCoy et al. 2008). Indeed, altered hormone concentrations are the defining characteristic, in many cases, of endocrine disruption. Six of seven studies on fish and amphibians document strong trends or significantly (five studies) altered sex hormone concentrations associated with atrazine exposure (Table 6). Although many of these studies were conducted in the field and are therefore correlative, the consistency of these results across studies suggests that atrazine alters sex hormone production and should be considered an endocrine-disrupting chemical. A more thorough understanding of the effects of atrazine on hormone concentrations will require more detailed studies that account for the inherent variability of endocrine system processes.

Effects on reproductive success. Reproductive success is strongly linked to population persistence and is likely one of the most important end points in toxicologic studies. Five studies that evaluated the effects of atrazine on measures of reproductive success met our metaanalysis requirements (Table 6). Two studies on adult fish, Pimephales promelas, found no significant effect of atrazine on number of eggs produced, fertilization success, proportion of hatchlings, or larval development. However, one of these studies (Bringolf et al. 2004) found several nonsignificant, adverse trends (Table 6). Two of three studies on amphibians found no effects of atrazine on hatching success, whereas one showed reduced hatching success and delayed hatching (Table 6). Given the mixed results, the effect of atrazine on reproductive success needs to be studied more thoroughly.

Effects of atrazine on fish and amphibian vitellogenin. Vitellogenin is an egg yolk precursor protein produced in the livers of female fish and amphibians. Estrogens induce vitellogenin synthesis in both males and females in vivo, and quantification of vitellogenin is now an accepted screening test for estrogenic effects of chemicals (Scholz and Mayer 2008). None of the five studies (four on fish) found significant effects of atrazine on circulating or whole-body concentrations of vitellogenin [see Supplemental Material, Table S2 (doi:10.1289/ehp.0901164.S1)]. Hence, these data do not support the hypothesis that atrazine is strongly estrogenic to fish.

Effects of atrazine on fish and amphibian aromatase. Cytochrome p450 aromatase catalyzes the conversion of androgens to estrogens in gonads and is critical for maintaining a balance between these sex hormone classes. Hayes et al. (2002) hypothesized that decreases in testosterone associated with atrazine exposure in their study could be driven by an atrazine-induced increase in aromatase and a concomitant increase in the conversion of testosterone and other androgens to estrogens. This hypothesis seemed reasonable because atrazine was known to increase aromatase in human cancer cell lines and in alligator gonadal-adrenal mesonephros (Crain et al. 1997; Sanderson et al. 2000). However, since 2002, several studies have explicitly tested whether atrazine increases aromatase in fish and amphibians, and only one of six studies included in our meta-analysis found that atrazine was associated with increased aromatase gene expression [see Supplemental Material, Table S2 (doi:10.1289/ehp.0901164.S1)].

Effects of atrazine on fish and amphibian populations and Although there are too few studies examining the effects of atrazine on freshwater vertebrate populations to warrant meta-analysis, and virtually all community-level studies infer--rather than test for--indirect effects (Rohr and Crumrine 2005), the effects of atrazine on populations and communities warrants a brief discussion. Any chemical that affects physiology, growth, development, reproduction, survival, or species interactions can affect population and community dynamics (Clements and Rohr 2009; Rohr et al. 2006a). However, the effects of contaminants might not result in immediate population declines because the survivors of chemical exposure frequently have less competition for resources, thus providing density-mediated compensation for adverse effects of the chemical (Rohr et al. 2006b). Demonstrating that a factor is the cause of any population decline is, indeed, incredibly difficult (Rohr et al. 2008a). Rohr et al. (2006b) revealed significant and delayed declines in Ambystoma barbouri salamander populations at 4, 40, and 400 [micro]g/L atrazine, above and beyond the counteracting effects of density-mediated compensation. Although this study provided greater ecologic realism than many studies on atrazine, caution should be taken extrapolating these effects to populations in nature because this study was conducted in laboratory terraria. There is certainly a need for controlled studies on the effects of pesticides on wildlife populations.

Several studies have examined the effects of atrazine on amphibian and fish communities (Boone and James 2003; de Noyelles et al. 1989; Kettle 1982; Rohr and Crumrine 2005; Rohr et al. 2008c). Many of these studies reported alterations in fish or amphibian growth and abundance that seem to be caused by atrazine-induced changes in photosynthetic organisms (reviewed by Giddings et al. 2005; Solomon et al. 2008). At ecologically relevant concentrations, atrazine is expected to have a bevy of indirect effects by altering the abundance of periphyton, phytoplankton, and macrophytes (Huber 1993; Solomon et al. 1996). However, none of these studies distinguish between direct and indirect effects of atrazine on fish or amphibians.

There are several field studies comparing amphibian populations or species richness between atrazine-exposed and unexposed habitats (Bonin et al. 1997; Du Preez et al. 2005; Knutson et al. 2004). All of these studies are correlational, and none thoroughly considered or ruled out alternative hypotheses for the observed patterns.

Caveats. We would be remiss not to mention some caveats regarding this meta-analysis. First, a problem with many meta-analyses is the "file-drawer" effect. This refers to the fact that researchers tend to place the results of experiments showing no effects in their file drawer, and many journals tend to publish fewer studies showing no effects than those with effects (Gurevitch and Hedges 1993; Osenberg et al. 1999). This might be less of a problem in studies on pesticides because these chemicals are designed to kill biota; thus in many cases, the null hypothesis might be an effect rather than the absence of one. Additionally, a substantial industry contingent works to ensure that both significant and nonsignificant effects of chemicals get published. Indeed, in the review of atrazine by Solomon et al. (2008), there were approximately 63 cases where atrazine had significant adverse effects and 70 cases where atrazine had no significant effects (Rohr JR, McCoy KA, unpublished data), suggesting that the file-drawer effect is unlikely to be strongly biasing submission and publication of nonsignificant atrazine results. However, we cannot completely discount the possibility that the file-drawer effect generated a bias toward greater publication of significant effects of atrazine.

Another admonishment is that some of the end points in this meta-analysis were not independent of one another. For example, we tallied multiple end points from a single study despite the possibility that they might not be entirely independent.

Finally, we must consider the findings of this meta-analysis on atrazine relative to alternative strategies for weed control. If the alternative to atrazine is another chemical, then we should ideally compare the effects of atrazine to the replacement chemical. In fact, atrazine might be less detrimental to freshwater vertebrates than a replacement herbicide. If the alternative to atrazine does not entail a chemical replacement, then the effects revealed here might indeed be disconcerting. However, we also cannot ignore the benefit, if any, that atrazine provides. Interestingly, several studies estimate that atrazine increases corn yields by only 1-3% (reviewed by Ackerman 2007). To adequately evaluate any chemical, we should ideally conduct a thorough cost-benefit analysis that considers the focal chemical and alternatives to its use and is based on comprehensive and accurate knowledge [see Ackerman (2007) for a review and critique of atrazine cost-benefit analyses].

Conclusions

As in past reviews, we found little evidence that atrazine consistently causes direct mortality of freshwater vertebrates at ecologically relevant concentrations, but there is evidence that atrazine might have adverse indirect ecologic effects. However, in contrast to a previous review on atrazine (Solomon et al. 2008), we unveiled consistent effects of atrazine at ecologically relevant concentrations for many other response variables in our meta-analysis. The discrepancy between our findings and the conclusions of previous reviews could be partly a function of differences in criteria for including studies in the group used to draw general conclusions about atrazine effects. Past reviews (e.g., Solomon et al. 2008) did not clearly define their inclusion criteria, did not make it clear which studies affected their conclusions (or how they came to their conclusions), and regularly dismissed significant effects of atrazine.

Here we reveal that, for freshwater vertebrates, atrazine consistently reduced growth rates, had variable effects on timing of metamorphosis that were often nonmonotonic, elevated locomotor activity, and reduced antipredator behaviors. Amphibian and fish immunity was reliably reduced by ecologically relevant concentrations of atrazine, and this was regularly accompanied by elevated infections. Atrazine exposure induced diverse morphologic gonadal abnormalities in fish and amphibians and was associated with altered gonadal function, such as modified sex hormone production. This suggests that atrazine should be considered an endocrine-disrupting chemical. Finally, we do not have a thorough appreciation of the reproductive repercussions of atrazine.

Several end points had enough well-conducted studies to warrant more sophisticated meta-analyses based on effect sizes (e.g., growth, timing of metamorphosis, activity, immunity, infections, gonadal abnormalities). Meta-analyses based on effect sizes can provide parameter and standard errors estimates and thus can be useful for probabilistic risk assessment and for predicting atrazine effects.

Although we found consistent effects of atrazine on freshwater vertebrates, the consequences of these effects remain uncertain. We know little about how atrazine-induced changes in vertebrate growth, somatic development, behavior, immunity, gonadal development, or physiology affect reproduction, populations, gene frequencies, or communities. However, it was Sir Austin Bradford Hill who wisely stated in his address to the Royal Society of Medicine in 1965 that
 All scientific work is incomplete [and] ... liable to be upset or
 modified by advancing knowledge. That does not confer upon us freedom
 to ignore the knowledge we already have, or to postpone action that
 it appears to demand at a given time. (Hill 1965)


Whatever action is taken in the re-evaluation of atrazine by the U.S EPA, we strongly encourage regulators to consider the consistent effects of atrazine on various taxa and to weigh these effects against any benefits atrazine provides and the alternatives to atrazine use.

CORRECTION

Corrections have been made from the original manuscript published online: Criteria for identifying results showing "substantial trends" has been clarified; the number of studies has been corrected in the text; and the "effect direction" for relevant studies has been corrected in Tables 1, 3, and 5.

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Jason R. Rohr and Krista A. McCoy

Department of Integrative Biology, University of South Florida, Tampa, Florida, USA

Address correspondence to J.R. Rohr, University of South Florida, Department of Integrative Biology, SCA 110, 4202 East Fowler Ave., Tampa, FL 33620 USA. Telephone: (813) 974-0156. Fax: (813) 974-3263. E-mail: jasonrohr@gmail.com

Supplemental Material is available online (doi:10.1289/ehp.0901164.S1 via http://dx.doi.org/).

We thank the Rohr lab, M. McCoy, and anonymous reviewers for comments on this work.

Funds were provided by grants from the National Science Foundation (DEB 0516227), the U.S. Department of Agriculture (NRI 2006-01370 and 2009-35102-0543), and the U.S. Environmental Protection Agency STAR grant R833835) to J.R.R.

The authors declare they have no competing financial interests.

Received 2 July 2009; accepted 23 September 2009.
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Author:Rohr, Jason R.; McCoy, Krista A.
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Date:Jan 1, 2010
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