Aquatic Toxicology 154 (2014) 230239

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Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Atrazine reduces reproduction in Japanese medaka (Oryzias latipes)

Diana M. Papoulias a, , Donald E. Tillitt a , Melaniya G. Talykina b ,
Jeffrey J. Whyte a,1 , Catherine A. Richter a
a
US Geological Survey, Columbia Environmental Research Center, 4200 New Haven Rd., Columbia, MO 65251, United States
b
Institute of Biology of Inland Waters, Russian Academy of Sciences, 152742 Borok, Nekouz, Yaroslavl, Russia

a r t i c l e i n f o a b s t r a c t

Article history: Atrazine is an effective broadleaf herbicide and the second most heavily used herbicide in the United
Received 12 December 2013 States. Effects along the hypothalamuspituitarygonad axis in a number of vertebrate taxa have been
Received in revised form 16 May 2014 demonstrated. Seasonally elevated concentrations of atrazine in surface waters may adversely affect
Accepted 19 May 2014
shes, but only a few studies have examined reproductive effects of this chemical. The present study was
Available online 27 May 2014
designed to evaluate a population endpoint (egg production) in conjunction with histological (reproduc-
tive stage, gonad pathology) and biochemical (aromatase activity, sex hormone production) phenotypes
Keywords:
associated with atrazine exposure in Japanese medaka. Adult virgin breeding groups of one male and
Herbicides
Egg production
four females were exposed to nominal concentrations of 0, 0.5, 5.0, and 50 g/L (0, 2.3, 23.2, 231 nM)
Genotoxicity of atrazine in a ow-through diluter for 14 or 38 days. Total egg production was lower (3642%) in all
atrazine-exposed groups as compared to the controls. The decreases in cumulative egg production of
atrazine-treated sh were signicant by exposure day 24. Reductions in total egg production in atrazine
treatment groups were most attributable to a reduced number of eggs ovulated by females in atrazine-
treated tanks. Additionally, males exposed to atrazine had a greater number of abnormal germ cells. There
was no effect of atrazine on gonadosomatic index, aromatase protein, or whole body 17 -estradiol or
testosterone. Our results suggest that atrazine reduces egg production through alteration of nal matu-
ration of oocytes. The reduced egg production observed in this study was very similar to our previously
reported results for fathead minnow. This study provides further information with which to evaluate
atrazines risk to sh populations.
Published by Elsevier B.V.

1. Introduction Aquatic vertebrate and invertebrate populations in lotic, lentic,

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine)
is an effective broadleaf herbicide and the most heavily used herbi-
cide in the United States after glyphosate. Atrazine use comprised
17% by weight of total pesticide use in 2009 with approximately
65 million lbs (30 million kgs) applied to crops (Thelin and
Stone, 2013). It enters surface waters predominantly in runoff
from crop lands, generally peaking seasonally with rainfall and
application (Lerch et al., 2011). Despite atrazines relatively low
potential for vertical distribution and bioaccumulation through
aquatic trophic levels, concerns exist that populations of aquatic
organisms exposed to atrazine are adversely affected.
Corresponding author. Tel.: +1 573 999 1788.
E-mail address: dpapoulias@usgs.gov (D.M. Papoulias).
1
Present address: University of Missouri, Division of Animal Sciences, Columbia,
MO 65211, USA.
wetland, and marine environments are annually exposed to water-
borne atrazine at concentrations ranging from 1 to 10 g/L (Belden
et al., 2012; Blevins, 2011; Casara et al., 2012; Cochran, 2011; Du
Preez et al., 2005; Gilliom et al., 2006; Pennington et al., 2001;
Shutler and Marcogliese, 2011; Stoeckel et al., 2012) and seasonally
at some locations atrazine can be as high as 50 g/L (Lerch et al.,
2011). Water body concentrations are site-specic and dependent
on factors such as soil permeability, precipitation, and watershed
area among others (Stone and Gilliom, 2012; Stoeckel et al., 2012).
Sediments may be a source of chronic exposure although few stud-
ies with atrazine-contaminated sediments have been conducted
(Kolpin et al., 2012; Phewnil et al., 2012).
Reviews have described both effects and the absence of effects
at multiple levels of biological organization on aquatic orga-
nisms after exposure to environmentally relevant concentrations
of atrazine (Rohr and McCoy, 2010; Solomon et al., 2008). Ambi-
guity among study results has led to controversy regarding
the risk atrazine presents with respect to health of aquatic

http://dx.doi.org/10.1016/j.aquatox.2014.05.022
0166-445X/Published by Elsevier B.V.
 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239
populations. A lack of clearly dened mechanisms for atrazines
effects contributes to on-going debates. Uncertainty notwithstand-
ing, the proposed physiological and biochemical pathways that
atrazine targets involve receptors of the reproductive endocrine
system (Cooper et al., 2007; Fan et al., 2007; Hayes et al., 2011;
Suzawa and Ingraham, 2008).
Only a few studies have investigated the effect of atrazine
directly or indirectly on sh reproduction. Generally, evidence for
deleterious effects is apparent but is questioned because a clear
mechanistic understanding is lacking. Fathead minnow (Pimephales
promelas) and cunner (Tautogolabrus adspersus) exposed to atrazine
produced fewer eggs than controls but the lack of a monotonic
doseresponse and low-dose effects are perplexing (Mills, 2006;
Tillitt et al., 2010). Two studies following EPAs 21-d reproduc-
tion protocol showed total cumulative egg production in atrazine
exposure tanks trending below controls. However, exposure time
may have been too short to achieve signicant differences (Bringolf
et al., 2004; Battelle, 2005). Observed reduced bluegill (Lepomis
macrochirus) reproduction in atrazine-treated ponds may have
been a direct effect or an indirect trophic effect (Kettle et al., 1987).
A few additional studies report atrazine affects key sh repro-
ductive behaviors (Saglio and Trijasse, 1998; Moore et al., 2007;
Shenoy, 2012).
Given atrazines potential to adversely affect reproduction,
we conducted a study to further understand population-level
implications of the use of atrazine, a purported reproduc-
tive endocrine disruptor. Our objectives were to evaluate
egg production and additional reproductive effects along the
hypothalamuspituitarygonad axis on male and female medaka
sh (Oryzias latipes) as measured by a suite of biological endpoints.
We chose medaka because of its mode of reproduction, ease of
egg collection, and because a great deal of information on medaka
reproduction exists.
2. Methods
2.1. General approach
Mature, virgin medaka sh were tested at three concentrations
of atrazine in a static renewal system. Adult survival and egg pro-
duction were monitored daily. Tissue samples were collected at
three time-points: prior to exposure, at the mid-point of the expo-
sure period, and at the end of the exposure period. Tissues were
analyzed for steroid hormones, gonad and body weight, aromatase
activity (brain and ovary), and gonad histopathology.
2.2. Experimental design
The study was designed to test for signicant differences in
effects between medaka sh exposed to 0.5 g/L (2.3 nM), 5.0 g/L
(23.2 nM), and 50 g/L (231 nM) atrazine and those exposed to a
solvent control with no atrazine (0.02% acetone). The experimen-
tal unit was a single tank containing four females and one male in
replicates of six, 6.5-L tanks for each of the 3 time points. The tanks
were placed in a diluter water bath with four tanks to a row and 14
rows. The diluter delivery system did not allow for complete ran-
domization of tank placement however, each row was randomly
assigned to an atrazine dose or the control. Tanks were randomly
assigned to a position and one of the three time points within each
row. Plumbing and tank materials were glass or Teon to minimize
chemical interferences or adsorption of atrazine.
2.3. Animal care and feeding
Virgin, mature d-rR medaka of approximately the same size
(mean (SD); female n = 24, 0.57 g (0.13); male n = 6, 0.64 g (0.10)),
231
from the Columbia Environmental Research Centers stock (orig-
inally obtained as a gift from Dr. Akihiro Shima, 1995), were
randomly assigned to a tank at a ratio of one male to four females for
a total of ve sh in a tank. Test sh were stocked into tanks prior to
the beginning of atrazine exposures to acclimate the sh to the test
system. Acclimation was terminated approximately one week after
stocking when all females had produced some eggs. Fish were fed
live brine shrimp nauplii twice daily. Tanks were siphoned clean
once daily. Water quality was monitored weekly throughout the
test and maintained within ASTM standards for oxygen, pH, hard-
ness, alkalinity, and total ammonia (ASTM, 2004). Animals were
held on a 14L:10D h photoperiod at 25 + 1 C. The water in a tank
was renewed once every 24 h. The study area was isolated to mini-
mize general disturbances and the sides of each tank were covered
with opaque contact paper to prevent interactions of sh tank to
tank.
2.4. Atrazine exposure and water analysis
Stock solutions of atrazine (98% purity, Fluka Chemical, Dorset,
UK) were prepared in a 60% acetone:40% water solution in amber
bottles and stored at 4 C until use in the diluter. Atrazine con-
centrations in all the tanks were measured weekly with n = 12
tanks (days 110) or n = 6 tanks (days 1435) using enzyme-linked
immunosorbent assay (ELISA) kits (Abraxis, Warminster, PA). The
method detection limit (MDL) for the atrazine ELISA procedure
was 0.05 g/L of water. Conrmatory analysis was performed on
atrazine stock preparations and selected tank water samples by
gas chromatography (Jimenez et al., 1997). Briey, water sam-
ples were extracted using methylene chloride; the extract dried
with sodium sulfate and ltered through glass bers; volume
reduced to 0.1 mL in methyl tertiary butyl ether (MtBE). Triph-
enylphosphate (Chem Service Inc., 500 ng/mL in MtBE) was added
as an instrumental internal standard so that a nal concentra-
tion 0.25 ng/L injected with samples or blanks. The extracts were
analyzed by gas chromatographic/nitrogen phosphorus detector
(GC/NPD) and quantied by PerkinElmers TotalChromTM worksta-
tion chromatography data software. Samples for GC/NPD analysis
were taken on three of 12 days that water was collected. Each of
those sample sets were comprised of triplicate water samples from
each of the treatment groups (0, 0.5, 5, and 50 g/L). Quality con-
trol samples were analyzed with each sample set and included:
atrazine-spiked water, matrix (tap) water blank, and a procedural
blank.
2.5. Egg collection
Tanks were monitored daily for adult mortalities and egg pro-
duction. Females were inspected for oviposition within 2 h of the
beginning of the light period. Females with eggs were gently dip-
netted from the tanks, eggs stripped off, and the female returned to
the tank. Tank walls and siphoned waste waters were also inspected
for loose eggs. Eggs were collected from each tank, placed in a Petri
dish with exposure water, held for 24 h in an incubator at 25 1 C,
then inspected for viability and counted.
2.6. Fish collection, histological and biochemical assays
Medaka were euthanized on collection days (day 0, day 14 and
day 38) with tricaine methanelsulfonate (MS-222, Sigma, St. Louis,
MO) and sh were weighed to the nearest 0.001 g. At each of the
testing time points, all sh in a tank were sampled between 19:00
and 24:00 h to minimize diurnal effects of reproductive cycle hor-
mones. Brains, livers, and gonads were dissected out and preserved
accordingly (see below) for a particular assay. Gonads and livers
were weighed to the nearest 0.0001 g. Aromatase protein content
232 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239
was measured using a tritiated water assay in fresh samples of brain
and ovary of two female sh from each replicate of each treatment
according to the methods of Orlando et al. (2002). 17-estradiol and
testosterone were measured by RIA on all the eviscerated carcasses
of medaka (Heppell and Sullivan, 2000).
2.7. Histology
Gonads of medaka were examined histologically to determine
reproductive stage and to evaluate pathological lesions. The whole
ovary of one female from each replicate of each treatment and
the testis of one male from three replicates of each treatment at
each time point were preserved in Davidsons solution. Prepara-
tion of tissues followed standard histological techniques (Luna,
1968). Briey, tissues were rinsed in two changes of 10 mM HEPES
buffer (pH 7.4) and dehydrated by immersion in graded aqueous
solutions ranging from 50% to 100% ethanol. This was followed
by immersion in xylene and subsequent inltration with parafn.
Tissue blocks were sectioned at 5 m thickness using a standard
microtome. Four sections 20 steps apart were taken at the approx-
imate midpoint of the ovary then mounted on glass slides and
stored at room temperature until staining. In preparation for stain-
ing, the longitudinal sections were dewaxed with xylene and then
rehydrated to water by immersion in graded aqueous solutions
containing decreasing amounts of ethanol ranging from 100% to
0%. Ovaries were stained with Harris hematoxylin and eosin (H &
E) and testes with Geimsa or H & E for histological analysis under
a compound microscope. Two readers each evaluated a section
of ovary to quantify the oogenic stages including pre-vitellogenic
(stage II), early vitellogenic (stage III; central germinal vesicle (GV)),
mid-late vitellogenic (stage IV; GV moving toward animal pole),
and mature (stage V; GV at the animal pole) (Iwamatsu et al.,
1988). The oocytes in each section were counted, percentages cal-
culated, and an average determined for each sh based on the two
sections evaluated. One-half or more of each testis was serial sec-
tioned and evaluated by a single reader for stage of development
and pathological lesions. Classication of testis reproductive stages
generally followed that described in Johnson et al. (2009). Medaka
testes were classied as follows: tubules containing spermatogonia
singly or in clusters predominate (stage II); spermatocytes preva-
lent (stage III); mostly spermatids with some spermatozoa in duct
(stage IV); and duct greatly expanded and lled with spermato-
zoa (stage V). Clastogenic abnormalities (e.g., bridges, chromosome
fragments, micronuclei) were enumerated for all stages of mitoic
spermatogonia but primarily at anaphase and telophase (Izyumov
and Talykina, 2007). The percent of cells with chromosomal abnor-
malities were identied on 1000 spermatogonial cells in each
shs two testicular lobes and averaged per sh. Abnormalities
were visually identied by direct observation by a reader with oil
immersion at 1000 magnication on an Olympus CX31 compound
microscope.
2.8. Statistics
All of the statistical analyses were conducted using SAS sta-
tistical software (SAS , Cary NC) with the probability of a type I
error set at 5% (p 0.05). The statistical unit is the tank. Although
tanks were randomly assigned a position in a row and rows were
randomly assigned to a treatment, tank position could not be
completely randomized throughout the waterbath diluter system
due to constraints on delivery of the test solutions. However, for
each dose level there were three rows allowing for broad spa-
tial distribution of the tanks throughout the diluter water bath.
Cumulative mean egg counts per tank were compared with a SAS
mixed model procedure with repeated measures and least square
means contrasts for differences between control and treatments.
1600
Control
Cumulative mean eggs/Breeding tank
1400 0.05 ug/L
5.0 ug/L
50 ug/L
1200
1000
*
800
600
400
200
0
0 5 10 15 20 25 30 35 40
Exposure period (d)
Fig. 1. Mean cumulative egg production (number/tank) of medaka exposed to
atrazine. Cumulative egg counts were compared with SAS Statistical Software GLM
procedure and least square means (p = 0.05). The rst day in which cumulative egg
numbers were signicantly different from controls is designated with an asterisk.
Mean egg production rates (eggs/tank with spawn/day) were
compared using GLIMMIX procedures of SAS with a Pois-
son distribution. Weekly spawning rates (no. of tanks with
eggs/treatment/week) were compared using GLIMMIX procedure
with a logit distribution and the total number of spawns per treat-
ment was compared using GLIMMIX procedures and the likelihood
of spawning with a binomial distribution. Contrasts of steroid
concentrations, aromatase activity in females, abnormal spermato-
gonia, histological abnormalities, and GSI were conducted using
GLM procedures with least square means comparisons for analy-
sis of variance or co-variance, or KruskalWallis non-parameteric
analysis of ranks where assumptions of normality or heterogeneity
of variances were violated. Ratio data (e.g., E/T) were log trans-
formed and proportional data (e.g., GSI, abnormal spermatogonia,
percent dead embryos) were arcsine transformed for analysis.
Data for abnormal spermatogonia were tested for within time-
treatment differences with a Students t-test. When no differences
existed, the data from sh collected at different times for a treat-
ment were pooled for analysis. Comparisons between the controls
and treatments of the mean percent of pre-vitellogenic oocytes
in ovary sections were done using an odds ratio and chi-square
statistic.
3. Results
3.1. Exposure and adult mortality
Atrazine exposure remained fairly constant and near nominal
concentrations for the entire course of the experiment (Table 1).
Mean atrazine concentrations in tank water over the 38-d expo-
sure were <MDL, 0.56 (0.02), 5.63 (0.10), and 51.5 (1.7) in the
control, low, medium and high atrazine dose groups, respectively
(Table 1). Water concentrations of atrazine measured by the ELISA
were veried to be in good correspondence with measurements
made by GC/NPD (see Supplemental information). Metabolites of
atrazine were not targeted for analysis due to the ow-through
nature of the exposure systems. No adult mortality was observed
in any of the treatment groups.
3.2. Egg production and spawning
Egg production (cumulative mean number of eggs/tank) was
reduced at all concentrations of atrazine (Fig. 1). Cumulative mean
egg production was 1449 eggs at 0 g/L, 885 eggs at 0.5 g/L, 805
eggs at 5.0 g/L, and 769 eggs at 50 g/L. Reductions in cumulative
 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239 233

Water concentrations (g/L) of atrazine in medaka exposure tanks. Mean and standard deviation of atrazine ELISA determinations from each exposure tank with n = 12 (days 110) or n = 6 (days 1435), except Day 13 treatment
80

(eggs/tank with a spawning event/d)

Mean (SE)b

5.63 (0.10)
0.56 (0.02)
Control

51.5 (1.7)
0.5 ug/L
70 5.0 ug/L *

<MDL

Mean egg production

50 ug/L

60 * * *

5.79 (0.83)
0.58 (0.41)
50

48.6 (6.1)
Day 35

<MDL
40

30

65.12 (8.4)
7.35 (0.65)
0.68 (0.05)
20
Day 31

<MDL

10
0 1 2 3 4 5 6 7

Weeks exposed
6.61 (0.67)
0.60 (0.08)

57.1 (7.7)
Day 28

<MDL

Fig. 2. Mean + SE egg production (eggs/tank with a spawning event/day) of medaka

exposed to atrazine. Mean egg production was calculated as the mean of the daily
number of eggs produced per tank with a spawning event over a given week. Sta-
tistical analysis of mean egg production rates was conducted with SAS Statistical
5.15 (0.45)
0.36 (0.06)

Software using GLIMMIX procedure with a Poisson distribution and a critical value of
49.7 (5.0)

p = 0.05. The weeks for which daily egg production in treated tanks was signicantly
Day 24

<MDL

different from controls is designated with an asterisk.

mean egg production rates were signicantly different (p 0.05)

5.39 (0.47)
0.55 (0.09)

45.4 (5.2)

beginning day 24 for the 0.5 and 5.0 g/L treatment groups and
Day 21

<MDL

day 25 for the 50 g/L treatment group and remained different

to the end of the study. Cumulative total numbers of eggs pro-
Exposure day

duced in each treatment at the end of the study were 10,228 eggs at
0 g/L, 6524 eggs at 0.5 g/L, 5923 eggs at 5.0 g/L, and 6059 eggs
4.60 (0.51)
0.50 (0.07)

46.0 (1.8)

at 50 g/L. This corresponds to reductions in eggs produced of 36%

Day 19

<MDL

(0.5 g/L), 42% (5.0 g/L), and 41% (50 g/L) relative to the control
treatment.
Brood groups in all tanks produced eggs at least 1 day during the
4.24 (0.13)
41.07 (5.5)
0.36 (0.06)

experimental period. Weekly rates of egg production, as a mean of

Day 17

<MDL

daily egg production in tanks with eggs, were signicantly reduced

in all atrazine treatments beginning week 3 through week 6 of
the exposure (Fig. 2). Although there were no statistically signif-
6.11 (0.41)
40.9 (16.1)
0.55 (0.09)

icant reductions in spawning events (days with eggs produced in

a tank; Supplemental information), there was a total of 64, 59, and
Day 14

<MDL

59 tank observations when no eggs were produced in the 0.5, 5.0,

and 50 g/L treatments, respectively, compared to 47 observations
when no eggs were produced in the control tanks. Histograms of
5.30 (0.46)
0.48 (0.10)

Method detection limit (MDL) was 0.05 g atrazine/L for ELISA measurements.
47.7 (7.1)

daily tank egg production show treated tanks were skewed toward
Day 10

<MDL

a lower rate of production (Fig. 3). Average daily egg production in

Treatment mean and standard error (SE) across entire exposure period.

the control tanks ranged from 0 to 70 eggs with 4050 eggs being
the modal rate (Fig. 3). Atrazine-treated tanks produced no more
than 40 eggs per day with 2030 eggs produced with the great-
4.89 (0.40)
0.52 (0.09)

46.1 (6.4)
0 g/L in which one sample was lost, thus n = 5 for that treatment.

est frequency (Fig. 3). Mean percent mortality + SD of the embryos

<MDL
Day 7

24 h after collection was, 44 + 23, (0.5 g/L), 41 + 19 (5.0 g/L), and

45 + 20 (50 g/L) and was not affected by atrazine exposure relative
to the control group (35 + 18) (Supplemental information).
5.25 (0.40)
0.62 (0.09)

53.3 (6.3)
<MDL
Day 3

3.3. Steroids and aromatase activity

Nominal concentration treatment groups.

Atrazine did not signicantly affect steroid hormone concen-

6.94 (0.74)
68.3 (10.5)
0.74 (0.11)

trations in either sex at either sampling time (Tables 2 and 3). No

signicant differences in E/T ratios were observed between control
<MDLc
Day 1

and treated sh at either collection time point (Tables 2 and 3).

Gonad and brain aromatase protein content (CYP19 isoforms) in
female medaka were not signicantly different among atrazine
Treatmenta (g/L)

treatments or controls at either 14 or 38 days.

3.4. Testiscular staging

Table 1

Mean GSI values were not signicantly different among treat-

0.5
5.0
50
0

ments (Table 2). Testes in all sh at all sampling times including

234 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239

20
0 g/L 20

15 15
Count

10 10

5 5

0 0
20
20

15
15
Count

10 10

5 5

0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

Mean number of eggs produced daily in a tank

Fig. 3. Frequency histograms of daily egg production in medaka exposed to four concentrations of atrazine. Ranges of egg production rates (X-axis) reect the means of daily
tank production counted over the 38-d experimental period (Y-axis).

at the time of stocking were either at stage III or IV. Thirty-three
percent of medaka testes at stocking (day 0) were at stage III and
66% were at stage IV (Supplemental information) and all control
sh at both 14 d and 38 d were at stage IV. Conversely, a maximum
of one out of three sh sampled from all atrazine treatments was
at stage IV at day 14 and two of three sh were at stage IV after 38
days (Table 2).
3.5. Testicular genotoxicity and pathology
The percent of abnormal mitotic events (labeled as percent
aberrant spermatogoina in Table 2) were numerically greater than
controls at all concentrations of atrazine for the combined time
points (p = 0.0076). The majority of abnormalities were associated
with delay or failure of chromosomes to reach the equatorial plate

Table 2
Male end points: mean concentrations (SD) of estradiol (E2 ) and testosterone (T), E/T ratio, gonad somatic index (GSI), aberrant spermatogonia, and testis histopathology in
medaka exposed to atrazine for 14 or 38 days. Values with asterisks (* ) are signicantly different from controls (p < 0.05).

Day Dose (g/L)

n Control n 0.5 n 5.0 n 50

Estradiol (ng/g)a 14 6 3.2 (4.4) 6 2.4 (1.6) 6 1.7 (1.0) 6 2.4 (2.0)
38 6 2.6 (1.7) 6 1.7 (0.8) 5 1.9 (0.7) 6 1.8 (1.2)
Testosterone (ng/g)a 14 5 1.8 (1.3) 6 1.5 (1.5) 6 1.1 (1.1) 6 1.4 (0.9)
38 5 1.7 (1.5) 5 2.6 (4.0) 5 9.7 (14.0) 6 0.8 (0.8)
E/T ratio 14 5 3.5 (4.4) 6 2.4 (1.2) 4 3.2 (1.8) 5 2.2 (1.3)
38 5 2.4 (1.6) 5 2.2 (2.1) 4 2.8 (4.9) 6 4.1 (3.7)
GSI (%)b 14 6 0.5 (0.3) 6 0.5 (0.6) 5 1.6 (1.9) 6 1.3 (2.3)
38 6 0.3 (0.2) 6 0.4 (0.1) 6 1.4 (2.8) 6 0.4 (0.2)
Testes stagingc
III 14 2 0 2 2 3 2 3 2
IV 14 2 0 1 1
III 38 3 0 3 1 3 1 3 1
IV 38 3 2 2 2
Spermatogoniad
Aberrant (%) 8 10 (5) 6 16 (5)* 6 29 (22)* 5 20 (8)*
Gonad 14 2 200 2 110 3 021 3 120
Histopathologye 38 3 210 3 300 3 120 3 021
a
Estradiol and testosterone were determined by RIA (Heppell and Sullivan, 2000) reported as ng/g body weight and standard deviation (SD).
b
GSI = [gonad weight/(body weight gonad weight)] 100.
c
Gonad staging is reported as the mode.
d
Data from 2 or 3 time points were pooled for analysis. Abnormalities consisted of chromosome bridges, fragments, and micronuclei.
e
Numbers of sh with no, minimal, or moderate pathology are shown in the XYZ format.
 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239 235

Table 3
Female end points: mean concentrations (SD) of estradiol (E2 ), testosterone (T), E/T ratio, gonad somatic index (GSI), egg staging, and gonad and brain aromatase activity in
medaka exposed to atrazine for 14 or 38 days. Values with an asterisk (* ) are signicantly different than control values.

Day Dose (g/L)

n Control n 0.5 n 5.0 n 50

a 14 6 7.2 (4.1) 6 5.0 (2.1) 6 5.8 (3.7) 6 6.9 (4.0)
Estradiol (ng/g)
38 6 7.0 (6.1) 6 3.7 (1.4) 6 7.9 (8.4) 6 4.4 (2.0)
a 14 6 6.2 (9.9) 6 1.3 (1.2) 6 2.5 (2.2) 6 1.7 1.3)
Testosterone (ng/g)
38 6 2.7 (2.4) 6 2.6 (2.4) 6 1.5 (1.0) 6 2.6 (4.0)
E/T ratio 14 6 5.2 (4.0) 6 6.7 (4.7) 6 4.2 (3.2) 6 7.9 (7.7)
38 6 6.0 (9.2) 6 4.8 (6.1) 6 5.5 (2.8) 6 7.0 (7.3)
GSI (%) 14 6 4.4 (2.1) 6 4.4 (1.5) 6 3.8 (2.6) 6 6.5 (4.2)
38 6 5.3 (3.3) 6 4.0 (2.2) 6 4.5 (3.3) 6 3.4 (2.4)
Oocyte stagingb
II (%) 14 5 59 (12) 5 70 (9) 6 63 (22) 6 61 (12)
III (%) 14 28 (4) 21 (13) 24 (9) 27 (15)
IV (%) 14 11 (9) 9 (15) 11 (16) 9 (11)
V (%) 14 3 (4) 0 (0) 2 (6) 3 (4)
II (%) 38 3 79 (14) 5 60 (21)* 4 60 (18)* 4 71 (15)
III (%) 38 9 (1) 19 (8) 19 (8) 19 (11)
IV (%) 38 10 (12) 18(19) 15 (9) 8 (7)
V (%) 38 2 (3) 3 (4) 6 (11) 1 (3)
Gonad aromatasec (pmol/mg protein) 14 6 16 (5) 6 13 (5) 6 14 (6) 6 11 (3)
38 6 15 (8) 6 22 (5) 6 13 (8) 6 12 (9)
Brain aromatasec (pmol/g protein) 14 5 9 (6) 6 7 (4) 6 14 (7) 5 13 (7)
38 5 10 (4) 6 13 (8) 5 17 (11) 5 18 (17)
a
Estradiol and testosterone were determined by RIA (Heppell and Sullivan, 2000) and reported as ng/g body weight. GSI = [gonad weight/(body weight gonad
weight)] 100.
b
Egg staging was determined on histological sections. Previtellogenic(II) were undeveloped, perinucleolar oocytes at various stages or perinucleolar and cortical alveoli
oocytes with no vitelline granules; vitellogenic were vitellogenic oocytes containing moderate numbers of vitelline granules(III) or fully developed oocytes with an abundant
amount of vitelline granules(IV); and mature were fully developed oocytes with germinal vesicles which had migrated to the vegetal pole(V).
c
Aromatase activities were determined as described (Orlando et al., 2002.

in late anaphase and early telophase, irregular movement of chro-
mosomes to the poles, and exclusion of lagging chromosomes with
formation of micronuclei (Supplemental information). Testicular
lesions were limited to germ cell syncytia and tissue degeneration
(Supplemental Information). In the ten control and 0.5 g atrazine-
exposed males, only two sh showed minimal lesions whereas, in
ten out of the twelve 5.0 and 50 g/L atrazine-exposed males lesion
severity was considered minimal or moderate (Table 2).
3.6. Ovarian staging and pathology
Mean GSI values were not signicantly different among treat-
ments (Table 3). Pre-vitellogenic and early vitellogenic stage
oocytes predominated in ovary sections prior to the beginning
of the exposures (Supplemental information). This condition per-
sisted in unexposed ovary sections (control) through the rst 14 d
of the experiment (Table 3). However, by day 38, pre-vitellogenic
oocytes comprised the majority of the control ovary sections
(Table 3). The composition of ovary sections of medaka exposed
to atrazine at 14 and 38 d of exposure was similar to the unex-
posed sh after 14 days (Table 3). There were signicantly fewer
pre-vitellogenic oocytes and more vitellogenic oocytes in sh from
the 0.5 and 5.0 g/L treatments compared to the controls 38 d
after atrazine exposure (p = 0.0036). No pathological lesions were
observed in the ovaries.
4. Discussion
Atrazine has been reported to have effects on the reproductive
systems of organisms from ve separate vertebrate taxa (mam-
mals, birds, amphibians, reptiles, and sh) (Cooper et al., 2007; Rohr
and McCoy, 2010; de la Casa-Resino et al., 2012; Neuman-lee et al.,
2013). In seven freshwater and marine species of sh, atrazine has
demonstrated effects on reproductive physiology, behavior, or out-
comes (Fortin et al., 2008; Rohr and McCoy, 2010). Here we present
the rst evidence of reproductive effects of atrazine on Japanese
medaka with additional genotoxic effects on male germ cells.
4.1. Egg production
The total number of eggs produced by medaka exposed to
atrazine fell below egg production of unexposed sh by the third
week of exposure ultimately resulting in 3642% fewer eggs being
produced by atrazine-exposed sh. Initially, the pattern of egg pro-
duction among the tanks was similar. Mean daily egg production in
the unexposed tanks tended to be greater, more variable, and fol-
lowed a tri-modal periodicity, whereas tank production tended to
be lower, less variable, and there was a attened slightly bi-modal
pattern in the atrazine exposure tanks (Supplemental information).
Moreover, the patterns were slightly desynchronized. The egg pro-
duction pattern observed in the control tanks likely reects the
normal pattern. A similar pattern with three peaks of production
over the course of a 39-d breeding period was observed by Leaf et al.
(2011). Rate of egg production, as calculated per female, in unex-
posed tanks was variable but similar to that reported by others
(Egami, 1959; Grady et al., 1991; Leaf et al., 2011). Extreme vari-
ability in individual medaka daily egg production has been noted
in rearing trials of 24 d duration (Davis et al., 2002). Egg produc-
tion can also vary seasonally with the greatest numbers of eggs
produced at mid-season and fewer eggs being produced at the
beginning and end of the season (Grady et al., 1991; Leaf et al.,
2011). During the 38 d of this study, a decline in egg production
(all treatments) at the end of the study was not observed however,
the fewest eggs were produced the rst week of the experiment
(Supplemental information). Medaka were rst-time spawners in
our study and therefore initial production would be expected to be
low; constant temperature and photoperiod removed any seasonal
effect. The multi-modal pattern of egg production we report has not
been previously described for medaka. That such a pattern would
develop is reasonable given that medaka females typically release
236 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239
eggs for several days then may pause for a day or two before resum-
ing oviposition. Under conditions wherein all sh are exposed to the
same constant environmental conditions and sh are in breeding
groups where hormonal and behavioral cues will be similar, it is
possible that a cyclical pattern such as we observed could develop.
Approximately study day 24, egg production in atrazine tanks
fell below that in the control tanks and remained depressed.
Although very few studies have investigated atrazine effects on
realized fecundity in sh species, reduced egg production has
been previously associated with atrazine exposure. Kettle et al.
(1987) explained their observed decrease in bluegill reproduction
in ponds treated with 20 and 500 g/L atrazine as an indirect effect
resulting from atrazines direct toxicity to macrophytes. However,
the design of that study was insufcient to dismiss an atrazine
effect on bluegill reproduction. Bringolf et al. (2004) reported a
trend of fewer total eggs produced by fathead minnow at 5 and
50 g/L atrazine but these results and their observed reduction
in eggs/spawn/female were not statistically signicant. However,
three males in unspecied treatments were misidentied at stock-
ing and were found to be females at the conclusion of the study.
These tanks were not removed from analyses. A reanalysis of
the data accounting for the additional eggs produced by these
females may change the conclusion (R. Bringolf, pers. comm.).
Design deciencies confounded interpretation of a third study
wherein fathead minnow breeding groups were exposed to two
concentrations of atrazine (Battelle, 2005). Mean fecundity as egg
production/female/day was 22% lower for the 25 and 250 g/L
atrazine groups compared to the control (Battelle, 2005). This result
was not statistically signicant. However, according to the authors,
there was very low power to detect a difference if one existed.
Moreover, at the end of the study two males, one in a control tank
and another in a low dose tank, were found to have undeveloped
gonads; the tanks were not removed from analyses. Whether or not
tank egg production was affected by these non-reproductive males
is unknown.
The study design of Tillitt et al. (2010) attempted to account
for some of the shortcomings of earlier atrazine studies through
increased statistical power, censoring of tanks with sex ratio stock-
ing errors, extended exposure time, and recovery of unattached
eggs in tank waste water. The methods for the fathead minnow
study reported by Tillitt et al. (2010) matched the testing of medaka
reported here and used the same exposure system. Mean cumula-
tive egg production of fathead minnow in that study was reduced at
all concentrations of atrazine relative to the control just as it was for
medaka in the present study (Tillitt et al., 2010). There were addi-
tional similarities in effects on the two species, but also differences.
For example, egg production in atrazine-treated medaka and fat-
head minnow signicantly diverged from controls at week three
and there was no adverse effect on egg survival. Unlike fathead
minnow, atrazine did not effect a reduction in medaka spawning
frequency, instead clutch size was reduced.
A test exposure period of 21 d as proscribed by the U.S. Envi-
ronmental Protection Agencys 21-d reproduction assay protocol
would have resulted in no signicant effect of atrazine on medaka
and fathead minnow egg production (Tillitt et al., 2010). If the
medaka and Tillitt et al. (2010) fathead minnow studies had been
conducted under a 21-d protocol the results would have been sim-
ilar to the 21-d Bringolf et al. (2004) and Battelle (2005) fathead
minnow studies which showed trends in reduced fecundity but no
signicant effect. The 21-d sh reproductive assay has been shown
to successfully identify effects on fecundity in a large number of
studies (Dang et al., 2011). However, results from a 21-d exposure
may not adequately reect adverse effects on total seasonal egg
production of fractional spawners such as medaka with highly vari-
able inter-female fecundity (Davis et al., 2002) or fathead minnows
that cycle approximately every 3 days to produce 1626 clutches
in a season (Gale and Buynik, 1982). In some cases, longer expo-
sure periods that encompass a greater portion of the spawning
period and greater numbers of replicates to account for among sh
variability may be warranted to reduce the possibility of Type II
errors.
4.2. Oocyte follicle maturation
The prevailing hypothesis explaining atrazines mode of action
centers on an increase in estrogen as a result of increased aromatase
gene expression and activity. Evidence for this hypothesis has come
principally from mammalian and amphibian studies (Hayes et al.,
2010; Fa et al., 2013) and the nding by Suzawa and Ingraham
(2008) that juvenile zebrash acutely exposed to atrazine increase
aromatase gene expression. There has been little evidence from
in vivo sh studies that exposure to atrazine increases aromatase
protein and the evidence that atrazine modulates steroid concen-
trations is conicting (Moore and Waring, 1998; Span et al., 2004;
Battelle, 2005; Nadzialek et al., 2008; Salaberria et al., 2009; Tillitt
et al., 2010). The medaka results presented here also do not support
an effect of atrazine on aromatase or steroids. However, in these
medaka, steroids were measured in whole bodies not circulating
plasma as is typical.
Supporting research has identied additional targets along
steroidogenic pathways where atrazine can have modulatory
effects most notably resulting in increased cAMP levels (Fan et al.,
2007; Suzawa and Ingraham, 2008; Gunderson et al., 2011; Jin et al.,
2013). Thus, organism-level reports of feminization and demas-
culinization attributed to atrazine can be reasonably linked to
estrogenic or anti-androgenic consequences of atrazine exposure.
Recent data also demonstrate atrazines interactions at multiple
points within biochemical pathways fundamental to gametogene-
sis and ovulation (Fan et al., 2007; Suzawa and Ingraham, 2008;
Kucka et al., 2012; Fa et al., 2013). However, despite research link-
ing atrazine to failed ovulation in rats, explanations of how atrazine
could disrupt ovulation in lower vertebrates are less familiar and
accepted (Cooper et al., 2007).
The means by which atrazine may prevent ovulation are dis-
tinct from, yet consistent with, the mechanism by which atrazine
may increase aromatase. In sh and other species, ovulation is pre-
ceded by oocyte follicle maturation whereby the pituitary signals
the follicle to produce maturation inducing hormone (MIH) and
maturation promoting factors (Nagahama and Yamashita, 2008).
Production of MIH requires a shift in production of C18 and C19
steroids (e.g., sex hormones) to production of C21 steroids (e.g.,
MIHs) within an individual follicle. Effectively, vitellogenesis stops,
the oocyte stops growing, and meiosis resumes in preparation for
ovulation. A key requirement in the pathway to oocyte matura-
tion is that cAMP levels increase prior to MIH production, then
decrease subsequent to MIH binding at the oocyte surface (Pace
and Thomas, 2005; Nagahama and Yamashita, 2008). Phosphodie-
sterase (PDE) degrades the second messenger cAMP production in
the ovarian follicle. Without PDE, cAMP accumulates in the folli-
cle and prevents resumption of meiosis (Haider, 2003). Exposure
to atrazine inhibits PDE resulting in an increase in cAMP levels
(Kucka et al., 2012). Therefore, exposure of reproductive females to
atrazine could result in abnormally high levels of cAMP in mature
oocyte follicles which in turn would prevent resumption of meiosis
and subsequent ovulation.
Additional evidence of atrazines potential to inhibit ovulation
exists beyond an hypothesized effect of cAMP on nal oocyte mat-
uration. Thomas and Sweatman (2008) reported atrazine reduced
resumption of meiosis (aka germinal vesicle breakdown) in Atlantic
croaker (Micropogonias undulatus) oocyte follicles in vitro. Through
competitive binding studies, they showed that atrazine competed
with MIH at its binding site on the oocyte. In mammals, the results
 D.M. Papoulias et al. / Aquatic Toxicology 154 (2014) 230239
from several research studies suggested that atrazine interfered
with the pre-ovulatory luteinizing hormone (LH) surge (reviewed
in Cooper et al., 2007). Subsequently, it was found that brain was the
site for atrazine effects and not pituitary gonadotropins (Foradori
et al., 2009). More recently, Fa et al. (2013) proposed that atrazine
targets granulosa cells specically, where atrazine-induced up-
regulation of a transcription factor interferes with a biochemical
cascade necessary for ovulation.
In addition to reduced fecundity, female sh experiencing sup-
pressed oocyte maturation and ovulation might be expected to
have altered GSIs and a different distribution of oocyte stages com-
pared to normally ovulating females. If females are ovulating less
frequently, males may not be engaged in spawning and therefore
spermiation may be reduced or germ cell development may be
affected and as a consequence testicular mass may be different in
atrazine-treated males compared to control males. Female medaka
exposed to atrazine in the present study did not show increases
in GSI. Ovarian histology in 0.5 and 5.0 g/L atrazine-treated sh
showed relatively fewer pre-vitellogenic oocytes than vitellogenic
oocytes when compared to control sh and male GSI tended to
be greater when compared to control sh. Results from previous
studies, that have investigated atrazines effect on sh egg pro-
duction are either equivocal or supportive of an adverse effect
of atrazine on ovulation. Generally, trends of reduced fecundity,
effects on male GSI, and atypical distribution of ovarian or testicular
staging in atrazine-treated sh were observed and notably there
was greater variability in these measurements compared with con-
trols (Bringolf et al., 2004; Battelle, 2005; Tillitt et al., 2010). These
indirect measures of suppressed ovulation may not show statisti-
cal differences from controls, but the trends are consistent among
studies and between species. Moreover, effects occurring at the
level of the oocyte follicle could mute or mask measures at higher
levels (i.e., organ, egg production). This would be particularly true
for sequential, batch-spawning species such as fathead minnow
and medaka with asynchronous oocyte development.
4.3. Germ cell genotoxicity
Atrazine exposure of medaka at concentrations >0.5 g/L
increased the number of abnormal mitoses in spermatogonial cells
by two to three times the number in unexposed sh. This is the
rst report demonstrating a link between atrazine-induced chro-
mosomal abnormalities and aberrant mitotic events in germ cells
of sh. Early reports of atrazines genotoxic effects in animals were
equivocal (Brusick, 1994; reviewed in Freeman and Rayburn, 2004).
More recently, atrazine has been shown to increase the number of
micronuclei in rat liver cells (Campos-Pereira et al., 2012) but whole
cell clastogenicity was not elevated over negative controls in anu-
ran tadpoles (Freeman and Rayburn, 2004). Fish studies are scarce.
The results of three of four studies on separate sh species using the
micronuclei test and or the comet assay were positive for atrazine-
related DNA damage in erythrocytes, gill, or liver cells (de Campos
Ventura et al., 2008; Zhu et al., 2011; Cavas, 2011; Santos and
Martinez, 2012). Thus, the majority of studies which have tested
for genotoxic effects of atrazine in sh at environmentally relevant
concentrations tend to support genotoxicity at low atrazine con-
centrations. The survival and developmental consequences to the
shes offspring of chromosomally defective sperm are not known.
Apical effects measured in the present study did not pro-
portionately increase or decrease with increasing atrazine water
concentrations. Atrazine-reduced medaka egg production and
increased genotoxicity at all three concentrations tested but not in
a dose-dependent manner. Fathead minnow egg production was
similarly affected by atrazine (Tillitt et al., 2010). Low-dose and
nonmonotonic effects of atrazine have previously been reviewed
by Vandenberg et al. (2012) and such effects have also been
237
demonstrated for many chemicals (Calabrese and Blain, 2011). A
combination of the pleiotropic effects, the complex feedback loops
along the brain-pituitary-gonad axis, and the homeostatic mecha-
nisms that support reproductive success likely contribute to the
difculty in reconciling exposure and effects of atrazine. Future
application of advancedomics technologies may prove valuable
in furthering an understanding of atrazines effects on sh repro-
duction.
Disclaimer
Any use of trade, product, or rm names is for descriptive
purposes only and does not imply endorsement by the U.S. Gov-
ernment.
Acknowledgments
Support for this work was provided by the USGS Environmental
Contaminants Biology Program. The authors thank James Candrl,
Mandy Annis, Diane Nicks, James L. Zajicek Vanessa Velz, Rachel
Claunch, Justin Buckler, Matt Keuss, Eugene Greer, Doug Hardesty,
Robin Lipkin, and Dina Boyt for their contributions to the con-
duct of this study; Dr. Mark Ellerseick, University of Missouri, for
statistical analysis; Dr. Jeffrey Wolf, Experimental Pathology Lab-
oratories, Sterling VA, for pathological analysis of gonads; and Dr.
Adria Elskus, USGS for reviewing an earlier draft of this manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.aquatox.2014.05.022.
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