Pharmaceuticals and Personal Care Products in the Environment

GROWTH AND DEVELOPMENT OF TADPOLES (XENOPUS LAEVIS) EXPOSED TOSELECTIVE SEROTONIN REUPTAKE INHIBITORS, FLUOXETINE AND SERTRALINE,

THROUGHOUT METAMORPHOSIS

DEANNA E. CONNERS,{ EMILY D. ROGERS,{ KEVIN L. ARMBRUST,{ JEONG-WOOK KWON,{ and

MARSHA C. BLACK*{{Department of Environmental Health Science, University of Georgia, Athens, Georgia 30602, USA

{Mississippi State Chemical Laboratory, Mississippi State University, Mississippi State, Mississippi 39762, USA

(Received 30 September 2008; Accepted 17 February 2009)

Abstract—Selective serotonin reuptake inhibitors (SSRIs) are widely prescribed drugs that are present in sewage effluents and surfacewaters. The objective of the present study was to determine whether low environmentally relevant concentrations of the SSRIsfluoxetine and sertraline could impair growth and development in tadpoles of the African clawed frog (Xenopus laevis) and to evaluateif such effects may be caused by a disruption of the neuroendocrine system. Tadpoles were exposed to SSRIs at concentrations of 0.1,1, and 10 mg/L for 70 d throughout metamorphosis. No effects on deformities were observed. Tadpoles exposed to fluoxetine (10 mg/L)and sertraline (0.1, 1, and 10 mg/L) exhibited reduced growth at metamorphosis. Tadpoles exposed to sertraline (0.1 and 1 mg/L)exhibited an acceleration of development as indicated by an increase in the time to tail resorption. The effects of SSRIs on growth anddevelopment in tadpoles were likely driven by reduced food intake. Reduced feeding rates were observed in SSRI-exposed tadpoles,and nutritional status can influence growth and development in amphibians via effects on the neuroendocrine system. Only sertralinewas capable of causing developmental toxicity in tadpoles at environmentally relevant concentrations. These data warrant additionalresearch to characterize the risks to human health and wildlife from pharmaceutical exposures.

Keywords—Anurans Endocrine disruption Fluoxetine Pharmaceuticals Sertraline

INTRODUCTION

The selective serotonin reuptake inhibitors (SSRIs) fluox-

etine and sertraline are widely prescribed drugs for the

treatment of depression, obsessive compulsive behaviors,

anxiety, and premenstrual dysphoric disorder in humans.

Selective serotonin reuptake inhibitors are an important class

of emerging environmental contaminants along with many

other pharmaceuticals and personal care products [1]. Pres-

ently, the risks associated with environmental exposures to

pharmaceuticals have not been well-characterized.

Low concentrations of SSRIs (#0.1 mg/L) have been

detected in sewage effluents and surface waters [2,3]. Johnson

et al. [4] estimated that predicted environmental concentra-

tions for surface waters may be as high as 0.2 mg/L for

fluoxetine and 0.6 to 1.2 mg/L for sertraline. Fluoxetine and

sertraline were also detected as contaminants in fish tissue [5].

In pregnant women taking prescriptions, these chemicals are

known to contaminate breast milk (fluoxetine ,2–384 mg/L;

sertraline 7–207 mg/L) and infant plasma (fluoxetine ,1–

265 mg/L; sertraline 0–1.5 mg/L) [6,7].

Developmental abnormalities were observed in fish [8]

exposed to low environmentally relevant concentrations of

fluoxetine. Adverse effects on development (e.g., reduced

growth) have been observed in neonatal rats exposed to

fluoxetine [9] and sertraline [10]. In humans, research indicates

that exposure to SSRIs during gestation or via breast milk can

cause developmental toxicity in infants (e.g., hypoglycemia,

respiratory distress, low birth weights, and reduced postnatal

growth) [6,11].

The mechanisms by which SSRIs may cause adverse

developmental effects are currently unknown. The objective

of the present study was to determine whether low environ-

mentally relevant concentrations of fluoxetine and sertraline

can impair growth and development in tadpoles of the African

clawed frog (Xenopus laevis) and to evaluate if such effects may

be caused by a disruption of the neuroendocrine system.

African clawed frogs are ideal organisms to investigate the

ecological and human health implications of exposures to low

concentrations of SSRIs in the environment. Xenopus laevis are

aquatic frogs that are easy to culture and breed in the laboratory,

and standardized protocols exist to explore the effects of

xenobiotics on growth and development of embryos [12] and

tadpoles [13]. Ecologically, frogs are an important test species to

use in toxicity tests because of environmental concern over

chemically induced deformities and declining amphibian popu-

lations [14,15]. In regards to human health, frogs are an

important model for studies of postembryonic organ develop-

ment [16] and chemically mediated thyroid dysfunction [17,18].

Current research indicates that the SSRI fluoxetine may

affect the thyroid system of mammals either by decreasing the

amount of thyrotropin-releasing hormone (TRH) produced by

the hypothalamus or by elevating the concentration of

triiodothyronine (T3), the more potent of two active thyroid

hormones (T3 and T4), in extrathyroidal tissues via effects on

the activities of iodothyronine deiodinases type II and type III

[19,20]. Fluoxetine is also known to affect behaviors controlled

by the production of corticotropin-releasing factor (CRF) in

the hypothalamus of fish and newts [21]. Interestingly, in frogs

CRF is the thyrotrophic hormone responsible for pituitary

production of thyroid-stimulating hormone (TSH), whereas in

mammals TSH release is controlled via TRH [22]. Together* To whom correspondence may be addressed (mblack@uga.edu).Published on the Web 7/2/2009.

Environmental Toxicology and Chemistry, Vol. 28, No. 12, pp. 2671–2676, 2009’ 2009 SETAC

Printed in the USA0730-7268/09 $12.00 + .00

2671

these data suggest that there may be a variety of mechanisms

by which SSRIs can interact with the hypothalamus–pituitary–

thyroid axis or the hypothalamus–pituitary–interrenal axis in

frogs and interfere with growth and development (Fig. 1).

Nonendocrine effects (i.e., neurological) may also be an

important contributor to the developmental toxicity of SSRIs

(e.g., serotonin controls left–right patterning in frog and chick

embryos [23] and serotonin plays a trophic role in digestive

processes such as food intake [24] and peristalsis [25]).

In the present study, a randomized block design was used to

expose tadpoles to fluoxetine and sertraline (0.1, 1, and 10 mg/L)

for 70 d throughout development. Biological end points that

were analyzed included mortality, deformities, growth, and

development. Both fluoxetine and sertraline caused develop-

mental toxicity in frogs, but only sertraline was capable of

producing effects at low environmentally relevant concentra-

tions. The type and timing of adverse effects suggest that SSRIs

may disrupt the neuroendocrine system of developing tadpoles.

MATERIALS AND METHODS

Frog breeding

All frog care complied with the University of Georgia’s

Animal Use Guidelines. Two certified breeding pairs of adult

Fig. 1. Hormonal control over morphological traits in developing Xenopus laevis tadpoles and locations where selective serotonin reuptake inhibitors(SSRIs) may interfere with metamorphosis based on amphibian endocrinology information [15,22,39,40] and on information on SSRI and endocrineinteractions [19–21]. CRF 5 corticotropin-releasing factor; TRH 5 thyrotropin-releasing hormone; TSH 5 thyroid-stimulating hormone; ACTH 5adrenocorticotropic hormone; PRL 5 prolactin; TH 5 thyroid hormones T3 and T4.

2672 Environ. Toxicol. Chem. 28, 2009 D.E. Conners et al.

frogs (Xenopus laevis) were obtained from Carolina Biological

Supply Company. Frogs were not fed prior to breeding and

were held for 9 d in a 40-L flow-through aquarium containing

dechlorinated tap water (23uC) (density of four frogs per

1,800 cm3 of water) (12:12 h light/dark cycle). Breeding was

conducted by methods similar to those outlined by the

American Society for Testing and Materials [13]. Briefly,

female frogs (9 and 10 cm in length) were injected with 1,000

International Units (IU) of sterile chorionic gonadotropin

(1 ml total volume) (ChorulonH Intervet, Schering-Plough

Animal Health) via the dorsal lymph sac, and frogs were

placed into individual breeding chambers containing 10 L

FETAX solution (21uC) (water depth of 6 cm above plastic

mesh bottom). Then, male frogs (7 and 8 cm in length) were

injected with 500 IU of chorionic gonadotropin (0.5 ml total

volume) and placed in breeding chambers along with females.

Breeding was allowed to proceed overnight for 14 h. Fertilized

embryos were gently flushed with a plastic transfer pipette into

a glass aquarium containing 40 L of aerated FETAX solution

(23uC). Embryos were allowed to hatch and develop for 3 d

prior to toxicity testing. By 3 d, tadpoles that had reached

Nieuwkoop and Faber [26] stages 42 and 43 of development

and were 7.0 to 8.0 mm in length were used to start the toxicity

tests.

Tadpole exposures

Exposure of tadpoles to SSRIs was conducted similar to the

protocol outlined by Opitz et al. [13], who validated a sensitive

method to detect endocrine-disrupting effects of xenobiotics

on the thyroid system. Fluoxetine was purchased as fluoxetine

hydrochloride (98.7% purity, Spectrum Chemicals and Labo-

ratory Products), and sertraline was purchased as sertraline

hydrochloride (98% purity, Toronto Research Chemicals).

Tadpoles were exposed to fluoxetine and sertraline at

concentrations of 0.1, 1, and 10 mg/L in FETAX solution

(pH 8.0 6 0.2; alkalinity 57 6 7 mg/L; hardness 104 6 7 mg/

L; data are means 6 standard deviation). A negative control

(FETAX solution) and a positive control (10 mg/L ammonium

perchlorate, which is a thyroid hormone inhibitor) were also

used in the experiment. Three replicate glass aquaria were used

for each treatment (n 5 3), except for the positive control (n 5

1). Glass aquaria contained 10 L of aerated test solution, and

28 tadpoles were allocated randomly to each aquarium.

Tadpoles were exposed to SSRIs for 70 days (12:12 h light/

dark cycle). Water in aquaria was renewed by 75% three times

per week. One time each week, prior to a water change, water

was analyzed for temperature (21.4 6 0.6uC), dissolved oxygen

(7.65 6 0.24), conductivity (1,421 6 44 ms), and total ammonia

nitrogen (0.7 6 0.2 mg/L) (data in parentheses are means 6

standard deviation).

Tadpoles were fed daily during exposures. Dry Nasco

Xenopus Frog BrittleTM for tadpoles was mixed with FETAX

solution in a blender (10 g of food to 100 ml of FETAX

solution) and dispensed into aquaria at a rate of 0.5 ml for

exposure days 1 to 7, 1.0 ml for exposure days 8 to 16, 2.0 ml

for exposure days 17 to 24, and 1.0 ml for exposure days 25 to

70. Feeding rates were adjusted during the experiment in an

attempt to have little to no food left remaining on the bottom

of aquaria the morning after feeding. However, this adjust-

ment was only successful for control tadpoles. Tadpoles

exposed to SSRIs ate less food than control tadpoles; hence

more uneaten food was observed on the bottom of these

aquaria.

Concentrations of SSRIs in water and tadpole tissue were

determined periodically throughout the experiment. On expo-

sure days 21 and 39, water samples (50 ml) were collected from

each replicate aquarium and composited (150 ml total volume)

both before and after water changes. Tadpole tissues were

sampled on exposure day 44 (composite of six tadpoles per

treatment, two tadpoles from each replicate aquarium). All

samples were stored frozen (220uC) until analyzed by liquid

chromatography–mass spectrometry (LC/MS). Water samples

from 1 and 10 mg/L treatments were analyzed by direct

injection onto the LC/MS apparatus, whereas water samples

from 0.1 mg/L treatments were analyzed by LC/MS after solid-

phase extraction (SPE, Bakerbond OctadecylH C18, 1,000 mg/

6 ml, Malinckrodt Baker). The recovery range for SPE water

samples was 83.5 to 87.2%. Tadpole tissues were extracted in

acetonitrile/methanol (1:1, v/v). Tissues (0.3 g) were homoge-

nized in 11 ml of acetonitrile/methanol and centrifuged. Then,

supernatants were decanted, evaporated to 1 ml, and com-

bined with 1 ml of the mobile phase solution (0.1% formic acid

in water/0.1% formic acid in acetonitrile, 1:1, v/v). Samples

were sonicated and filtered (0.45-mm Whatman polytetrafluo-

roethylene syringe filters) prior to injection onto the LC/MS

apparatus. The recovery range for tissue samples was 65 to

71%. Composited tissue samples were run in triplicate.

Analyses of adverse effects

Tadpoles were observed daily for mortalities and weekly for

deformities and stage of development. On exposure day 30,

eight tadpoles were sampled destructively from each aquarium

to determine hind limb growth and body mass of prometa-

morphs. Tadpoles were captured with a mesh net and placed in

a lethal solution of 0.2% FinquelH MS-222 (tricaine methane-

sulfonate, Argent Laboratories) for approximately 2 min.

Hind limb length was quantified with a dissecting microscope

by use of an ocular micrometer. Then, four tadpoles were

processed individually to determine body mass, and four

tadpoles were archived (220uC) for later analyses outside the

scope of the present study. On exposure day 44, six tadpoles

were sampled destructively from each aquarium (described

above) to quantify forelimb emergence and tissue concentra-

tions of SSRIs (described previously). When tadpoles com-

pleted metamorphosis, the time to tail resorption was

recorded, and they were processed individually to determine

body mass. Body mass was quantified by drying tadpoles at

100uC for 60 h in a drying oven, then samples were dessicated

for 24 h prior to determining dry weights on an analytical

balance.

Statistics

Data were analyzed at a significance level of p , 0.05.

Fisher’s exact test was used to evaluate the effects of SSRI

exposures on deformities in tadpoles. The effects of SSRI

exposures on growth (body mass) and development (hind limb

length, forelimb emergence, and time to tail resorption) were

evaluated with analysis of variance (ANOVA) by use of

Systat’ version 11 software (Systat Software). All data were

checked for normality (Shapiro–Wilk’s test) and homogeneity

of variance (Bartlett’s test) prior to use in ANOVAs, and data

were transformed if necessary to meet the assumptions of the

statistical tests. When an ANOVA indicated that there were

significant differences among treatments, Dunnett’s multiple

comparison procedure was used to determine which SSRI

treatments differed significantly from the control treatment.

Effects of pharmaceuticals on tadpole growth and development Environ. Toxicol. Chem. 28, 2009 2673

RESULTS

Tadpole exposures

Concentrations of SSRIs were measured in water and

tadpole tissues (Table 1). Concentrations of fluoxetine and

sertraline in water varied during the course of the static-

renewal experiment. Four hours after a water change,

fluoxetine concentrations were 0 to 19% lower than nominal

concentrations. By 48 h after a water change, fluoxetine

concentrations were 70 to 80% lower than nominal concen-

trations. Four hours after a water change, sertraline concen-

trations were 29 to 40% lower than nominal concentrations.

By 48 h after a water change, sertraline concentrations were 15

to 87% lower than nominal concentrations.

On exposure day 44, tadpoles accumulated higher concen-

trations of sertraline than fluoxetine. Pharmaceutical concen-

trations in tadpoles exposed to 10 mg/L sertraline were sixfold

higher than concentrations in tadpoles exposed to 10 mg/L

fluoxetine.

Analyses of adverse effects

Mortality. No significant effects on mortality were observed

in tadpoles exposed to fluoxetine and sertraline for 70 d. The

experiment mortality rate was 18 6 2%. The majority of

mortalities occurred during the first 7 d of exposures (8 6 1%).

During the remaining time of the experiment (i.e., exposure

days 8 to 70), the weekly mortality rate ranged from 0 to 3%.

Deformities. No significant increases in deformities were

observed in tadpoles exposed to fluoxetine and sertraline. A

low number of deformities were observed in all exposure

treatments, including the controls (Table 2). Deformities

observed included bent tails, edemas, missing or partial eyes,

and missing limbs. The overall prevalence for the total number

of deformities was 3.6%. Slight but insignificant increases in

bent tails were observed in tadpoles exposed to 10 mg/L

sertraline (p 5 0.12).

Growth. No significant effects on growth were observed in

prometamorphic tadpoles exposed to fluoxetine and sertraline

for 30 d (data not shown). Body mass among prometamorphic

tadpoles from different treatments was remarkably similar and

averaged 0.024 6 0.001 g dry weight (n 5 21 aquaria).

Significant effects on growth were observed in fluoxetine (F

5 4.702; df 5 3; p 5 0.006) and sertraline (F 5 5.299; df 5 3; p

5 0.003) exposed tadpoles that had completed metamorphosis

(Fig. 2). Reduced growth was observed in tadpoles exposed to

10 mg/L fluoxetine and 0.1, 1, and 10 mg/L sertraline. Reduced

growth was also observed in tadpoles exposed to 1 mg/L

fluoxetine, but data were not statistically significant. Growth

was reduced by approximately 35% in fluoxetine and sertraline

exposed tadpoles.

Development. No significant effects on development were

observed in tadpoles exposed to fluoxetine and sertraline

during early stages of metamorphosis. On exposure day 30,

hind limb length averaged 5.0 6 0.22 mm in tadpoles from all

treatments. On exposure day 44, the percentage of tadpoles

completing forelimb emergence averaged 54.0 6 3.4% for all

treatments.

Significant effects on development were observed in

tadpoles exposed to sertraline (F 5 5.025; df 5 3; p 5 0.030)

during later stages of metamorphosis (Fig. 3). Development

was accelerated in tadpoles exposed to 0.1 and 1 mg/L

sertraline. These tadpoles completed tail resorption 10 d

earlier than control tadpoles. No effects on tail resorption

were observed in tadpoles exposed to 10 mg/L sertraline.

Table 1. Concentrations of fluoxetine and sertraline measured inwater and tadpole tissue. Water samples were collected at 4 and 48 hafter a water change, and data are medians of samples collected onexposure days 22 and 40. Tissue samples are from tadpoles that were

collected on day 44 of exposures

Treatment

Nominalconcentration

in water (mg/L)

Measured concentrations

Water(mg/L) (4 h)

Water(mg/L) (48 h)

Tissue(ng/g wet wt)

Fluoxetine 0.1 0.15 0.02 ,LODa

1.0 0.89 0.24 ,LODa

10.0 8.07 3.00 0.36 6 0.04Sertraline 0.1 0.06 0.09 ,LODa

1.0 0.67 0.16 0.18 6 0.0010.0 7.08 1.26 2.1 6 0.12

a Less than the limit of detection (LOD) of 0.05 ng/g wet weight.

Table 2. Incidence of cumulative deformities (%) in tadpoles exposed to fluoxetine and sertraline for 70 d. Data are percentages calculated fromprobabilities for the number of deformities observed among the 84 tadpoles that were exposed in each treatment

Treatment Concentration (mg/L) Bent tails Edema Missing or partial eyes Missing or deformed limbs Total deformities

Control 0 0.0 1.2 1.2 0.0 2.4Fluoxetine 0.1 1.2 1.2 0.0 1.2 3.6

1.0 2.4 3.5 0.0 0.0 6.010.0 2.4 0.0 0.0 0.0 2.4

Sertraline 0.1 0.0 1.2 0.0 0.0 1.21.0 1.2 0.0 0.0 0.0 1.2

10.0 3.6 1.2 0.0 1.2 6.0

Fig. 2. Effects of fluoxetine and sertraline on growth (body mass) intadpoles exposed to selective serotonin reuptake inhibitors throughoutmetamorphosis. Data are means 6 standard error, n 5 3 aquaria pertreatment. Asterisks indicate significant differences from controls (p, 0.05).

2674 Environ. Toxicol. Chem. 28, 2009 D.E. Conners et al.

Although not significant, a similar acceleration of develop-

ment was observed in tadpoles exposed to 1 mg/L fluoxetine.

One aquarium of tadpoles was exposed to 10 mg/L

ammonia perchlorate during the experiment as a positive

control. None of the tadpoles exposed to ammonia perchlorate

had completed metamorphosis when the experiment ended

(i.e., 70 d of exposure).

DISCUSSION

Many environmental contaminants have been inadequately

tested for developmental toxicity, especially toxicity that may

result from a disruption of the nervous system or the endocrine

system. In the present study, we observed that the pharma-

ceuticals fluoxetine and sertraline can cause developmental

toxicity in tadpoles. Adverse effects observed included reduced

growth and an acceleration of metamorphosis. No effects on

deformities were observed in tadpoles exposed to SSRIs.

Developmental abnormalities have been observed in fish [8]

exposed to fluoxetine, and birth defects are an important

human health concern for SSRI use during pregnancy. All

tadpoles from this experiment exhibited a low incidence of

deformities, and no significant increase in deformities was

observed among SSRI-exposed tadpoles. These results are

largely consistent with research on fish [8] and epidemiological

studies of infants [27,28] that show that any potential SSRI-

induced defects would be rare and difficult to detect.

Reduced growth was observed in tadpoles that were exposed

to high concentrations of fluoxetine and low, moderate, and high

concentrations of sertraline throughout metamorphosis. Effects

on growth were likely related to reduced rates of feeding among

SSRI-exposed tadpoles, which may have been mediated by an

increase in serotonin levels. Selective serotonin reuptake

inhibitors act by inhibiting the reuptake of serotonin in

presynaptic nerve cells, thereby increasing the extracellular levels

of the neurotransmitter available for binding to postsynaptic

receptors. Elevations in serotonin are known to dramatically

inhibit food intake in mice [24]. In fish and amphibians,

serotonin-mediated neuronal activity may stimulate CRF-

controlled behaviors of the hypothalamus [21], and CRF has

an inhibitory effect on foraging in tadpoles [29].

Reduced growth is a serious sign of developmental toxicity

for both humans and wildlife. Body size of frogs at meta-

morphosis is an important determinant for adult fitness, and

larger body weights may enable frogs a greater ability to

escape predation, seek out new territories, and mate more

successfully [30,31]. A possible corollary of reduced growth in

frogs to human health is low birth weight. Low birth weights

are a leading adverse health outcome for children that can

cause complications later in life such as an increased risk for

chronic illnesses and impaired cognitive development [32].

Although exposures to high concentrations of SSRIs during

gestation can cause low birth weights in infants [6], the

implications for human health of the present results observed

in frogs exposed to low concentrations of SSRIs remain

unclear, especially in light of the proposed mechanism that

SSRIs reduce foraging behavior in tadpoles.

Acceleration of metamorphosis (i.e., increased rate of tail

resorption) was observed in tadpoles that were exposed to low

and moderate concentrations of sertraline. A similar but

insignificant trend was observed in tadpoles exposed to low

concentrations of fluoxetine. Similar to the proposed mechanism

for reduced growth effects, the acceleration observed in

metamorphosis may also be related to reduced food intake in

SSRI-exposed tadpoles. Amphibian larvae are known to

respond to decreased food supplies by initiating metamorphosis

at earlier stages of development as an adaptive response to

escape stressful habitats [22,33,34]. Hence, SSRI exposures may

have chemically tricked tadpoles into earlier metamorphosis by

eliciting false signals to the neuroendocrine system that the

environment was deficient in food. Presently, the molecular

mechanisms by which SSRIs may disrupt the neuroendocrine

system of tadpoles are unknown, but the effects that we observed

on metamorphosis may be related to a stimulation of CRF-

controlled activities of the hypothalamus–pituitary–thyroid axis

or the hypothalamus–pituitary–interrenal axis (Fig. 1).

Interestingly, an acceleration of metamorphosis was not

observed in tadpoles exposed to high concentrations of fluoxetine

or sertraline. Somehow, these tadpoles lost the plasticity to

respond to the nutritional cues elicited by SSRI exposures.

Exposure–response relationships that display stimulation at low

concentrations and inhibition at high concentrations are known

as hormesis [35]. Similar developmental responses have been

observed in tadpoles exposed to atrazine [36]. A variety of

mechanisms may exist for explaining the observed hormetic effect

in SSRI-exposed tadpoles. One plausible mechanism may be that

tadpoles exposed to high concentrations of SSRIs had lower

numbers of either serotonin receptors or CRF receptors, which

would dampen any functional response to elevated serotonin

levels. Negative feedbacks created by a down-regulation of

receptor expression are a common mechanism for the develop-

ment of drug tolerance [37].

In the present study, sertraline was more effective at

causing developmental toxicity in tadpoles than fluoxetine.

Limited data suggest that sertraline may be more potent and

more bioaccumulative than fluoxetine. Henry et al. [38]

demonstrated that sertraline was more potent than fluoxetine

at causing acute mortality in Ceriodaphnia dubia. In the present

study, we observed that tadpoles bioaccumulated sertraline to

higher concentrations than fluoxetine. Adverse effects of

sertraline on tadpole growth and development were observed

at low environmentally relevant concentrations.

The widespread presence of pharmaceuticals in water,

coupled with the ability of pharmaceuticals to have effects on

organisms at low concentrations, warrants additional research.

Further, such research should entail a better characterization

Fig. 3. Effects of fluoxetine and sertraline on development (time to tailresorption) in tadpoles exposed to selective serotonin reuptakeinhibitors throughout metamorphosis. Data are means 6 standarderror, n 5 3 aquaria per treatment. Asterisks indicate significantdifferences from controls (p , 0.05).

Effects of pharmaceuticals on tadpole growth and development Environ. Toxicol. Chem. 28, 2009 2675

of risks to both people and wildlife and an evaluation of risk

management options that would be most effective at reducing

environmental exposures.

Acknowledgement—We thank Samantha Burton for assistance withwater quality analyses and adult frog care. This research wassupported by a U.S. Environmental Protection Agency (U.S. EPA)Science to Achieve Results grant to M.C. Black and K.L. Armbrust(Grant R829006). This research has not been subject to U.S. EPAreview and therefore does not necessarily reflect the view of theAgency.

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