TOXICOLOGICAL SCIENCES 121(2), 292–302 (2011)
doi:10.1093/toxsci/kfr069
Advance Access publication March 23, 2011
Triclosan and Thyroid-Mediated Metamorphosis in Anurans:
Differentiating Growth Effects from Thyroid-Driven Metamorphosis in
Xenopus laevis
Douglas J. Fort,*,1 Michael B. Mathis,* Warren Hanson,* Chelsea E. Fort,* Lisa T. Navarro,† Robert Peter,‡ Claudia Büche,†
Sabine Unger,§ Sascha Pawlowski,{ and James R. Plautzk
*Fort Environmental Laboratories, Stillwater, Oklahoma 74074; †Syngenta Crop Protection, Basel 4058, Switzerland; ‡Intertek Expert Services, Basel 4002,
Switzerland; §BASF Schweiz AG, Basel 4057, Switzerland; {BASF SE, Ludwigshafen 67056, Germany; and kDSM Nutritional Products 1, Basel 4002,
Switzerland
1
To whom correspondence should be addressed at Fort Environmental Laboratories, 515 South Duncan Street, Stillwater, OK 74074. Fax: (405) 533-1250.
E-mail: [email protected].
Received December 16, 2010; accepted March 11, 2011
In a previously reported study, we used a standard metamorphosis anuran model to assess potential effect of the antibacterial agent triclosan (TCS) on normal prometamorphic
Xenopus laevis. Results indicated that environmentally relevant
TCS concentrations did not alter the normal course of thyroidmediated metamorphosis in this standard anuran model. However, to examine potential effects of TCS exposure during
premetamorphosis and to distinguish between effects on metamorphosis and effects on growth, a longer term TCS exposure
study was conducted. Standard Nieuwkoop and Faber (NF) stage
47 X. laevis larvae were exposed for 32 days (ca. NF stage 59–60)
via flow-through to four different concentrations of TCS: < 0.2
(control), 0.8, 3.1, 12.5, or 50.0 mg TCS/l. Primary endpoints were
survival, hind limb length, body length (whole; snout-to-vent),
developmental stage, wet whole body weight, thyroid histology,
plasma thyroid hormone (TH) concentrations, TH receptor beta
(TRb), and type II and III deiodinase (DI-2 and DI-3) expression.
Endpoints measured to evaluate effects on thyroid-mediated
metamorphosis including developmental stage, thyroid histology,
TRb expression, DI-2 and DI-3 expression, and thyroid gland
3,5,3#,5#-tetraiodothyronine (T4) and plasma T4 and 3,5,3#triiodothyronine (T3) levels were not affected by TCS exposure.
However, increased larval growth based on whole body length
(0.78, 12.5, and 50 mg TCS/l), snout-vent length (3.1 and 12.5 mg
TCS/l), and whole body weight (0.8, 12.5, and 50.0 mg TCS/l) was
observed following 32-day TCS exposure. These results indicated
that TCS exposure during pre- and prometamorphosis increased
larval growth but did not alter the normal course of metamorphosis in X. laevis. The increased growth associated with TCS
exposure was not unexpected and is generally consistent with the
presence of reduced bacterial stressors in culture.
Key Words: triclosan; anuran; metamorphosis; thyroid; growth;
Xenopus laevis.
The thyroid gland in most amphibians develops during late
embryogenesis (Dodd and Dodd, 1976; Regard, 1978). In
Xenopus laevis, the thyroid develops from a thickening of the
pharyngeal epithelium around Nieuwkoop and Faber (NF)
stage 35 (hatching) (Nieuwkoop and Faber, 1994). Following
division of the thyroid, follicular development is present by NF
stage 44. A functional thyroid gland with approximately 20
follicles is present by NF stage 53. As the prometamorphosed
larvae mature, follicular development continues, resulting in
growth of the gland. Concurrently, thyroid hormone (TH)
synthesis and secretion into the circulatory system increases in
preparation for metamorphosis. After metamorphosis is
complete, the thyroid gland regresses in size, which may be
responsible for the reduced levels of circulating TH after
metamorphosis is complete. Two naturally occurring THs,
3,5,3#,5#-tetraiodothyronine (T4) and 3,5,3#-triiodothyronine
(T3) have been identified. Based on years of experimental
evidence, there is little question today concerning the causative
effect of TH on amphibian metamorphosis (Allen, 1929, 1916;
Brown et al., 1995; Dodd and Dodd, 1976; Gudernatsch, 1912;
Leloup and Buscagalia, 1977; Tata, 1968; White and Nicoll,
1981). Three primary morphological changes occur during
anuran metamorphosis: (1) resorption or regression of tissue or
organ systems that have primary function only in the larval life
stage; (2) the remodeling of larval organ systems to their adult
form, which are suitable only for the adult; and (3) de novo
development of tissues in the adult that are not required by the
larvae.
Pharmaceutical and personal care products (PPCPs) are
potential sources of xenobiotics found in both aquatic and
terrestrial environments (Kolpin et al., 2002). Triclosan (TCS)
(5-chloro-2-[2,4-dichlorophenoxy] phenol) is a broad-spectrum
antimicrobial used in a variety of over-the-counter drug
products, which include antigingivitis toothpaste, antibacterial
Ó The Author 2011. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
For permissions, please email: [email protected]
EFFECT OF TRICLOSAN EXPOSURE ON ANURAN METAMORPHOSIS AND GROWTH
hand soaps, first aid antiseptics, as well as in personal care
products such as deodorants. Down-the-drain consumer waste is
the primary source of TCS in the environment, as municipal
wastewater treatment facilities (WWTFs) removed at least 70%
as the result of biodegradation and 20–25% due to physical
sorption (Bester, 2005, 2003; Heidler and Halden, 2006;
Sabaliunas et al., 2003; Singer et al., 2002; Thompson et al.,
2005; Waltman et al., 2006). Recently, Bock et al. (2010)
evaluated the fate and partitioning of TCS in WWTFs using
a probabilistic fugacity model. This model predicted that 84–
92% of TCS is removed by WWTFs with 40% removed due to
physical sorption and 48% due to biodegradation. TCS levels
measured in surface water generally range from 0.1 to 0.7 lg
TCS/l (Bester, 2005; United States Environmental Protection
Agency [USEPA], 2008).
To date, studies examining the potential endocrine-disrupting
capacity of TCS in aquatic organisms have been less than
conclusive. Foran et al. (2000) evaluated the effects of TCS
exposure on Oryzias latipes (medaka) development, concluding
that TCS was not estrogenic but potentially weakly androgenic.
However, these conclusions were based solely on nonsignificant
changes in gender ratios and fin length. Ishibashi et al. (2004)
reported weak vitellogenin induction in male medaka and
X. laevis exposed to TCS, suggesting a weak estrogenic response.
Structural similarities in TCS and TH have prompted further
investigation into potential thyroid axis disruption capacity of
TCS (Crofton et al., 2007; Veldhoen et al., 2006). Veldhoen
et al. (2006) suggested that TCS was capable of modulating THdependent gene expression and disrupting postembryonic Rana
catesbeiana (bullfrog) development. Several studies have been
conducted to evaluate whether TCS affects TH homeostasis via
downstream mechanisms. Hanioka et al. (1996) and Jinno et al.
(1997) found that TCS induced cytochrome P-450 2B1/2 and
hepatic ethoxy-resorufin-O-deethylase and pentoxy-resorufin-Odeethylase activity. Schuur et al. (1998) determined that TCS
inhibited iodothyronine sulfonation in rats in vitro. TCS has also
been shown to inhibit 3-phosphoadenosine 5-phosphosulfatesulfotransferase and UDP glucuronosyltransferase in human
liver fractions (Wang et al., 2004). Most recently, You (2004)
found that TCS induced glucuronidase activity, which is
important in maintaining TH homeostasis (Capen, 1994; DeVito
et al., 1999).
Based on the indication that TCS could disturb thyroid axis
function, leading to altered metamorphosis in amphibians,
further evaluation of the effects of TCS on anuran metamorphosis in the standard model, X. laevis, was performed
(Fort et al., 2010). Because the studies by Veldhoen et al.
(2006) used a premetamorphic model, which was not actively
engaged in metamorphosis, the study by Fort et al. (2010) was
conducted using a prometamorphic model to determine if
a similar response occurred during metamorphosis. The study
by Fort et al. (2010) was critically reviewed by Helbing
et al. (2011) with response by Fort et al. (2011). Prometamorphosis refers to the stages of development in which active
293
metamorphosis occurs prior to metamorphic climax, whereas
premetamorphosis refers to the larval stages prior to the onset
of prometamorphosis. Results from the initial prometamorphic
study (Fort et al., 2010) indicated that environmentally relevant
TCS concentrations did not alter the normal course of thyroidmediated metamorphosis in prometamorphic X. laevis exposed
to TCS for 21 days. However, to examine potential effects of
TCS exposure during premetamorphosis (stages prior to the
onset of active metamorphosis) a 32-day study was conducted
using NF stage 47 larvae. The study was performed to further
evaluate and confirm potential effects of TCS on anuran
metamorphosis and distinguish between effects on metamorphosis from effects on growth. The primary endpoints
were hind limb length, body length (whole and snout-to-vent
[SVL]), developmental stage, wet weight, thyroid histology,
and daily measure of mortality. In addition, whole thyroid
tissue and plasma samples were collected for endogenous TH
(thyroxine [T4] and triiodothyronine [T3]) analysis. Hind limb
(deiodinase) and tail fin tissue samples were collected during
the exposure to measure effects on TH receptor beta (TRb) and
type II and III deiodinase (DI-2 and DI-3) expression. Results
from these studies are presented in this report.
MATERIALS AND METHODS
Materials
Triclosan. The test chemical, TCS, lot number 60240CL5, was provided
by Ciba, Inc., now part of BASF (Basel, Switzerland) and was used throughout
the study. TCS was > 99.9% pure, as confirmed by the Certificate of Analysis.
Stock solution preparation and test concentrations. A working stock
solution of TCS was used to prepare the test concentrations. The stock solution
was prepared in a 19-l glass carboy by dissolving TCS in approximately 4 l of
dilution water at 50°C using a hot plate and high-capacity top stirrer. After
cooling, the carboy was topped off with dilution water to a total volume of 18 l.
Based on the previous 21-day exposure study (Fort et al., 2010), the same target
concentrations were used: control (< 0.2), 0.8, 3.1, 12.5, and 50.0 lg/l TCS.
Culture (dilution) water and laboratory control. Dechlorinated (charcoal
filtered) tap water was used as culture or dilution water. The dechlorinated
laboratory water was prepared by passing tap water through a four-filter system;
a multimedia filter to remove suspended solids in the feed water; a 10$
pretreatment filter (5 lm) to remove any additional solids; a 3.6 cf activated
virgin carbon treatment filter to remove chlorine, ammonia, and higher
molecular weight organics; and a 5-lm polishing filter to remove any carbon
particles from the carbon treatment phase. The dechlorinated tap water also
served as the laboratory control water. The temperature, pH, and dissolved
oxygen (DO) of the culture water were monitored daily during testing. Other
culture water quality characteristics measured at least once during the 32-day
study were conductivity, hardness, alkalinity, ammonia-nitrogen, and residual
oxidants. Trace contaminants, to include organics (volatile and semivolatile)
and inorganics (metals, nitrates/nitrites, fluoride, and iodide), were analyzed
annually.
Test Animal
The test species for this study was the South African Clawed Frog (X.
laevis), from which NF stage 47 larvae were used in the metamorphosis assay.
Culture and animal husbandry. The X. laevis larvae used for this study
were obtained from an in-house culture (originally purchased from Xenopus I,
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FORT ET AL.
Dexter, MI) where adults were injected with human chorionic gonadotropin to
induce reproduction. All larvae that were used as test organisms were derived
from the same clutch (spawn). The embryos were held at 21°C ± 1°C for 4 days
to allow for hatching and development to NF stage 47.
MS source was Turbulon Spray in negative mode with an MS/MS multiple
reaction monitoring scan at IS: 4200 V, temperature: 450°C, EP: 10, and
dwell time: 100 ms. The mean percent recovery of TCS from spiked analytical
reference samples was 89.6 ± 3.8%.
Feeding. Larvae were fed Sera Micron (Sera GmbH, Heinsberg, Germany)
throughout the preexposure period (after NF stage 45/46) and during the entire
test period of 32 days. Sera Micron, a commercially available tadpole food that
has been shown to support proper growth and development of X. laevis larvae,
is a fine particulate that stays suspended in the water column for a long period
of time and is subject to washing out with the flow. Therefore, the total daily
amount of food was divided into smaller portions and fed twice daily. Initially,
300 mg Sera Micron per tank was fed twice per day (total ¼ 600 mg/day) for
the first week of exposure. During the course of the study, the total daily food
ration was increased approximately 15% per week to accommodate larval
growth. Feeding was continued at a frequency of twice per day on Monday
through Friday and once per day at twice the volume on weekends. Sera Micron
was fed as a stock solution and freshly prepared in the laboratory every other
day at a confirmed density of 60 mg Sera Micron/ml dilution water.
Test Animal Selection
Exposure System
The route of TCS exposure was abiotic via culture media, which is the most
appropriate method for aquatic organisms and readily water soluble test
materials. A flow-through diluter system (Benoit Mini-Diluter; Environmental
Consulting and Testing, Superior, WI) was used in the performance of the
amphibian metamorphosis assay exposure (Benoit et al., 1982). The system
consisted of water contact components of glass (aquaria), stainless steel (diluter
housing and water bath), and Teflon (tubing responsible for test material
delivery). Exposure tanks were glass aquaria (with approximate measurements
of 22.5 3 14.0 3 16.5 cm deep) equipped with standpipes that resulted in an
actual tank volume of 4.0 l and minimum water depth of 10–15 cm. The system
was capable of supporting five exposure concentrations and a control, with up
to four replicates per treatment. The flow rate to each tank was 25 ml/min,
which provided a complete volume replacement every 2.7 h. Fluorescent
lighting was used to provide a photoperiod of 12 h light and 12 h dark at an
intensity that ranged from 600 to 1000 lux (lumens/m2) at the water surface.
Water temperature was maintained at 21°C ± 1°C, pH maintained between 6.5
and 8.5, and the DO concentration > 3.5 mg/l (> 40% of air saturation) in each
test chamber.
Chemical Analysis
Temperature, pH, DO, and light intensity were measured daily to monitor
diluter system performance. The solubility, purity, and stability (volatility and
degradation rate) of TCS were determined prior to starting the in-life assay.
Based on this information, the frequency of preparation of fresh stock solutions
was determined. Exposure concentrations were also analyzed from treatment
samples collected from the mini-diluter at test startup, at least weekly thereafter,
and at test takedown. TCS analysis of the test solutions and stock solutions was
performed using liquid chromatography-mass spectrometry (LC-MS) with
isotope dilution (Agilent 1100 high performance liquid chromatography
[HPLC] with API 4000 Triple Quadrupole MS).
Test solutions from each concentration were sampled for TCS during the
diluter system equilibration phase, at in-life study day 0, approximately once
per week during in-life study, and at test termination. In addition, each new
stock solution was sampled and analyzed for TCS. Immediately following
collection, the pH in all test solutions was adjusted to < 2.0 with phosphoric
acid, spiked with a 13C6-TCS internal standard (10 ng/ll in methanol) (Ciba,
Inc.), and stored at 4°C. Due to the biodegradability of TCS in aqueous
conditions, analysis was performed as soon as possible following collection.
Samples of the TCS test concentrations were extracted with methanol (1:1 [vol/
vol]) and analyzed by LC-MS. The stock solution extracts were diluted 1:100
(vol/vol) and analyzed by LC-MS.
A Phenomex Synergy Max RP 75 3 2 mm (4 lm particle) HPLC column
was used with a flow rate of 300 ll/min at room temperature. The eluents
consisted of water with 0.1% (vol/vol) formic acid (A) and methanol (B). The
When a sufficient number of the preexposure population of X. laevis reached
developmental stage 47 (5–7 days posthatch at 21°C ± 1°C), larvae were
transferred to a pooling tank containing dilution water. Individual larvae were
randomly removed from the pooling tank by scooping with a small strainer and
anesthetized in a transparent chamber (e.g., 100 mm Petri dish containing 100
mg of tricaine methanesulfonate [MS-222]/l dilution water buffered with 200
mg/l of sodium bicarbonate). Animals were carefully handled during this
transfer in order to minimize handling stress and to avoid injury.
The developmental stage of the animals was then determined by using
a binocular dissection microscope. In order to reduce the ultimate variability in
developmental stage, it is important that this staging be conducted as accurately
as possible. The primary developmental landmark for selecting NF stage 47
organisms is the presence of the hind limb bud (Nieuwkoop and Faber, 1994).
The morphological characteristics of the hind limbs were examined under the
microscope.
Larvae that met the stage criterion were transferred to a holding tank
containing 100% culture (dilution) water and allowed to recover from the
anesthesia. After recovery, the selected larvae were randomly distributed (no
more than 80 per treatment) to exposure treatment tanks containing 4.0 l
treatment solution until each tank contained 20 larvae (five larvae per liter
density). Each treatment tank was then inspected for animals with abnormal
appearance (e.g., injuries, abnormal swimming behavior, etc.). Overtly unhealthy
appearing larvae were removed from the treatment tanks and replaced with larvae
newly selected from the holding tank at test initiation. Treatment tanks were
properly labeled and randomly assigned to a position in the exposure system in
order to account for possible variations in temperature and light intensity.
Test Method
The randomly selected NF stage 47 larvae were exposed to four test
concentrations and a dilution water control. Each test concentration and control
was evaluated in quadruplicate, with 20 organisms per replicate. Once larvae
were placed in the exposure system, mortality observations were made daily
and any dead larvae were immediately removed. On day 12, five larvae
randomly selected per replicate were anesthetized in MS-222, plasma collected
for T3 and T4 analyses, thyroid glands collected for T4 analysis, hind limbs
collected for deiodinase activity, and tails collected for TRb gene expression
measurements. The test was terminated on day 32, at which time all test
animals were anesthetized in MS-222, staged for development, weighed,
digitally photographed for measurement, and visually observed for dysmorphology. Larvae were randomly selected (five per replicate), euthanized, and
reserved for possible histology. An additional five anesthetized larvae were
randomly selected from the remaining animals in each replicate treatment (100
animals) for blood serum and thyroid gland tissue collection for T3 and T4
analyses and tail tissue for TRb and deiodinase gene expression measurements.
Critical test parameters and experimental conditions for the in-life study are
presented in Table 1.
Biological Endpoints
The primary endpoints of the metamorphosis assay were mortality,
developmental stage (NF), growth (determined by length and weight), and
thyroid histology. Gross morphology (physical appearance at test takedown)
was a secondary endpoint. In addition, whole thyroid tissue and serum samples
were collected at test termination for analysis of endogenous T3 (serum only)
and T4. Tail biopsies were collected for measurement of TRb, DI-2, and DI-3
expression. Table 1 provides an overview of the measurement endpoints and
the corresponding observation time points.
Whole-body morphometric endpoints. More specifically, all test tanks
were checked daily for dead larvae and the numbers were recorded for each
EFFECT OF TRICLOSAN EXPOSURE ON ANURAN METAMORPHOSIS AND GROWTH
295
TABLE 1
Experimental Conditions—32-Day Amphibian Pre-/Prometamorphosis Assay
Test substance
Test system (species)
Initial larval stage
Exposure period
Larvae selection criteria
80’023/X target stock concentration/target test concentration
Measured stock concentration/measured test concentration
Exposure system
Exposure route
Preexposure flow rate/exposure flow rate
Primary endpoints/determination days
Mortality
Developmental stage
Hind limb length
Whole body length
Snout-vent length
Wet body weight
Thyroid histology
Secondary endpoint/approximate
Gross morphology
determination days
Additional endpoints/approximate
T4/T3 analysis
determination days
TRb expression
Deiodinase activity
Dilution water/laboratory control
Larval density
Test solution/test vessel
Replication
Acceptable mortality rate in controls
Thyroid fixation for histology
Feeding
Lighting
Water temperature
PH
DO concentration
Analytical chemistry water sample schedule
IRGASAN DP 300 (TCS), 99.9% purity
Xenopus laevis larvae
NF stage 47
32 days (exposure day 0 ¼ NF stage 47)
Developmental stage and optional total length
1.65 mg/l/0.8, 3.1, 12.5, and 50.0 lg/l
1.50 mg/l/0.3, 1.3, 5.9, and 29.6 lg/l
Flow-through mini-diluter
Abiotic exposure via culture media
50/25 ml/min
Daily
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 32 (NF stage 59/60)
Day 12 (NF stage 51), and day 32 (NF stage 59/60) on tails
Day 12 (NF stage 51) on hind limbs and day 32 (NF stage
59/60) on tails
Dechlorinated tap water (charcoal filtered)
20 Larvae per test vessel (5 larvae/l)
4 l (10–15 cm water height)
Four replicates per test concentration and control
10%
Number fixed
Five per replicate (randomly selected)
Region
Head
Fixative/preservative
Davidson’s fixative/10% (wt/vol) formalin (buffered)
Food
Sera Micron
Frequency/amount
Twice daily/360–840 mg/day per tank
Photoperiod
12 h Light:12 h dark
Intensity
600–1000 lux (measured at water surface)
21°C ± 1°C
6.5–8.5
> 3.5 mg/l (> 40% air saturation)
Once per week during PE and E phases (minimum of eight sample events)
Note. PE, pre-exposure; E, exposure.
tank. Dead animals were removed from the test tank as soon as observed. The
developmental stage of X. laevis larvae were determined using generally
accepted staging criteria from Nieuwkoop and Faber (1994). Differentiation of
the hind limbs is under control of THs and are major developmental landmarks
already used in the determination of developmental stage. Hind limb
development was used qualitatively in the determination of developmental
stage but was considered as a quantitative endpoint in the present study by
normalizing hind limb length to SVL. Therefore, hind limb length was
measured as an endpoint to detect effects on the thyroid axis. All length
measurements were based on digital photographs of the organisms from each
treatment. Two different approaches to assess larval growth were used: (1)
measurement of whole body length and (2) measurement of SVL. Both length
parameters were assessed to produce a solid database for a comparison
concerning their utility to reflect changes in larval growth.
Determinations of wet body weight were used to assess possible effects of
TCS on the growth rate of larvae in comparison to the control group. Although
measurements of larval wet weight may provide a more precise estimation of
larvae growth, weight determinations during the course of the study would be
technically more difficult to obtain. Compared with length measurements, the
removal of adherent water for weight determinations could cause more stressful
conditions for larvae, including possible skin damage. Therefore, wet weight
measurements were only performed on day 12 subsamples prior to tissue
sampling and on all remaining larvae at test termination (day 32).
Although developmental stage and hind limb length are important endpoints
to evaluate exposure-related changes in metamorphic development, developmental delay cannot, by itself, be considered a diagnostic indicator of thyroid
axis modulation. Some changes may only be observable by routine
histopathological analysis. In summary, developmental stage and hind limb
length endpoints were used as morphological measures of metamorphosis,
whereas whole body length, SVL, and wet whole body weight endpoints were
used as a morphological measure of growth.
Biochemical and gene expression endpoints. T4 and more specifically T3
control metamorphic processes in amphibians, including X. laevis. Blood and
thyroid gland tissue samples were collected at days 12 and 32 for T3 and T4
analyses. TH was measured by using newly customized ELISA kits from
GenWay (San Diego, CA). Samples were collected for T3 and T4 analyses at
exposure day 12 and at test conclusion. Plasma was collected via aortic
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FORT ET AL.
puncture, and whole thyroid glands were dissected and homogenized in
amphibian Ringers/assay buffer (pH 8.1). The homogenized tissue samples
were then centrifuged at approximately 9000 3 g and the supernatant collected
for cross-reactivity. Serum and thyroid glands that could not be processed and
analyzed at test takedown were snap frozen in liquid nitrogen and stored at
80°C for subsequent analysis. Mean percent recoveries of T3- and T4-spiked
analytical reference samples were 96.2 ± 2.1 and 92.8 ± 3.7%, respectively.
Measurement of TRb and peripheral deiodinase expression during metamorphosis represents a downstream biomarker of thyroid axis activity at
a molecular level. TRb expression has been used as an early indicator of
disruption of metamorphic events in amphibians and the thyroid axis.
Measurement of DI-2 assists in evaluation of the conversion of T4 to the
more potent metamorphic inducers T3 in the tissues. Measurement of DI-3
represents a means of evaluating the deactivation of THs by the conversion of
T3 to diiodothyronine and the conversion of T4 to reverse T4. Tissue samples
at exposure days 0, 12, and 32 were collected for examination of TRb and
deiodinase expression, respectively.
PCR methodologies for specifically measuring TRb (Fort et al., 2010;
Veldhoen and Helbing, 2001) and deiodinase (Dubois et al., 2006; Lehigh
Shirey et al., 2006) gene expression changes, as the result of exposure to
potential endocrine-disrupting chemicals in X. laevis hind limb and tail
biopsies, were used. Tissue samples were preserved in RNAlater (Ambion,
Inc., Austin, TX) prior to gene expression analysis. Each biopsy was
homogenized in 300 ll TRIzol using a Brinkman Polytron microhomogenizer
(Fisher Scientific, Houston, TX) and total RNA isolated, resuspended in
RNase-free water (Invitrogen, Burlington, Ontario), and stored at 80°C prior
to complementary (cDNA) preparation. A 20-lg sample of glycogen was added
during RNA isolation to maximize RNA recovery (Zhang et al., 2006). The
isolated RNA (1.0 lg) from each sample was annealed with 500 ng of random
hexamer oligonucleotide and cDNA produced with Superscript II reverse
transcriptase (Invitrogen) in accordance with manufacturer specifications. The
produced cDNA was diluted at least 25-fold prior to amplification.
PCR amplification was performed using a Mini-Opticon system (BioRad,
Hercules, CA) with TRb- and deiodinase-specific primers (Invitrogen) in
accordance with manufacturer’s recommendations and Fort et al. (2010). Amplified
DNA products were separated on a 2% agarose gel, visualized by ethidium bromide
staining on a Chemi-Imager 4000 (Alpha InnoTech, San Leandro, CA), and
quantified by accompanying software. Amplifications were optimized to a linear
range of detection. Furthermore, densitometric data were normalized to control
gene, ribosomal protein L8 (rpL8), which is a suitable marker of integrity.
Kruskal-Wallis (KW) ANOVA on ranks (nonparametric data sets) accompanied
by appropriate post hoc comparison tests (p ¼ 0.05). Statistical evaluation of
frequency distributions of developmental stages in the control and treatments
was performed using a Mann-Whitney U-test (p ¼ 0.05) as described by Fort
et al. (2011) and USEPA (2009). The 5th and 95th percentiles for median
developmental stage data sets were also determined and reported.
Thyroid morphometric endpoints. Measurement of various thyroid gland
measurements was made following the termination of exposure on day 32.
Measurements including thyroid area, follicle count, follicular cell height,
follicular area, colloid content per specimen, and colloid content per follicle
were collected to further evaluate potential effect of TCS exposure on thyroid
gland function and assist in separating growth effects from developmental
effects on metamorphosis.
Developmental stage. The median developmental stages
obtained for the control, 0.3, 1.3, 5.9, and 29.6 lg/l TCS
measured treatments following 32 days of TCS exposure were
NF stages 60, 59, 59, 59, and 59, respectively (Table 2).
Overall, the frequencies of developmental stage obtained
following 32 days of exposure to TCS in each of the treatment
concentrations were not different from control (Mann-Whitney
U, p > 0.05). SVL normalized hind limb lengths in each of the
treatment concentrations were not significantly different from
the control (ANOVA, p ¼ 0.192).
Thyroid pathology. The heads of stage-matched NF 59 larvae were cut
from the body in a transverse plane just caudal to the eyes. Decalcification was
not necessary to remove bone at the time of gross trimming. The head was
processed using an automatic tissue processor and then embedded in paraffin
with the cut side down. Rough bilateral sections were made until the thyroid
glands were observed. When the thyroid glands were reached, three 32-lm step
sections were made and the step sections were mounted on a slide. The slides
were then stained with hematoxylin and eosin. The histopathological evaluation
of the thyroid glands included review of potential changes in the glands as
hypertrophy of follicular cells, hyperplasia of thyroid follicles, size of thyroid
follicles, and degree of colloid accumulation in the thyroid follicles,
a qualitative assessment as to size of the thyroid gland.
Data Analysis
With the exception of developmental stage data, apical morphology
endpoints were analyzed using one-way ANOVA (parametric data sets) or
RESULTS
TCS Measurement
Measured TCS concentrations of the control and each test
concentration (target concentration in parenthesis) were < 0.2
(below detection limit) lg/l (control), 0.3 (0.78), 1.3 (3.1), 5.9
(12.5), and 29.6 (50.0) lg/l, reported as time-weighted average
concentrations based on eight sampling events per test
concentration during the 32-day test period (Tables 2–5).
Control
A summary of morphological endpoint data for the dilution
water control is presented in Table 2. During the 32-day
exposure period initiated at NF stage 47, the mean control
survival frequency was 100% and the median control NF
developmental stage was 60 (Table 2). Developmental stage
distribution, whole body, hind limb and snout-vent lengths, and
whole body weight were consistent with historical control data
(Fort et al., 2010) (Table 2). Control larvae thyroid gland
histology appeared normal (Table 4).
TCS Treatments
Mortality. Negligible mortality was observed during the test
and ranged from 0.0% in the control and 1.3, 5.9, and 29.6 lg/l
TCS treatments to 1.3% in the 0.3 lg/l TCS treatment (Table 2).
Growth. Mean whole body length, snout-vent length, and
whole body weight data for day 32 are provided in Table 2.
Whole body length and weight of larvae exposed to 0.3, 5.9,
and 29.6 lg/l TCS were significantly greater than the
laboratory control (KW-ANOVA, Dunn’s method, p < 0.05
and ANOVA, Bonferroni t-test, p 0.001, < 0.001, and 0.004,
respectively). Mean SVLs of larvae exposed to the 1.3 and 5.9
lg/l TCS were significantly greater than control larvae (KWANOVA, Dunn’s method, p < 0.05).
297
EFFECT OF TRICLOSAN EXPOSURE ON ANURAN METAMORPHOSIS AND GROWTH
TABLE 2
Summary of Morphological Endpoint Data—32-Day Amphibian Pre-/Prometamorphosis Assay with TCS
Test concentration
Measured
meana
Mean
mortality
Target
lg/l
lg/l
SEM
0.0 (Control)
0.78
3.1
12.5
50.0
< 0.2
0.3
1.3
5.9
29.6
0.00
0.08
0.22
0.95
3.93
Median stageb
NF
60
59
59
59
59
(58–62)
(58–61)
(58–62)
(58–61)
(58–61)
Mean whole
body length
Mean snoutvent length
Mean hind
limb lengthc
Mean whole
body weight
%
SEM
cm
SEM
cm
SEM
cm
SEM
g
SEM
0.00
1.30
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
6.16
6.65d
6.43
6.68d
6.72d
0.15
0.14
0.10
0.13
0.06
1.799
1.904
2.03d
2.13d
1.98
0.15
0.15
0.04
0.11
0.15
0.93
0.86
0.76
0.66
0.70
0.15
0.07
0.01
0.05
0.08
1.09
1.27e
1.15
1.30e
1.31e
0.02
0.05
0.04
0.09
0.07
a
Reported values are based on means of eight sampling events per concentration with SEM. Test concentrations measured as less than method detection limit
(MDL, 0.2 lg/l) were reported as half the MDL (0.1 lg/l) for calculation purposes.
b
The 5th and 95th percentiles for the median stage data are report below each median in parenthesis.
c
Mean hind limb length was normalized to the snout-vent length.
d
Significantly greater than control (KW-ANOVA on Ranks, Dunn’s method, p < 0.05).
e
Significantly greater than control (ANOVA, Bonferroni t-test, p < 0.05).
Gross morphology. Examination of terminal stage larvae
did not reveal grossly observable changes, indicating no
external adverse effects from exposure to the study chemical
concentrations.
Thyroid pathology. None of the specimens examined from
the control treatments displayed abnormal thyroid gland
histology (Table 3). A TCS concentration-related increase in
the occurrence of minimal thyroid gland hypertrophy and
minimal thyroid gland vascular congestion was observed.
Diagnosis of minimal gland hypertrophy was based on visual
inspection with peer review suggesting that the thyroid glands
were generally larger than found in control specimens. The
increase in thyroid gland size was accompanied by the
TABLE 3
Summary of Histological Findings—Amphibian Pre-/Prometamorphosis Assay
Measured TCS
Concentrationa
lg/l
SEM
Specimens Examined
n
Thyroid pathology
n
< 0.2 (Control)
0.00
10
0.3
0.08
10
1.3
0.22
10
5.9
0.95
10
29.6
3.93
10
0
1
3
1
2
3
5
3
8
6
Histological observations
Minimal
Minimal
Minimal
Minimal
Minimal
Minimal
Minimal
Minimal
Minimal
Minimal
thyroid
thyroid
thyroid
thyroid
thyroid
thyroid
thyroid
thyroid
thyroid
thyroid
gland
gland
gland
gland
gland
gland
gland
gland
gland
gland
hypertrophyb
congestionc
hypertrophyb
congestionc
hypertrophyb
congestionc
hypertrophyb
congestionc
hypertrophyb
congestionc
a
Reported values are based on means of eight sampling events per concentration with SEM. Test concentrations measured as less than method detection limit
(MDL, 0.2 lg/l) were reported as half the MDL (0.1 lg/l) for calculation purposes.
b
Diagnosis of gland hypertrophy was based on visual inspection with peer review suggesting that the thyroid glands were generally larger than found in control
specimens (see Table 4). The increase in thyroid gland size was accompanied by the observation that the larger glands contained larger follicles and an increased
number of follicles per gland. The increase in gland and follicle size was not accompanied by follicular hypertrophy or hyperplasia. The lack of follicular
hypertrophy or hyperplasia was suggested to be the result of both the minimal nature of the response and the increased body size of the treated specimens. Further
examinations of the relationship between specimen growth and thyroid gland size and physiology are provided in Tables 4 and 5.
c
Vascular congestion was characterized by the presence of dilated blood vessels within the interstitium of the thyroid glands. Because a co-occurrence of
vascular congestion and thyroid hypertrophy occurred, thyroid gland enlargement could not be attributed to increased vascularization and appeared to be a function
of the size of specimens examined.
298
FORT ET AL.
FIG. 1 (a) Photomicrograph of bilateral sections of thyroid glands from
representative exposure day 32 control Xenopus laevis. (b) Close-up
photomicrograph of bilateral sections of thyroid glands from representative
exposure day 32 control X. laevis.
observation that the larger glands contained larger follicles and
an increased number of follicles per glands. The increase in
gland and follicle size was not accompanied by follicular
hypertrophy or hyperplasia. The lack of follicular hypertrophy
or hyperplasia was suggested to be the result of both the
minimal nature of the response and the increased body size of
the treated specimens. Vascular congestion was characterized
by the presence of dilated blood vessels within the interstitium
of the thyroid glands. Because a co-occurrence of vascular
congestion and thyroid hypertrophy occurred, thyroid gland
enlargement could not be attributed to increased vascularization and appeared to be a function of the size of specimens
examined. Photomicrographs of bilateral thyroid sections from
control and 29.6 lg/l TCS-treated larvae documenting the
normalcy of the thyroid glands are provided in Figures 1a and 2c.
TH analysis. No detectable plasma THs (< 0.02 lg/dl T4
and < 0.4 lg/dl T3) were measured at exposure day 12 (ca. NF
stage 51). Results of analysis of T4 in thyroid glands and T3
and T4 in plasma samples collected on exposure day 32 from
FIG. 2 (a) Photomicrograph of bilateral sections of thyroid glands from
representative Xenopus laevis exposed to 29.6 lg/l TCS for 32 days. (b) Closeup photomicrograph of bilateral sections of thyroid glands from representative
X. laevis exposed to 29.6 lg/l TCS for 32 days. (c) Close-up photomicrograph
of bilateral section of thyroid gland from representative X. laevis exposed to
29.6 lg/l TCS for 32 days.
stage-matched laboratory control and TCS-treated larvae are
presented in Figures 3a and 3b, respectively. Lower serum T4,
but not T3, levels in samples collected from the 0.3 (ANOVA,
Bonferroni t-test, p ¼ 0.012 [serum T4] and 0.181 [serum T3])
and 1.3 lg/l (ANOVA, Bonferroni t-test, p ¼ 0.004 [serum T4]
EFFECT OF TRICLOSAN EXPOSURE ON ANURAN METAMORPHOSIS AND GROWTH
FIG. 3 (a) T4 concentrations in day 32 exposure Xenopus laevis thyroid
glands in stage-matched specimens compared with day 32 control. No
statistical differences observed relative to control (ANOVA, p ¼ 0.166). (b)
Plasma T4 and T3 concentrations in exposure day 32 X. laevis in stage-matched
specimens compared with day 32 control), ‘‘*’’ confers statistical difference
from the control (ANOVA, Bonferroni t-test, p < 0.05).
and 0.181 [serum T3]) TCS-treated larvae were found
compared with laboratory control samples. No change in
serum T3 levels or thyroid gland T4 levels in the TCS-treated
larvae was found compared with laboratory control samples
(ANOVA, p ¼ 0.166).
TRb and deiodinase expression. Evaluation of TRb and
DI-2 and DI-3 expression in hind limb (exposure day 12 DI
only) and tail fin biopsies from stage-matched TCS exposure
day 12 and day 32 specimens relative laboratory control larvae
is presented in Figures 4a and 4b, respectively. No effect of
TCS exposure on TRb (ANOVA, p ¼ 0.881) and DI-2 and DI3 (KW-ANOVA, p ¼ 0.936 and 0.240) expression in exposure
day 12 specimens was observed in tail (TRb) and hind limb
(deiodinase) tissues relative to the control. In addition, no
changes in either TRb (ANOVA, p ¼ 0.196) or deiodinase
299
FIG. 4 (a) Mean folds of expression of TRb and deiodinase (DI)-2 and DI3 expression in exposure day 12 Xenopus laevis tail (TRb) and hind limb (DI)
tissue in stage-matched specimens compared with exposure day 12 control
specimens. Concentrations represent measured values at exposure day 12. No
statistical differences were observed compared with control (ANOVA, p ¼
0.881 [TRb] and KW-ANOVA, p ¼ 0.936 and 0.240, respectively [DI-2 and
DI-3)]. (b) Mean folds of expression of TRb and deiodinase (DI)-2 and DI-3
expression in exposure day 32 X. laevis tail tissue in stage-matched specimens
compared with exposure day 32 control specimens. No statistical differences
were observed compared with control (ANOVA, p ¼ 0.196 [TRb] and KWANOVA, p ¼ 0.265 and 0.386, respectively [DI-2 and DI-3]).
(KW-ANOVA, p ¼ 0.265 and 0.386, respectively) expression
in tail fin tissue were noted in biopsies collected from the TCS
treatments at exposure day 32 relative to the control.
Thyroid gland metrics and relationship to larval growth. A
quantitative evaluation of the thyroid glands in control and
TCS-treated specimens was performed to evaluate the relationship between thyroid gland size and anatomical/physiological
characteristics and larval growth. A summary of exposure day
32 thyroid gland analytical measurements is provided in Table
4. Based on this examination, the mean thyroid gland size of
larvae exposed to 0.3, 5.9, and 29.6 (ANOVA, Bonferroni ttest, p ¼ 0.015, 0.001, and 0.043) lg/l TCS, but not the 1.3
300
FORT ET AL.
TABLE 4
Summary of Thyroid Measurement Data—32-Day Amphibian Pre-/Prometamorphosis Assay with TCS
Measured TCS
Mean
Mean
Mean follicle
Mean
Mean colloid
Mean colloid
Concentrationa
Thyroid area
Follicle count
Cell height
Follicle area
Area per animal
Area per follicle
lg/l
SEM
mm2
SEM
n
SEM
lm
SEM
lm2
SEM
lm2
SEM
lm2
SEM
< 0.2
0.3
1.3
5.9
29.6
0.00
0.08
0.22
0.95
3.93
0.144
0.180b
0.175
0.189b
0.176b
0.01
0.01
0.01
0.01
0.01
10.1
12.2
11.6
12.4
11.3
0.66
0.91
0.61
1.0
0.70
14.94
13.94
12.69c
11.08c
13.33c
0.14
0.12
0.12
0.12
0.12
10,327
9900
9518
11,600
9662
635.4
702.4
618.1
1116
983.1
4048
4035
4281
4900
4686
418.2
564.4
446.1
538.5
708.0
461.8
398.0
421.6
551.2
554.6
68.1
62.7
62.7
154.3
179.9
a
Reported values are based on means of eight sampling events per concentration with SEM. Test concentrations measured as less than method detection limit
(MDL, 0.2 lg/l) were reported as half the MDL (0.1 lg/l) for calculation purposes.
b
Significantly greater than control (ANOVA, Bonferroni t-test, p < 0.05).
c
Significantly less than control (KW-ANOVA, Dunn’s test, p < 0.05).
(ANOVA, Bonferroni t-test, p ¼ 0.053) lg/l TCS-treated
larvae, was significantly larger than glands from control
specimens. No differences in the number of follicles (ANOVA,
p ¼ 0.267), mean follicle size (KW-ANOVA, p ¼ 0.235),
mean colloid content per animal (KW-ANOVA, p ¼ 0.521),
and mean colloid content per follicle (KW-ANOVA, p ¼
0.850) were found between the TCS-treated larvae and control
larvae. Mean follicular cell height decreased (1.3, 5.9, and 29.6
lg/l TCS) with increasing TCS concentration (KW-ANOVA,
Dunn’s test, p < 0.05). Because no change in epithelial cell
shape or structure was noted, changes in follicular cell height
were attributed to growth.
Multiple comparison correlation analysis of thyroid gland
measurements and growth parameters is provided in Table 5.
The only positive correlation between growth endpoints and
analytical measurement of thyroid anatomy/physiology was
mean thyroid area and whole body length (Pearson correlation,
r2 ¼ 0.77, p ¼ 0.037). A negative correlation between mean
follicular cell height and mean SVL was detected (Pearson
correlation, r2 ¼ 0.99, p ¼ 0.002), but this relationship was
not detected with the other measures of growth. None of the
analytical measures of thyroid function (physiology), such as
the number of follicles, follicle size, and most importantly
colloid per animal and colloid per follicle, were correlated
with larval growth. These results suggested that the slightly
increased size of the thyroid gland, decreasing follicular cell
height, and increased vascularization associated with increasing TCS exposure concentration were simply a function of
increased growth and not a direct effect on the thyroid gland.
DISCUSSION
Anuran metamorphosis is separated into three distinct
periods: premetamorphosis, prometamorphosis, and metamorphic climax. Premetamorphosis refers to a period of embryonic
and early larvae development that takes place without TH.
Some advanced morphological developments occur during this
stage including hind limb bud development. More specific
morphogenesis, such as differentiation of the digits and
rapid growth (elongation) of the hind limbs, occurs during
prometamorphosis. Biochemically, prometamorphosis is
TABLE 5
Multiple Comparison Analysis of Growth and Thyroid Data—32-Day Amphibian Pre-/Prometamorphosis Assay with TCS
Mean whole body length
Mean snout-vent length
Mean whole body weight
Mean thyroid
area
Mean follicle
count
Mean follicle
cell height
Mean follicle
area
Mean colloid
area per animal
Mean colloid
area per follicle
0.901
0.037
0.841
0.075
0.843
0.073
0.812
0.095
0.742
0.151
0.749
0.145
0.594
0.291
20.988
0.002
0.581
0.305
0.500
0.391
0.504
0.387
0.433
0.466
0.627
0.258
0.811
0.096
0.688
0.199
0.405
0.499
0.456
0.440
0.528
0.361
Note. Hypothesis testing was performed using Pearson product moment correlation, p > 0.050. The top number is the correlation coefficient, and the bottom number
is the p value. The pairs of variables with positive correlation coefficients and p values below 0.050 tend to increase together. For the pairs with negative correlation
coefficients and p values below 0.050, one variable tends to decrease, whereas the other increases. For pairs with p values greater than 0.050, no significant relationship
between the two variables exists. Values in bold represent a significant positive or negative correlation between the two associated variables.
EFFECT OF TRICLOSAN EXPOSURE ON ANURAN METAMORPHOSIS AND GROWTH
characterized by rising concentrations of endogenous TH. The
final period is metamorphic climax, in which a surge of TH
triggers the final processes associated with metamorphosis,
including forelimb development and resorption of the tail.
Drastic internal transformations at the organ system, tissue, and
biochemical levels are also taking place during prometamorphosis and metamorphic climax.
The thyroid axis represents one potential target for environmental chemicals. Environmental agents, toxicants, natural
products, and complex mixtures can alter metamorphosis
by interacting with the thyroid axis. Furthermore, the complexity
of the thyroid axis yields many different possible mechanisms of inhibiting metamorphic processes in amphibians at
differing biochemical and molecular levels. Thus, the use of
amphibians as representatives of the chordates to screen for
thyroid-disrupting chemicals or chemical mixtures is not
unreasonable.
In the present study, significant differences in developmental
stages between the laboratory control and the TCS treatments
were not observed, no adverse effects on thyroid histology
were detected, and no changes in either thyroid gland T4 or
serum T3 and T4 levels were found. In addition, no changes in
TRb or DI-2 and DI-3 expression in tail tissue collected from
exposure day 32 larvae or hind limb (deiodinase) and tail
(TRb) tissue collected from exposure day 12 larvae exposed to
TCS were noted in this study in relation to control specimens.
Exposure to TCS did increase larval growth and generally
increased the overall size and vasculature of the thyroid glands
in the higher TCS concentrations. However, the general
increase in thyroid gland size was related to increased growth
in the TCS treatments. Not surprisingly, the increase in growth
observed was greater in the 32-day pre-/prometamorphosis
exposure than in original studies using 21-day prometamorphosis exposure format (Fort et al., 2010). Previous studies
(Fort et al., 2010 and Fort and Rogers, unpublished data) found
that antibiotic (penicillin/streptomycin) treatment of X. laevis
larvae during premetamorphic development slightly increased
larval growth, but exposure during prometamorphosis did not
increase metamorphic rate, cause histopathological changes in
the thyroid gland, alter serum or thyroid gland T4 levels, or
markedly alter TRb expression. Thus, the antibiotic/antibacterial agents studied appear to marginally accelerate premetamorphic growth by a nonthyroidal process but do not alter the
process of metamorphosis in X. laevis. The increased growth
associated with TCS exposure was not unexpected and is
generally consistent with the antimicrobial activity of TCS in
culture (Calabrese, 2008; Fort et al., 2010).
Because the Endocrine Disruptor Screening and Testing
program administered by the USEPA requires the use of a 21day exposure Amphibian Metamorphosis Assay (frog)
(USEPA, 2009), the importance of distinguishing between
effects on larval growth and effects on metamorphosis should
not be underestimated. Differentiating between the two effects
essentially determines if the test substance is endocrine active
301
within the thyroid axis in the case of metamorphosis or
potentially acts by a nonendocrine mode of action in the case
of growth. The inability to separate effects on amphibian
metamorphosis from growth effects leads to the potential for
falsely identifying thyroid-disrupting chemicals. In the case of
TCS, effects on whole body length, SVL, and whole (wet)
body weight, but not hind limb length, plasma THs, thyroid
gland THs, and TRb and deiodinase expression, clearly
indicated that TCS altered growth, but not thyroid-mediated
metamorphosis. In addition, further quantitative examination
of the thyroid gland was required to confirm that the subtle
effects on the thyroid were actually the result of increased
growth. Overall, this study demonstrated that significant
professional judgment may be required in evaluating results
from amphibian metamorphosis studies.
In conclusion, the present study provided evidence that TCS
does not alter the normal course of metamorphosis in the
standard anuran model, X. laevis, at the test concentrations
studied. Based on the battery of endpoints used in this study,
the no observed adverse effect concentration for disruption of
thyroid-driven metamorphosis was 29.6 lg/l TCS. However,
TCS exposure to X. laevis tadpoles during development was
capable of increasing growth. The effects on growth appeared
to be the result of nonthyroidal mechanisms and were generally
consistent with the presence of reduced bacterial stressors in
culture.
FUNDING
Ciba, Inc., now part of BASF; Oklahoma Center for the
Advancement of Science and Technology (6552).
ACKNOWLEDGMENTS
Research was conducted in compliance with the Animal
Welfare Act and other Federal statues and regulations relating
to animals and experiments involving animals.
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