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Examination of an amphibian metamorphosis assay under an individual-separated exposure system using Silurana tropicalis tadpoles
Ecotoxicology and Environmental Safety 86 (2012) 86–92
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Examination of an amphibian metamorphosis assay under an individual-separated exposure system using Silurana tropicalis tadpoles Masahiro Saka a,n, Noriko Tada a, Yoichi Kamata b a b
Kyoto Prefectural Institute of Public Health and Environment, Murakamicho 395, Fushimi-ku, Kyoto 612-8369, Japan Division of Microbiology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2012 Received in revised form 27 August 2012 Accepted 28 August 2012 Available online 12 October 2012
We examined the validity of an amphibian (Silurana tropicalis) metamorphosis assay (a 28-day semistatic test) under an individual-separated exposure system, where tadpoles were individually held in small glass beakers. We first conducted a comparative rearing experiment for 28 days between this exposure system and the traditional individual-grouped exposure system, both of which held 30 tadpoles (stages 49 and 50) in dechlorinated tap water (a control solution). The former system served to reduce interindividual variability in regard to three morphological measures (developmental stage, hind limb length, and total body length). Under this system, we tested thyroxine (T4, 1 mg/L) and propylthiouracil (PTU, 75 mg/L) for 28 days of exposure. The morphological data collected at 7-day intervals indicated that significant metamorphic acceleration and retardation were consistently induced in the tadpoles exposed to T4 and PTU, respectively. In addition, the thyroid glands of the tadpoles exposed to T4 and PTU clearly exhibited atrophy and hypertrophy accompanied with severe follicular cell hyperplasia, respectively. Our results are in agreement with the historical data generated from previous studies employing the traditional exposure system, thus indicating the validity of our alternative testing protocol. & 2012 Elsevier Inc. All rights reserved.
Keywords: Amphibian Metamorphosis assay Silurana tropicalis Thyroxine Propylthiouracil Exposure system
1. Introduction Amphibian metamorphosis, which is induced and regulated by thyroid hormones, accompanies drastic transformations in essentially every organ and tissue. Particularly in anurans, the process of morphological alteration caused by metamorphosis (tadpole-to-frog transformation) can be easily monitored by visual inspection. In addition, postembryonic development has been thoroughly studied as a biological model to elucidate thyroid functions (Shi, 2000). By exploiting these advantages, an in vivo assay to detect chemicals disrupting thyroid functions has been designed using Xenopus laevis, a worldwide experimental anuran species (Kloas, 2002; Kloas et al., 2003; OECD, 2004; Opitz et al., 2005). The experimental protocol of this assay has been standardized by OECD (2009). Meanwhile, similar metamorphosis assays have also been successfully developed using other anuran species, such as Silurana tropicalis (formerly called Xenopus tropicalis) (Mitsui et al., 2006) and Rana rugosa, an endemic species in Japan (Oka et al., 2009). In particular, S. tropicalis, which is phylogenetically related to X. laevis, has attracted attention as a new model species, due to its experimental superiority
n
Corresponding author. Fax: þ81 75 621 4164. E-mail address: [email protected] (M. Saka).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.08.034
over X. laevis: a shorter life cycle and a diploid genome that increase the practicability of use for multi-generational tests and genetic analyses (Song et al., 2003; Fort et al., 2004a, 2004b; Kashiwagi et al., 2010). However, traditional assays have practical difficulties when conducted by one or two individual researchers due to the large number of tadpoles required for the examination of morphological endpoints. As demonstrated by historical studies (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006; Oka et al., 2009), tadpoles, even within a control, tend to develop enhancing individual differences in growth and development during exposure periods. This tendency would become more conspicuous under individual-grouped exposure systems where 20–30 tadpoles are held together in a single test tank, because the tadpoles can easily interfere with each other during feeding. An evaluation of results yielded using amphibian metamorphosis assays is based largely on statistical comparisons of morphological data concerning tadpole development. Accordingly, such large within-group variability necessitates the use of a large number of tadpoles for control and chemical treatment groups. In addition, when including replication, the number of tadpoles increases two- to four-fold. Therefore, an exposure system to exclude interactions among tadpoles can serve to minimize interindividual variability of morphological data and thereby allow the number of tadpoles to be reduced. The simplest design fit for this purpose is
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an individual-separated exposure system in which each tadpole is held in a single test vessel. This system also enables individual identification without marking of the tadpoles, which is technically difficult (Rice et al., 1998). However, in the individual-separated exposure system, which requires as many test vessels as the total number of test tadpoles, the test vessel must be reduced in size due to the limited volume of the temperature-constant chamber. This exposure system inevitably reduces the amount of swimming space available for each tadpole. For certain anuran species, it has been suggested that tadpole development can be accelerated by gradually reducing aqueous space that may stimulate the release of thyrotropin, a thyroid-stimulating hormone (Denver, 1998; Denver et al., 1998). The anuran metamorphosis assay under the individual-separated exposure system may therefore not truly detect thyroid-disrupting chemicals, unless sufficient volume of test solution is provided for each tadpole during the test. The current work aimed to establish an amphibian metamorphosis assay practicable by one or two individual researchers, using S. tropicalis tadpoles as a test species. We first conducted a comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system in order to confirm that the former served to reduce the interindividual variability of the morphological without inducing developmental acceleration that would affect the test results. Subsequently, under the individual-separated exposure system, we tested thyroxine (T4, one of thyroid hormones) and propylthiouracil (PTU, an antithyroid chemical), both of which have been used as test chemicals in traditional metamorphosis assays. By comparing the results with the traditional data, we verified the validity of the amphibian metamorphosis assay under the individual-separated exposure system.
2. Materials and Methods
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Table 1 The experimental protocol of the metamorphosis assay using Silurana tropicalis tadpoles. Duration Temperature Light Diluent
Test procedure Group size Feeding Aeration
28 days 25 1C (with a deviation of 7 1 1C) 12-h light/12-h dark photoperiod using white fluorescent lamps Dechlorinated tap water (hardness, approximately 40 mg/L as CaCO3; pH, 6.5–7.5; dissolved oxygen, 95%–105% saturation at 25 1C; iodine concentration, approximately 4 mg/L) Semistatic test with solution renewal three times a week (Monday, Wednesday, and Friday) Thirty individuals for each group (a diluent control and chemical treatment groups) Daily with 1% (w/v) Sera Microns solution: 0.5 mL (days 0–6) and 1 mL (days 7–28) per individual None
solution for PTU was adjusted to approximately 7.0 by adding a small amount of 1 M HCl. Adjustment of pH was not done for the test solution of T4 because its pH dropped to nearly 7 due to 3 106-fold dilution. 2.3. Experiments 2.3.1. Assay protocol The current work consisted of two experiments that were conducted following the protocol shown in Table 1. Although OECD (2009) presented a standard protocol for conducting an amphibian metamorphosis assay under the traditional individual-grouped exposure system, the current experiments employing an individual-separated exposure system involved two major alterations: (1) the prolonged test duration from 21 to 28 days and (2) the use of total body length instead of snout–vent length as one of morphological endpoints. Most of historical data of amphibian metamorphosis assays have been yielded by employing the 28-day exposure duration and measuring total body length to monitor growth of tadpoles (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006; Oka et al., 2009). Since the validation of an individual-separated exposure system would be based largely on comparisons between the above historical data and those obtained from the current work, the two experiments were also performed during 28 days of exposure, using total body length as an indicator of tadpole growth.
2.1. Animal husbandry Adult pairs of S. tropicalis were obtained from the Institute of Amphibian Biology of Hiroshima University. The adult frogs were kept in polypropylene aquaria filled with dechlorinated tap water under the following conditions: water depth, 9 cm; water temperature, 25 7 1 1C; photoperiod, 12-h light/12-h dark; frog density, six frogs per 1800 cm2 of water surface area; feeding, thrice per week with a commercial diet for aquatic frogs (XL-2, Oriental Yeast, Tokyo, Japan). After an acclimatization period of 4–6 weeks, breeding was induced by injecting the frogs into the dorsal lymph sac with human chorionic gonadotropin (Wako Pure Chemical Industries, Osaka, Japan): each male received a small primer dose of 20 IU (7 days prior to mating) followed by a final dose of 200 IU (several hours prior to mating), and each female received a single dose of 300 IU (several hours prior to mating). Spawned eggs and developing tadpoles were reared in polypropylene aquaria filled with dechlorinated tap water under the following conditions: water depth, 7 cm; water temperature, 25 71 1C; photoperiod, 12-h light/12-h dark; tadpole density, approximately 200 individuals per 1800 cm2 of water surface area; feeding, daily with a commercial tadpole food (Sera Microns, Sera GmbH, Heinsberg, Germany) started on day 5 postfertilization. The amount of daily food was adjusted according to tadpole size so as to avoid water deterioration from excess food. The water was gently aerated during the rearing period. Two weeks after fertilization, tadpoles at stages 49 and 50 (Nieuwkoop and Faber, 1994) with a total body length of approximately 20 mm were used to start each experiment, which employed tadpoles derived from three different frog pairs. The care and treatment of all frogs and tadpoles, including the survivors after the completion of the experiments, complied with the current laws of Japan and the guidelines presented by the American Society of Ichthyologists and Herpeto logists (ASIH, 2004). 2.2. Test chemicals and preparation of test solutions Since both T4 and PTU (the highest grade available, Sigma-Aldrich, St. Louis, MO., USA) were hardly soluble in neutral water, these chemicals were first dissolved in 0.7 M NaOH and 1 M NaOH, respectively. These solutions (T4, 3000 mg/L; PTU, 90 g/L) were used as stock solutions. Test solutions (T4, 1 mg/L; PTU, 75 mg/L) were prepared by diluting (3 106-fold for T4 and 1200-fold for PTU) the stock solutions with a diluent, as described in Table 1. The pH of the test
2.3.2. Experiment 1: A comparative rearing experiment between two different exposure systems A rearing experiment involving no chemical treatment groups was conducted to compare tadpole development between an individual-separated exposure system and an individual-grouped exposure system. In the former system, each of 30 tadpoles was held in a 500-mL (53 cm2 11 cm) glass beaker containing 330 mL of the diluent (i.e., control solution). In the latter system, 30 tadpoles were held together in a 45-L glass aquarium (1600 cm2 28 cm) containing 10 L of the diluent. The two exposure systems were therefore designed to provide the same rearing conditions of tadpole loading (one individual per 330 mL) and water depth (6 cm). On days 0 and 28, each tadpole was inspected alive for developmental stage, hind limb length, and total body length on its ventral image captured using a digital microscope (CCD camera and controller, VH-6300; zoom lens, VH-Z05; Keyence, Osaka, Japan). The developmental stage was determined consulting the normal table of X. laevis (Nieuwkoop and Faber, 1994). The hind limb length (from the root of the thigh to the tip of the fourth toe) and total body length (from the tip of the snout to the end of the tail fin) were measured to the nearest 0.1 mm. The length measurement was made as if the objective was stretched straightforward. 2.3.3. Experiment 2: A trial experiment of the metamorphosis assay using T4 and PTU The metamorphosis assay was conducted under the individual-separated exposure system that held tadpoles individually in a 500-mL glass beaker containing 330 mL of the test solution. In this experiment, T4 (a thyroid system agonist; 1 mg/L) and PTU (an antithyroid substance; 75 mg/L) were tested. Each tadpole was inspected at 7-day intervals for developmental stage, hind limb length, and total body length using the same method as that described in experiment 1. In addition, observations without a microscope were made daily regarding mortality, abnormal behavior, and grossly visible malformations. At the termination of the test, all tadpoles were fixed (see below) and then (2 days after the fixation) weighed to the nearest 1 mg after removing adherent water with a dry paper towel. 2.4. Histological examinations of the thyroid glands At the termination of experiment 2, all tadpoles were inactivated by gradually cooling down the water temperature to approximately 15 1C, in order to suppress
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further progress of the tadpole development. The tadpoles were euthanized in a 200 mg/L tricaine methanesulfonate (Kanto Chemical, Tokyo, Japan) solution neutralized with a 0.1% (w/v) NaHCO3 solution, and then fixed in Mildforms 10 N (Wako Pure Chemical Industries, Osaka, Japan) for histological examinations of thyroid glands. The current work was a validation study using T4 and PTU, the histological effects of which on thyroid glands have been well-documented using X. laevis tadpoles by OECD (2007). Therefore, only five tadpole specimens were randomly selected from each chemical treatment group and the control group. The entire head involving the thyroid glands was amputated and embedded in paraffin after being dehydrated. The tadpole head in the paraffin block was oriented ventral to dorsal on a horizontal plane in the mold to allow for sectioning from the ventral face. Sectioning was conducted serially at 5 mm thickness, and the serial sections were stained with hematoxylin and then eosin. A section involving the central portion of the thyroid glands was selected and observed while consulting a thyroid histology atlas presented by OECD (2007).
2.5. Chemical analyses In experiment 2, fresh and used test solutions were examined for the concentrations of T4 and PTU at 7-day intervals. Fresh test solutions were sampled prior to dispensing into test beakers on days 0, 7, 14, and 21. Used test solutions in three test beakers randomly selected from each chemical treatment group were sampled on days 7, 14, 21, and 28, at which the used test solutions were allowed to be static for 3 days (i.e., from Friday to Monday). Similarly, pH and DO were also measured at 7-day intervals. For the T4 analysis, each test solution (100 mL) was loaded on a solid-phase extraction cartridge (OASYSs HLB 6cc, Waters, MA., USA). The T4 trapped in the cartridge was eluted with 5 mL of methanol containing 25% ammonia solution at 2% (v/v). The elution containing T4 was evaporated by a gentle flow of N2 gas and reconstituted in 1 mL of methanol containing formic acid at 2% (v/v). Standard T4 solutions (0.05–1 mg/L) for calibration were similarly treated using the solid-phase extraction method because the recovery of T4 through this method was approximately 70%. The T4 concentration was determined using a high-performance liquid chromatograph (ACQUITY Ultra Performance LC, Waters, MA., USA) connected to a tandem mass spectrometer (Quattro Premier XE, Waters, MA., USA), the operational conditions of which are shown in Table 2. For the PTU analysis, each test solution was filtered through a cellulose acetate filter with a mesh of 0.2 mm (DISMIC-25CS, Advantec MFC, CA., USA). The filtrate was diluted with acetonitrile and directly injected into a high-performance liquid chromatograph (LC-10A, Shimadzu, Kyoto, Japan) connected to a UV absorption detector (SPD-10A, Shimadzu, Kyoto, Japan), which was run following the operational conditions shown in Table 2.
2.6. Statistical analyses In experiment 1, the F-test was used to determine significant differences in dispersion of parametric data (hind limb length and total body length) between the two exposure systems. Significant differences in the means between the two exposure systems were determined using the t-test or Welch’s test. For nonparametric data (developmental stage data), the Ansari–Bradley test and the median test were used instead of the F-test and the t-test (or Welch’s test), respectively. In experiment 2, one-way analysis of variance followed by Dunnett’s test was used to compare parametric and homoscedastic data between the three groups (T4- and PTU-treatment groups and the control group). For nonparametric or parametric but heteroscedastic data, the Kruskal–Wallis test followed by the Steel test was applied. Those analyses were performed at the level of P o0.05 using the SYSTAT J version 11 (Hulinks, Tokyo, Japan).
3. Results 3.1. Experiment 1 Throughout experiment 1, one dead tadpole was observed in the individual-grouped exposure system, however, no other external abnormalities were observed in either systems. As shown in Fig. 1, the tadpoles reared under the individualseparated exposure system developed with significantly small dispersions in developmental stage (median and range¼stage 57 and stages 56–58), hind limb length (mean7SD¼9.86 7 1.61 mm), and total body length (mean7SD¼48.6 71.76 mm) compared with those reared under the individual-grouped exposure system (stage 56 and stages 54–64; 8.6073.99 mm; and 48.873.74 mm in the order presented above). No significant differences were found in the median or mean values of developmental stage, hind limb length, or total body length between the two exposure systems. Regarding the daily total amount of food consumption, apparent differences were not observed between the two exposure systems: during experiment 1, the green-colored water due to the daily feeding with Sera Microns became clear within 24 h in any of the 30 glass beakers (individual-separated exposure system) and the glass aquarium (individual-grouped exposure system). 3.2. Experiment 2 During experiment 2, pH and DO in the fresh test solutions sampled at 7-day intervals ranged from 6.7 to 7.4 and 96.2% to 105% saturation at 25 1C, respectively. In the used test solutions sampled at 7-day intervals, pH and DO ranged from 6.9 to 7.5 and 56.3% to 98.8% saturation at 25 1C, respectively. The concentrations of T4 and PTU in the fresh test solutions sampled at 7-day intervals ranged from 98.4% to 105% of the nominal concentration 1 mg/L (T4) and 106% to 114% of the nominal concentration 75 mg/L (PTU). In the used test solutions, the T4 concentration was maintained at approximately 80% of the nominal concentration on day 7, but then clearly dropped to approximately 30%–50% of the nominal concentration despite periodical solution renewals (Fig. 2). The PTU concentration in the used test solutions was consistently maintained near the nominal concentration (Fig. 2). Throughout experiment 2, no weakened or dead tadpoles or other external abnormalities, such as aberrant behavior or malformations, were observed in any of the control, T4- and PTUtreatment groups. As shown in Fig. 3, significant differences in the developmental stage and hind limb length were consistently observed among the three groups. The tadpoles exposed to T4 rapidly developed and reached metamorphic climax stages,
Table 2 The operational conditions of the high-performance liquid chromatographs for quantifying thyroxine (T4) and propylthiouracil (PTU). Apparatus parameters
Column Mobile phase Gradient profile Flow rate Oven temperature Injection volume Detector
Optimized conditions for each apparatus parameter T4
PTU
ACQUITY UPLC HSS C18, 2.1 mm id 100 mm length, 1.8 mm particle size (Waters, MA., USA) Solvent A: 0.5% (v/v) acetic acid Solvent B: acetonitrile % of solvent B: 20% (0–0.5 min); 20%–90% (0.5–3.5 min); 90% (3.5–6.5 min) 0.2 mL/min 40 1C 5 mL Electrospray ionization in positive mode monitoring precursor and product ions at 778 and 732 (m/z), respectively
Inertsil ODS-3, 4.6 mm id 150 mm length, 5 mm particle size (GL Sciences, Tokyo, Japan) 35% (v/v) acetonitrile containing 5% (v/v) acetic acid Isocratic mode 0.5 mL/min 40 1C 20 mL UV detection at 270 nm of wave length
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20
66
89
60
b
b
a
64
a
58 56 54 52
15 b a 10
5
Total body length (mm)
60
Hind limb length (mm)
Developmental stage
50 62
40 30 20 10
50 0
48 A
B
Day 0
A
0 A
B
Day 28
B
A
Day 0
B
A
Day 28
B
Day 0
A
B
Day 28
Rearing period Fig. 1. The developmental stage, hind limb length, and total body length of Silurana tropicalis tadpoles reared for 28 days under the individual-separated exposure system (A, n¼ 30) and the individual-grouped exposure system (B, initially n¼ 30 but reduced to 29 on day 28 due to the death of a tadpole). The developmental stage data are indicated as median (open circles) and mode (crosses) values with ranges (vertical bars). The data of hind limb length and total body length are presented as mean 7 SD. Between the two exposure systems, a significant difference (Po 0.05) indicated using different superscripts (a, b) was found only in the dispersion (or variance) on day 28 but not in the median (or mean) value of each morphological point.
100 % of nominal concentration
120 110 100 90 80 70 60 50 40 30 20 10 0
T4
90 80 70 60 50 40 30 20 10 0 Day 7
Day 14
Day 21
PTU
Day 7
Day 28
Day 14
Day 21
Day 28
Sampling date
* *
* * *
*
*
*
C T PCT PC TPCT PCT P Day 0
Day 7 Day 14 Day 21 Day 28
20
*
15
*
10 * 5 0
*
*
*
*
*
C TPCT PC TPCT PCT P Day 0
Day 7 Day 14 Day 21 Day 28
Total body length (mm)
66 64 62 60 58 56 54 52 50 48
Hind limb length (mm)
Developmental stage
Fig. 2. The concentrations of thyroxine (T4) and propylthiouracil (PTU) in the used test solutions sampled at 7-day intervals (n¼ 3 for each sampling) determined by the use of high-performance liquid chromatographs connected to a tandem mass spectrometer (T4) and a UV detector (PTU). The values (mean 7 SD) are presented as percentages of the nominal concentrations (T4 ¼ 1 mg/L; PTU ¼ 75 mg/L).
60 50 40 * *
30
* *
*
* *
20 10 0
CTPCTPCTPCTPCT P Day 0
Day 7 Day 14 Day 21 Day 28
Exposure period Fig. 3. The developmental stage, hind limb length, and total body length of S. tropicalis tadpoles exposed to the control solution, T4 (1 mg/L), and PTU (75 mg/L) at 7-day intervals. The symbols are the same as those in Fig. 1. The abbreviations C, T, and P represent the control, T4-treatment, and PTU-treatment groups, respectively. Each group consisted of 30 tadpoles. Significant differences (control vs. T4, PTU: P o 0.05) in the median (or mean) values are indicated by asterisks.
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exhibiting completely developed hind limbs, well elongated forelimbs, and degenerating tails on day 28. The development of the tadpoles exposed to PTU was obviously inhibited. These tadpoles remained at premetamorphic stages with poorly developed hind limbs even on day 28 when most of the tadpoles in the control group had reached prometamorphic stages or the initial metamorphic climax stage and exhibited well developed hind limbs. The mean total body lengths of the tadpoles exposed to T4 and PTU were significantly shorter than those of the control group at all time points with the exception of the PTU group on day 28. In the wet body mass at the termination of the test, a significant difference was found between the control group and the T4 group, but not found between the control group and the PTU group (Fig. 4). Comparative thyroid gland histology conducted among the three groups is shown in Fig. 5. The five tadpoles exposed to T4 uniformly showed thyroid gland atrophy: due to decreases in follicular size and amount of colloid, the overall glands size was reduced to approximately 1/3 in comparison with the control glands. According to the atlas presented by OECD (2007), the severity of the atrophy observed was ‘grade 2 (moderate)’. On the other hands, the thyroid glands of the five tadpoles exposed to PTU were more than two times as large as the control glands, uniformly exhibiting hypertrophy of ‘grade 4 (severe)’ (OECD,
700
Wet body mass (mg)
600 500 400 *
300 200 100 0
C
T Chemical treatment group
P
Fig. 4. The wet body mass of S. tropicalis tadpoles exposed to the control solution, T4 (1 mg/L), and PTU (75 mg/L) at the test termination (day 28). The abbreviations C, T, and P are the same as those in Fig. 3. The values are presented as mean 7SD with an asterisk indicating a significant difference in the mean values (control vs. T4: Po 0.05).
2007) with contact of both glands at the midline. The thyroid gland hypertrophy observed in the five tadpoles involved follicular cell hypertrophy of ‘grade 3 (severe)’ and follicular cell hyperplasia of ‘grade 3 (severe)’ (OECD, 2007).
4. Discussion The current study was conducted to establish an alternative testing protocol for an amphibian metamorphosis assay, which would be practicable to carry out by only one or two individual researchers. The subject of this study is therefore how to reduce practical burdens on testers. We addressed this issue by employing an individual-separated exposure system that would make tadpoles develop as uniformly as possible by excluding interference during feeding among the tadpoles. In other words, we expected that this exposure system would reduce within-group variability in tadpole growth and development and thereby allow group size, i.e. the number of test tadpoles, to be reduced. Our expectation was supported by the results of experiment 1. Apparent differences in the daily total amount of food consumption were not observed between the individual-separated and individual-grouped systems. However, the interindividual variability in the three morphological endpoints was significantly smaller under the former system than under the latter: the maximum and minimum values of developmental stage differed by only two stages under the former whereas by ten stages under the latter. In addition, the SD values in hind limb length and total body length under the former were less than half of those under the latter (Fig. 1). These facts suggest that the former system reduced the variability by equalizing the daily amount of food consumption among the tadpoles. We initially suspected that the former system, which used small-sized test vessels, might also induce developmental acceleration because gradually reducing swimming space can be a trigger to the release of thyrotropin in certain species of tadpoles (Denver, 1998; Denver et al., 1998). However, contrary to our suspicion, no significant differences in the median or mean values of the data observed for the three endpoints were found between the two exposure systems. As suggested by Rose (2005), tadpoles of fully aquatic anuran species, such as X. laevis and S. tropicalis, are not as sensitive to volumetric changes in water levels as other anuran species that are aquatic during larval stages but substantially terrestrial after metamorphosis. It is therefore concluded that the individualseparated exposure system has an experimental advantage of variability-reducing effects without developmental acceleration. In experiment 2, we tested T4 (1 mg/L) as a thyroid system agonist and PTU (75 mg/L) as an antithyroid substance using the metamorphosis assay under the individual-separated exposure system. In general, when conducting static and semistatic tests, attention should be given to the quality degradation of test solutions during the tests. Since experiment 2 was conducted
Fig. 5. Comparative thyroid gland histology of the S. tropicalis tadpoles in the control (left), T4-treatment (1 mg/L, center), and PTU-treatment (75 mg/L, right) groups. The abbreviations T, F, and Fc indicate thyroid glands, follicles, and follicular cells, respectively. Each scale bar corresponds to 0.1 mm.
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under a semistatic regime with small-sized test vessels, we examined pH, DO, and concentrations of test chemicals of the used test solutions that were allowed to be static for 3 days. The deviations of pH (6.9–7.5) and DO (56.3%–98.8% saturation at 25 1C) fulfilled the criteria determined by OECD (2009) in which the pH and DO should be maintained at 6.5 to 8.5 and 440% air saturation, respectively. As shown in Fig. 2, the T4 concentration clearly decreased as the tadpole development proceeded, and it dropped to nearly 30% of the nominal concentration (1 mg/L) at the test termination. This step-by-step decrease in the T4 concentration may be due to uptake of T4 by the tadpoles. It is likely that the amount of T4 absorbed through the surface of the tadpole body increased as they developed before reaching metamorphic climax stages. Thereafter, alterations in skin permeability occurring with metamorphosis (Nieuwkoop and Faber, 1994) may have relevance to the decrease of T4 in the test solutions. Alternatively, T4 in the test solutions might be absorbed into tadpole feces, because the amount of the tadpole feces seemed to increase since day 7 when the amount of daily food was doubled. On the other hand, the PTU concentration was maintained near the nominal concentration (75 mg/L, exceedingly high compared the nominal concentration of T4) throughout the test. Similar trends have also been reported in the previous studies that tested T4 (1 mg/L) and PTU (75 mg/L) under a semistatic regime with the same renewal interval as in our protocol, although these studies employed different anuran species: X. laevis (Kloas et al., 2003) and R. rugosa (Oka et al., 2009). These facts, coupled with our results, suggest limitations of semistatic tests in general, and therefore, the decrease in the T4 concentration that occurred during experiment 2 does not necessarily invalidate our testing protocol. During experiment 2, T4 was nontoxic to the S. tropicalis tadpoles, however, it consistently induced significant acceleration to metamorphosis, as indicated by significantly further advanced developmental stages, longer hind limb lengths, shorter total body lengths, and smaller wet body mass of the tadpoles exposed to T4 compared with those observed in the control group (Fig. 3). These effects promoting morphological alterations toward metamorphosis rather than tadpole growth (elongation of total body length) reflect the actions of T4. In contrast, PTU, which was also nontoxic to the S. tropicalis tadpoles, caused pronounced inhibitory effects on metamorphosis, as illustrated by the tadpoles showing extremely slow progress in developmental stage and poorly developed hind limbs without fully differentiated digits even on day 28 (Fig. 3). The mean total body length in the PTU group was significantly shorter than that in the control group on days 7–21 (Fig. 3). However, at the test termination, no significant difference was found between the two groups not only in the mean total body length but also in the mean wet body mass. Similar retardation of tadpole growth was also induced by PTU (75 mg/L) in the previous studies employing X. laevis (Kloas et al., 2003) and S. tropicalis (Mitsui et al., 2006). As a potent inhibitor of thyroid peroxidase, PTU blocks T4 synthesis in the thyroid gland in mammals (Cooper et al., 1983; Engler et al., 1982) and anurans (Robertson and Kelley, 1996). Apart from this action, PTU also inhibits extrathyroidal conversion of T4 to triiodothyronine (T3) and augments thyrotropin release in mammals, which is independent of the first effect (Geffner et al., 1975). However, such antithyroidal actions of PTU cannot account for the growth retardation observed in our study because the inhibition of metamorphosis arising from the blockade of T4 synthesis (and T4-to-T3 conversion) does not necessarily lead to tadpole growth inhibition (Opitz et al., 2005). For example, amitrole and ethylenethiourea, both of which act as an antithyroid chemical in X. laevis, do not affect tadpole growth at the concentrations that induce complete blockage of metamorphosis arising from T4 synthesis inhibition (Kloas et al., 2003). The growth retardation
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induced by PTU may therefore suggest that PTU has extrathyroidal and toxic side effects that have not yet been mechanistically ascertained (Kloas et al., 2003; Opitz et al., 2005). Neither total body length data nor snout-vent length data should be solely used to determine thyroid toxicity of test chemicals (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006). Rather, such length data (and also body mass data) should be used in conjunction with developmental stage and histopathology data in order to determine thyroid-disrupting activities (OECD, 2009). In amphibian metamorphosis assays, evaluation of non-thyroidal effects such as growth inhibition is also required to avoid false outcomes because such effects can also induce mild developmental retardation (OECD, 2009). The data of tadpole length and weight can provide useful information to assess possible effects of test chemicals on tadpole growth. Here, we must pay attention to the utility of total body length because tadpoles at stage 60 or further show signs of tail resorption and reshaping of the head, reducing tail length and snout–vent length and consequently reducing total body length (Nieuwkoop and Faber, 1994; Kloas et al., 2003). Under the traditional individualgrouped exposure system, many tadpoles, even in the control, showed development beyond stage 59 at the test termination (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006). It has therefore been proposed that the use of total body length should be limited to the initial 14 days (Opitz et al., 2005) or 21 days (Kloas et al., 2003; Mitsui et al., 2006) of exposure. Meanwhile, under the individual-separated exposure system, all tadpoles in the control solution did not develop beyond stage 59 both in experiments 1 and 2 due to small interindividual variability in developmental stage (Figs. 1 and 3). We substituted total body length for snout–vent length in order to compare our results with the historical data, and the above-described result suggests the utility of total body length until day 28 under the individualseparated exposure system. In addition, the prolonged metamorphosis assay with the exposure duration of 28 days may find other non-thyroidal effects of chemicals that cannot be detected within 21 days of exposure. In any of the three groups (the control, T4-, and PTU-treatment groups), the median or mean values of the morphological data obtained at 7-day intervals (Fig. 3) were quite similar to those reported by Mitsui et al. (2006), who developed an amphibian metamorphosis assay using S. tropicalis tadpoles under the traditional individual-grouped exposure system. In addition, the variability of the morphological data concerning the three endpoints was greatly reduced compared with that presented by Mitsui et al. (2006). For example, at the test termination (day 28), developmental stage of the tadpoles in the control ranged from stage 51 to stage 64 in the report by Mitsui et al. (2006), whereas from stage 56 to stage 59 in the current work (experiment 2). These comparative evaluations support the validity and superiority of the alternative testing protocol using the individualseparated exposure system. Although Mitsui et al. (2006) did not present any histological observations of thyroid glands, the thyroid gland atrophy observed in the tadpoles exposed to T4 (Fig. 5) is a typical symptom caused by exogenous T4 in X. laevis (OECD, 2007) and R. rugosa (Oka et al., 2009). According to these two previous studies, PTU causes thyroid gland hypertrophy and follicular cell hyperplasia in the above two anuran species. In our study, similar histological lesions were observed in the tadpoles exposed to PTU (Fig. 5). Therefore, our histological observations of the thyroid glands pathologically supported the metamorphosis-promoting and metamorphosis-inhibiting effects of T4 and PTU, respectively. Overall, the amphibian metamorphosis assay using S. tropicalis tadpoles was successfully validated even under the individualseparated exposure system, by testing T4 and PTU. The superiority
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of this alternative testing protocol is mainly due to the fact that it can be successfully performed by only one or two individual researchers. This testing protocol may therefore have the potential to positively contribute to the progress of ecotoxicological studies concerning the detection of chemicals with agonistic and antagonistic activities on the thyroid hormone axis. However, it should be noted that the current study tested only two chemicals that have strong effects on the thyroid system: T4 is a thyroid hormone itself and PTU is a well-known antithyroid substance. In general, effects of thyroid hormone-disrupting chemicals in the environment would not be as strong as those of the two chemicals tested. Further studies using other chemicals will be required to confirm whether our assay can detect slight but significant effects on the thyroid system.
5. Conclusions The current work examined an alternative testing protocol for amphibian (S. tropicalis) metamorphosis assays: an individualseparated exposure system that was expected to reduce interindividual variability in tadpole growth and development and thereby allow the number of test tadpoles to be reduced (n¼30 for each group). Our expectation was verified by the comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system. Using the new testing protocol, we tested the well-known thyroid system agonist T4 and antithyroid substance PTU for 28 days of exposure. Morphological data of the tadpoles exposed to T4 and PTU consistently indicated significant metamorphic acceleration and retardation, respectively. The thyroid glands of the tadpoles clearly showed histological alterations caused by T4 and PTU. Our results agree with the historical data generated from previous studies employing the traditional exposure system, thereby indicating the validity of the amphibian metamorphosis assay employing the individual-separated exposure system.
Acknowledgments The frogs S. tropicalis were supplied from the Institute of Amphibian Biology of Hiroshima University as a part of the National BioResource Project by the Japan Ministry of Education, Culture, Sports, Science, and Technology. We thank A. Kashiwagi, M. Takase, and Y. Yaoita for technical advice on the keeping and breeding of S. tropicalis. References ASIH, 2004. Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research. ASIH, Lawrence.
Cooper, D.S., Kieffer, J.D., Halpern, R., Saxe, V., Mover, H., Maloof, F., Ridgway, E.C., 1983. Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function. Endocrinology 113, 921–928. Denver, R.J., 1998. Hormonal correlates of environmentally induced metamorphosis in the western spadefoot toad Scaphiopus hammondii. Gen. Comp. Endocrinol. 110, 326–336. Denver, R.J., Mirhadi, N., Phillips, M., 1998. Adaptive plasticity in amphibianmetamorphosis: response of Scaphiopus hammondii tadpoles to habitat desiccation. Ecology 79, 1859–1872. Engler, H., Taurog, A., Dorris, M.L., 1982. Preferential inhibition of thyroxine and 3,5,30 -triiodothyronine formation by propylthiouracil and methylmercaptoimidazole in thyroid peroxidase-catalyzed iodination of thyroglobulin. Endocrinology 110, 190–197. Fort, D.J., Rogers, R.L., Thomas, J.H., Buzzard, B.O., Noll, A.M., Spaulding, C.D., 2004a. Comparative sensitivity of Xenopus tropicalis and Xenopus laevis as test species for the FETAX model. J. Appl. Toxicol. 24, 443–457. Fort, D.J., Thomas, J.H., Rogers, R.L., Noll, A., Spaulding, C.D., Guiney, P.D., Weeks, J.A., 2004b. Evaluation of the development and reproductive toxicity of methoxychlor using an anuran (Xenopus tropicalis) chronic exposure model. Toxicol. Sci. 81, 443–453. Geffner, D.L., Azukizawa, M., Hershman, J.M., 1975. Propylthiouracil blocks extrathyroidal conversion of thyroxine to triiodothyronine and augments thyrotropin secretion in man. J. Clin. Invest. 55, 224–229. Kashiwagi, K., Kashiwagi, A., Kurabayashi, A., Hanada, H., Nakajima, K., Okada, M., Takase, M., Yaoita, Y., 2010. Xenopus tropicalis: an ideal experimental animal in amphibia. Exp. Anim. 59, 395–405. Kloas, W., 2002. Amphibians as a model for the study of endocrine disruptors. Int. Rev. Cytol. 216, 1–57. Kloas, W., Opitz, R., Lutz, I., 2003. Ringtest: Effects of Pesticides and Other Chemicals on Thyroid System in the Amphibian Xenopus laevis. Federal Environmental Agency Research Report 20067409, Berlin, Germany. Mitsui, N., Fujii, T., Miyahara, M., Oka, T., Kashiwagi, A., Kashiwagi, K., Hanada, H., Urushitani, H., Santo, N., Tooi, O., Iguchi, T., 2006. Development of metamorphosis assay using Silurana tropicalis for the detection of thyroid systemdisrupting chemicals. Ecotoxicol. Environ. Saf. 64, 281–287. Nieuwkoop, P.D., Faber, J., 1994. Normal Table of Xenopus laevis (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis. Garland, New York. OECD, 2004. Detailed Review Paper on Amphibian Metamorphosis Assay for the Detection of Thyroid Active Substances. OECD, Paris. OECD, 2007. Guidance Document on Amphibian Thyroid Histology. OECD, Paris. OECD, 2009. Amphibian Metamorphosis Assay. OECD Guidelines for Testing of Chemicals. No. 231. OECD, Paris. Oka, T., Miyahara, M., Yamamoto, J., Mitsui, N., Fujii, T., Tooi, O., Kashiwagi, K., Takase, M., Kashiwagi, A., Iguchi, T., 2009. Application of metamorphosis assay to a native Japanese amphibian species, Rana rugosa, for assessing effects of thyroid system affecting chemicals. Ecotoxicol. Environ. Saf. 72, 1400–1405. ¨ Opitz, R., Braunbeck, T., Bogi, C., Pickford, D.B., Nentwig, G., Oehlmann, J., Tooi, O., Lutz, I., Kloas, W., 2005. Description and initial evaluation of a Xenopus metamorphosis assay for detection of thyroid system-disrupting activities of environmental compounds. Environ. Toxicol. Chem. 24, 653–664. Rice, T.M., Walker, S.E., Blackstone, B.J., Taylor, D.H., 1998. A new method for marking individual anuran larvae. Herpetol. Rev. 29, 92–93. Robertson, J.C., Kelley, D.B., 1996. Thyroid hormone controls the onset of androgen sensitivity in the developing larynx of Xenopus laevis. Dev. Biol. 176, 108–123. Rose, C.S., 2005. Integrating ecology and developmental biology to explain the timing of frog metamorphosis. Trends Ecol. Evol. 20, 129–135. Shi, Y-B., 2000. Amphibian Metamorphosis—From Morphology to Molecular Biology. Wiley-Liss, New York. Song, M.O., Fort, D.J., McLaughlin, D.L., Rogers, R.L., Thomas, J.H., Buzzard, B.O., Noll, A.M., Myers, N.K., 2003. Evaluation of Xenopus tropicalis as an alternative test organism for frog embryo teratogenesis assay—Xenopus (FETAX). Drug Chem. Toxicol. 26, 177–189.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Examination of an amphibian metamorphosis assay under an individual-separated exposure system using Silurana tropicalis tadpoles Masahiro Saka a,n, Noriko Tada a, Yoichi Kamata b a b
Kyoto Prefectural Institute of Public Health and Environment, Murakamicho 395, Fushimi-ku, Kyoto 612-8369, Japan Division of Microbiology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2012 Received in revised form 27 August 2012 Accepted 28 August 2012 Available online 12 October 2012
We examined the validity of an amphibian (Silurana tropicalis) metamorphosis assay (a 28-day semistatic test) under an individual-separated exposure system, where tadpoles were individually held in small glass beakers. We first conducted a comparative rearing experiment for 28 days between this exposure system and the traditional individual-grouped exposure system, both of which held 30 tadpoles (stages 49 and 50) in dechlorinated tap water (a control solution). The former system served to reduce interindividual variability in regard to three morphological measures (developmental stage, hind limb length, and total body length). Under this system, we tested thyroxine (T4, 1 mg/L) and propylthiouracil (PTU, 75 mg/L) for 28 days of exposure. The morphological data collected at 7-day intervals indicated that significant metamorphic acceleration and retardation were consistently induced in the tadpoles exposed to T4 and PTU, respectively. In addition, the thyroid glands of the tadpoles exposed to T4 and PTU clearly exhibited atrophy and hypertrophy accompanied with severe follicular cell hyperplasia, respectively. Our results are in agreement with the historical data generated from previous studies employing the traditional exposure system, thus indicating the validity of our alternative testing protocol. & 2012 Elsevier Inc. All rights reserved.
Keywords: Amphibian Metamorphosis assay Silurana tropicalis Thyroxine Propylthiouracil Exposure system
1. Introduction Amphibian metamorphosis, which is induced and regulated by thyroid hormones, accompanies drastic transformations in essentially every organ and tissue. Particularly in anurans, the process of morphological alteration caused by metamorphosis (tadpole-to-frog transformation) can be easily monitored by visual inspection. In addition, postembryonic development has been thoroughly studied as a biological model to elucidate thyroid functions (Shi, 2000). By exploiting these advantages, an in vivo assay to detect chemicals disrupting thyroid functions has been designed using Xenopus laevis, a worldwide experimental anuran species (Kloas, 2002; Kloas et al., 2003; OECD, 2004; Opitz et al., 2005). The experimental protocol of this assay has been standardized by OECD (2009). Meanwhile, similar metamorphosis assays have also been successfully developed using other anuran species, such as Silurana tropicalis (formerly called Xenopus tropicalis) (Mitsui et al., 2006) and Rana rugosa, an endemic species in Japan (Oka et al., 2009). In particular, S. tropicalis, which is phylogenetically related to X. laevis, has attracted attention as a new model species, due to its experimental superiority
n
Corresponding author. Fax: þ81 75 621 4164. E-mail address: [email protected] (M. Saka).
0147-6513/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2012.08.034
over X. laevis: a shorter life cycle and a diploid genome that increase the practicability of use for multi-generational tests and genetic analyses (Song et al., 2003; Fort et al., 2004a, 2004b; Kashiwagi et al., 2010). However, traditional assays have practical difficulties when conducted by one or two individual researchers due to the large number of tadpoles required for the examination of morphological endpoints. As demonstrated by historical studies (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006; Oka et al., 2009), tadpoles, even within a control, tend to develop enhancing individual differences in growth and development during exposure periods. This tendency would become more conspicuous under individual-grouped exposure systems where 20–30 tadpoles are held together in a single test tank, because the tadpoles can easily interfere with each other during feeding. An evaluation of results yielded using amphibian metamorphosis assays is based largely on statistical comparisons of morphological data concerning tadpole development. Accordingly, such large within-group variability necessitates the use of a large number of tadpoles for control and chemical treatment groups. In addition, when including replication, the number of tadpoles increases two- to four-fold. Therefore, an exposure system to exclude interactions among tadpoles can serve to minimize interindividual variability of morphological data and thereby allow the number of tadpoles to be reduced. The simplest design fit for this purpose is
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an individual-separated exposure system in which each tadpole is held in a single test vessel. This system also enables individual identification without marking of the tadpoles, which is technically difficult (Rice et al., 1998). However, in the individual-separated exposure system, which requires as many test vessels as the total number of test tadpoles, the test vessel must be reduced in size due to the limited volume of the temperature-constant chamber. This exposure system inevitably reduces the amount of swimming space available for each tadpole. For certain anuran species, it has been suggested that tadpole development can be accelerated by gradually reducing aqueous space that may stimulate the release of thyrotropin, a thyroid-stimulating hormone (Denver, 1998; Denver et al., 1998). The anuran metamorphosis assay under the individual-separated exposure system may therefore not truly detect thyroid-disrupting chemicals, unless sufficient volume of test solution is provided for each tadpole during the test. The current work aimed to establish an amphibian metamorphosis assay practicable by one or two individual researchers, using S. tropicalis tadpoles as a test species. We first conducted a comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system in order to confirm that the former served to reduce the interindividual variability of the morphological without inducing developmental acceleration that would affect the test results. Subsequently, under the individual-separated exposure system, we tested thyroxine (T4, one of thyroid hormones) and propylthiouracil (PTU, an antithyroid chemical), both of which have been used as test chemicals in traditional metamorphosis assays. By comparing the results with the traditional data, we verified the validity of the amphibian metamorphosis assay under the individual-separated exposure system.
2. Materials and Methods
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Table 1 The experimental protocol of the metamorphosis assay using Silurana tropicalis tadpoles. Duration Temperature Light Diluent
Test procedure Group size Feeding Aeration
28 days 25 1C (with a deviation of 7 1 1C) 12-h light/12-h dark photoperiod using white fluorescent lamps Dechlorinated tap water (hardness, approximately 40 mg/L as CaCO3; pH, 6.5–7.5; dissolved oxygen, 95%–105% saturation at 25 1C; iodine concentration, approximately 4 mg/L) Semistatic test with solution renewal three times a week (Monday, Wednesday, and Friday) Thirty individuals for each group (a diluent control and chemical treatment groups) Daily with 1% (w/v) Sera Microns solution: 0.5 mL (days 0–6) and 1 mL (days 7–28) per individual None
solution for PTU was adjusted to approximately 7.0 by adding a small amount of 1 M HCl. Adjustment of pH was not done for the test solution of T4 because its pH dropped to nearly 7 due to 3 106-fold dilution. 2.3. Experiments 2.3.1. Assay protocol The current work consisted of two experiments that were conducted following the protocol shown in Table 1. Although OECD (2009) presented a standard protocol for conducting an amphibian metamorphosis assay under the traditional individual-grouped exposure system, the current experiments employing an individual-separated exposure system involved two major alterations: (1) the prolonged test duration from 21 to 28 days and (2) the use of total body length instead of snout–vent length as one of morphological endpoints. Most of historical data of amphibian metamorphosis assays have been yielded by employing the 28-day exposure duration and measuring total body length to monitor growth of tadpoles (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006; Oka et al., 2009). Since the validation of an individual-separated exposure system would be based largely on comparisons between the above historical data and those obtained from the current work, the two experiments were also performed during 28 days of exposure, using total body length as an indicator of tadpole growth.
2.1. Animal husbandry Adult pairs of S. tropicalis were obtained from the Institute of Amphibian Biology of Hiroshima University. The adult frogs were kept in polypropylene aquaria filled with dechlorinated tap water under the following conditions: water depth, 9 cm; water temperature, 25 7 1 1C; photoperiod, 12-h light/12-h dark; frog density, six frogs per 1800 cm2 of water surface area; feeding, thrice per week with a commercial diet for aquatic frogs (XL-2, Oriental Yeast, Tokyo, Japan). After an acclimatization period of 4–6 weeks, breeding was induced by injecting the frogs into the dorsal lymph sac with human chorionic gonadotropin (Wako Pure Chemical Industries, Osaka, Japan): each male received a small primer dose of 20 IU (7 days prior to mating) followed by a final dose of 200 IU (several hours prior to mating), and each female received a single dose of 300 IU (several hours prior to mating). Spawned eggs and developing tadpoles were reared in polypropylene aquaria filled with dechlorinated tap water under the following conditions: water depth, 7 cm; water temperature, 25 71 1C; photoperiod, 12-h light/12-h dark; tadpole density, approximately 200 individuals per 1800 cm2 of water surface area; feeding, daily with a commercial tadpole food (Sera Microns, Sera GmbH, Heinsberg, Germany) started on day 5 postfertilization. The amount of daily food was adjusted according to tadpole size so as to avoid water deterioration from excess food. The water was gently aerated during the rearing period. Two weeks after fertilization, tadpoles at stages 49 and 50 (Nieuwkoop and Faber, 1994) with a total body length of approximately 20 mm were used to start each experiment, which employed tadpoles derived from three different frog pairs. The care and treatment of all frogs and tadpoles, including the survivors after the completion of the experiments, complied with the current laws of Japan and the guidelines presented by the American Society of Ichthyologists and Herpeto logists (ASIH, 2004). 2.2. Test chemicals and preparation of test solutions Since both T4 and PTU (the highest grade available, Sigma-Aldrich, St. Louis, MO., USA) were hardly soluble in neutral water, these chemicals were first dissolved in 0.7 M NaOH and 1 M NaOH, respectively. These solutions (T4, 3000 mg/L; PTU, 90 g/L) were used as stock solutions. Test solutions (T4, 1 mg/L; PTU, 75 mg/L) were prepared by diluting (3 106-fold for T4 and 1200-fold for PTU) the stock solutions with a diluent, as described in Table 1. The pH of the test
2.3.2. Experiment 1: A comparative rearing experiment between two different exposure systems A rearing experiment involving no chemical treatment groups was conducted to compare tadpole development between an individual-separated exposure system and an individual-grouped exposure system. In the former system, each of 30 tadpoles was held in a 500-mL (53 cm2 11 cm) glass beaker containing 330 mL of the diluent (i.e., control solution). In the latter system, 30 tadpoles were held together in a 45-L glass aquarium (1600 cm2 28 cm) containing 10 L of the diluent. The two exposure systems were therefore designed to provide the same rearing conditions of tadpole loading (one individual per 330 mL) and water depth (6 cm). On days 0 and 28, each tadpole was inspected alive for developmental stage, hind limb length, and total body length on its ventral image captured using a digital microscope (CCD camera and controller, VH-6300; zoom lens, VH-Z05; Keyence, Osaka, Japan). The developmental stage was determined consulting the normal table of X. laevis (Nieuwkoop and Faber, 1994). The hind limb length (from the root of the thigh to the tip of the fourth toe) and total body length (from the tip of the snout to the end of the tail fin) were measured to the nearest 0.1 mm. The length measurement was made as if the objective was stretched straightforward. 2.3.3. Experiment 2: A trial experiment of the metamorphosis assay using T4 and PTU The metamorphosis assay was conducted under the individual-separated exposure system that held tadpoles individually in a 500-mL glass beaker containing 330 mL of the test solution. In this experiment, T4 (a thyroid system agonist; 1 mg/L) and PTU (an antithyroid substance; 75 mg/L) were tested. Each tadpole was inspected at 7-day intervals for developmental stage, hind limb length, and total body length using the same method as that described in experiment 1. In addition, observations without a microscope were made daily regarding mortality, abnormal behavior, and grossly visible malformations. At the termination of the test, all tadpoles were fixed (see below) and then (2 days after the fixation) weighed to the nearest 1 mg after removing adherent water with a dry paper towel. 2.4. Histological examinations of the thyroid glands At the termination of experiment 2, all tadpoles were inactivated by gradually cooling down the water temperature to approximately 15 1C, in order to suppress
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further progress of the tadpole development. The tadpoles were euthanized in a 200 mg/L tricaine methanesulfonate (Kanto Chemical, Tokyo, Japan) solution neutralized with a 0.1% (w/v) NaHCO3 solution, and then fixed in Mildforms 10 N (Wako Pure Chemical Industries, Osaka, Japan) for histological examinations of thyroid glands. The current work was a validation study using T4 and PTU, the histological effects of which on thyroid glands have been well-documented using X. laevis tadpoles by OECD (2007). Therefore, only five tadpole specimens were randomly selected from each chemical treatment group and the control group. The entire head involving the thyroid glands was amputated and embedded in paraffin after being dehydrated. The tadpole head in the paraffin block was oriented ventral to dorsal on a horizontal plane in the mold to allow for sectioning from the ventral face. Sectioning was conducted serially at 5 mm thickness, and the serial sections were stained with hematoxylin and then eosin. A section involving the central portion of the thyroid glands was selected and observed while consulting a thyroid histology atlas presented by OECD (2007).
2.5. Chemical analyses In experiment 2, fresh and used test solutions were examined for the concentrations of T4 and PTU at 7-day intervals. Fresh test solutions were sampled prior to dispensing into test beakers on days 0, 7, 14, and 21. Used test solutions in three test beakers randomly selected from each chemical treatment group were sampled on days 7, 14, 21, and 28, at which the used test solutions were allowed to be static for 3 days (i.e., from Friday to Monday). Similarly, pH and DO were also measured at 7-day intervals. For the T4 analysis, each test solution (100 mL) was loaded on a solid-phase extraction cartridge (OASYSs HLB 6cc, Waters, MA., USA). The T4 trapped in the cartridge was eluted with 5 mL of methanol containing 25% ammonia solution at 2% (v/v). The elution containing T4 was evaporated by a gentle flow of N2 gas and reconstituted in 1 mL of methanol containing formic acid at 2% (v/v). Standard T4 solutions (0.05–1 mg/L) for calibration were similarly treated using the solid-phase extraction method because the recovery of T4 through this method was approximately 70%. The T4 concentration was determined using a high-performance liquid chromatograph (ACQUITY Ultra Performance LC, Waters, MA., USA) connected to a tandem mass spectrometer (Quattro Premier XE, Waters, MA., USA), the operational conditions of which are shown in Table 2. For the PTU analysis, each test solution was filtered through a cellulose acetate filter with a mesh of 0.2 mm (DISMIC-25CS, Advantec MFC, CA., USA). The filtrate was diluted with acetonitrile and directly injected into a high-performance liquid chromatograph (LC-10A, Shimadzu, Kyoto, Japan) connected to a UV absorption detector (SPD-10A, Shimadzu, Kyoto, Japan), which was run following the operational conditions shown in Table 2.
2.6. Statistical analyses In experiment 1, the F-test was used to determine significant differences in dispersion of parametric data (hind limb length and total body length) between the two exposure systems. Significant differences in the means between the two exposure systems were determined using the t-test or Welch’s test. For nonparametric data (developmental stage data), the Ansari–Bradley test and the median test were used instead of the F-test and the t-test (or Welch’s test), respectively. In experiment 2, one-way analysis of variance followed by Dunnett’s test was used to compare parametric and homoscedastic data between the three groups (T4- and PTU-treatment groups and the control group). For nonparametric or parametric but heteroscedastic data, the Kruskal–Wallis test followed by the Steel test was applied. Those analyses were performed at the level of P o0.05 using the SYSTAT J version 11 (Hulinks, Tokyo, Japan).
3. Results 3.1. Experiment 1 Throughout experiment 1, one dead tadpole was observed in the individual-grouped exposure system, however, no other external abnormalities were observed in either systems. As shown in Fig. 1, the tadpoles reared under the individualseparated exposure system developed with significantly small dispersions in developmental stage (median and range¼stage 57 and stages 56–58), hind limb length (mean7SD¼9.86 7 1.61 mm), and total body length (mean7SD¼48.6 71.76 mm) compared with those reared under the individual-grouped exposure system (stage 56 and stages 54–64; 8.6073.99 mm; and 48.873.74 mm in the order presented above). No significant differences were found in the median or mean values of developmental stage, hind limb length, or total body length between the two exposure systems. Regarding the daily total amount of food consumption, apparent differences were not observed between the two exposure systems: during experiment 1, the green-colored water due to the daily feeding with Sera Microns became clear within 24 h in any of the 30 glass beakers (individual-separated exposure system) and the glass aquarium (individual-grouped exposure system). 3.2. Experiment 2 During experiment 2, pH and DO in the fresh test solutions sampled at 7-day intervals ranged from 6.7 to 7.4 and 96.2% to 105% saturation at 25 1C, respectively. In the used test solutions sampled at 7-day intervals, pH and DO ranged from 6.9 to 7.5 and 56.3% to 98.8% saturation at 25 1C, respectively. The concentrations of T4 and PTU in the fresh test solutions sampled at 7-day intervals ranged from 98.4% to 105% of the nominal concentration 1 mg/L (T4) and 106% to 114% of the nominal concentration 75 mg/L (PTU). In the used test solutions, the T4 concentration was maintained at approximately 80% of the nominal concentration on day 7, but then clearly dropped to approximately 30%–50% of the nominal concentration despite periodical solution renewals (Fig. 2). The PTU concentration in the used test solutions was consistently maintained near the nominal concentration (Fig. 2). Throughout experiment 2, no weakened or dead tadpoles or other external abnormalities, such as aberrant behavior or malformations, were observed in any of the control, T4- and PTUtreatment groups. As shown in Fig. 3, significant differences in the developmental stage and hind limb length were consistently observed among the three groups. The tadpoles exposed to T4 rapidly developed and reached metamorphic climax stages,
Table 2 The operational conditions of the high-performance liquid chromatographs for quantifying thyroxine (T4) and propylthiouracil (PTU). Apparatus parameters
Column Mobile phase Gradient profile Flow rate Oven temperature Injection volume Detector
Optimized conditions for each apparatus parameter T4
PTU
ACQUITY UPLC HSS C18, 2.1 mm id 100 mm length, 1.8 mm particle size (Waters, MA., USA) Solvent A: 0.5% (v/v) acetic acid Solvent B: acetonitrile % of solvent B: 20% (0–0.5 min); 20%–90% (0.5–3.5 min); 90% (3.5–6.5 min) 0.2 mL/min 40 1C 5 mL Electrospray ionization in positive mode monitoring precursor and product ions at 778 and 732 (m/z), respectively
Inertsil ODS-3, 4.6 mm id 150 mm length, 5 mm particle size (GL Sciences, Tokyo, Japan) 35% (v/v) acetonitrile containing 5% (v/v) acetic acid Isocratic mode 0.5 mL/min 40 1C 20 mL UV detection at 270 nm of wave length
M. Saka et al. / Ecotoxicology and Environmental Safety 86 (2012) 86–92
20
66
89
60
b
b
a
64
a
58 56 54 52
15 b a 10
5
Total body length (mm)
60
Hind limb length (mm)
Developmental stage
50 62
40 30 20 10
50 0
48 A
B
Day 0
A
0 A
B
Day 28
B
A
Day 0
B
A
Day 28
B
Day 0
A
B
Day 28
Rearing period Fig. 1. The developmental stage, hind limb length, and total body length of Silurana tropicalis tadpoles reared for 28 days under the individual-separated exposure system (A, n¼ 30) and the individual-grouped exposure system (B, initially n¼ 30 but reduced to 29 on day 28 due to the death of a tadpole). The developmental stage data are indicated as median (open circles) and mode (crosses) values with ranges (vertical bars). The data of hind limb length and total body length are presented as mean 7 SD. Between the two exposure systems, a significant difference (Po 0.05) indicated using different superscripts (a, b) was found only in the dispersion (or variance) on day 28 but not in the median (or mean) value of each morphological point.
100 % of nominal concentration
120 110 100 90 80 70 60 50 40 30 20 10 0
T4
90 80 70 60 50 40 30 20 10 0 Day 7
Day 14
Day 21
PTU
Day 7
Day 28
Day 14
Day 21
Day 28
Sampling date
* *
* * *
*
*
*
C T PCT PC TPCT PCT P Day 0
Day 7 Day 14 Day 21 Day 28
20
*
15
*
10 * 5 0
*
*
*
*
*
C TPCT PC TPCT PCT P Day 0
Day 7 Day 14 Day 21 Day 28
Total body length (mm)
66 64 62 60 58 56 54 52 50 48
Hind limb length (mm)
Developmental stage
Fig. 2. The concentrations of thyroxine (T4) and propylthiouracil (PTU) in the used test solutions sampled at 7-day intervals (n¼ 3 for each sampling) determined by the use of high-performance liquid chromatographs connected to a tandem mass spectrometer (T4) and a UV detector (PTU). The values (mean 7 SD) are presented as percentages of the nominal concentrations (T4 ¼ 1 mg/L; PTU ¼ 75 mg/L).
60 50 40 * *
30
* *
*
* *
20 10 0
CTPCTPCTPCTPCT P Day 0
Day 7 Day 14 Day 21 Day 28
Exposure period Fig. 3. The developmental stage, hind limb length, and total body length of S. tropicalis tadpoles exposed to the control solution, T4 (1 mg/L), and PTU (75 mg/L) at 7-day intervals. The symbols are the same as those in Fig. 1. The abbreviations C, T, and P represent the control, T4-treatment, and PTU-treatment groups, respectively. Each group consisted of 30 tadpoles. Significant differences (control vs. T4, PTU: P o 0.05) in the median (or mean) values are indicated by asterisks.
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exhibiting completely developed hind limbs, well elongated forelimbs, and degenerating tails on day 28. The development of the tadpoles exposed to PTU was obviously inhibited. These tadpoles remained at premetamorphic stages with poorly developed hind limbs even on day 28 when most of the tadpoles in the control group had reached prometamorphic stages or the initial metamorphic climax stage and exhibited well developed hind limbs. The mean total body lengths of the tadpoles exposed to T4 and PTU were significantly shorter than those of the control group at all time points with the exception of the PTU group on day 28. In the wet body mass at the termination of the test, a significant difference was found between the control group and the T4 group, but not found between the control group and the PTU group (Fig. 4). Comparative thyroid gland histology conducted among the three groups is shown in Fig. 5. The five tadpoles exposed to T4 uniformly showed thyroid gland atrophy: due to decreases in follicular size and amount of colloid, the overall glands size was reduced to approximately 1/3 in comparison with the control glands. According to the atlas presented by OECD (2007), the severity of the atrophy observed was ‘grade 2 (moderate)’. On the other hands, the thyroid glands of the five tadpoles exposed to PTU were more than two times as large as the control glands, uniformly exhibiting hypertrophy of ‘grade 4 (severe)’ (OECD,
700
Wet body mass (mg)
600 500 400 *
300 200 100 0
C
T Chemical treatment group
P
Fig. 4. The wet body mass of S. tropicalis tadpoles exposed to the control solution, T4 (1 mg/L), and PTU (75 mg/L) at the test termination (day 28). The abbreviations C, T, and P are the same as those in Fig. 3. The values are presented as mean 7SD with an asterisk indicating a significant difference in the mean values (control vs. T4: Po 0.05).
2007) with contact of both glands at the midline. The thyroid gland hypertrophy observed in the five tadpoles involved follicular cell hypertrophy of ‘grade 3 (severe)’ and follicular cell hyperplasia of ‘grade 3 (severe)’ (OECD, 2007).
4. Discussion The current study was conducted to establish an alternative testing protocol for an amphibian metamorphosis assay, which would be practicable to carry out by only one or two individual researchers. The subject of this study is therefore how to reduce practical burdens on testers. We addressed this issue by employing an individual-separated exposure system that would make tadpoles develop as uniformly as possible by excluding interference during feeding among the tadpoles. In other words, we expected that this exposure system would reduce within-group variability in tadpole growth and development and thereby allow group size, i.e. the number of test tadpoles, to be reduced. Our expectation was supported by the results of experiment 1. Apparent differences in the daily total amount of food consumption were not observed between the individual-separated and individual-grouped systems. However, the interindividual variability in the three morphological endpoints was significantly smaller under the former system than under the latter: the maximum and minimum values of developmental stage differed by only two stages under the former whereas by ten stages under the latter. In addition, the SD values in hind limb length and total body length under the former were less than half of those under the latter (Fig. 1). These facts suggest that the former system reduced the variability by equalizing the daily amount of food consumption among the tadpoles. We initially suspected that the former system, which used small-sized test vessels, might also induce developmental acceleration because gradually reducing swimming space can be a trigger to the release of thyrotropin in certain species of tadpoles (Denver, 1998; Denver et al., 1998). However, contrary to our suspicion, no significant differences in the median or mean values of the data observed for the three endpoints were found between the two exposure systems. As suggested by Rose (2005), tadpoles of fully aquatic anuran species, such as X. laevis and S. tropicalis, are not as sensitive to volumetric changes in water levels as other anuran species that are aquatic during larval stages but substantially terrestrial after metamorphosis. It is therefore concluded that the individualseparated exposure system has an experimental advantage of variability-reducing effects without developmental acceleration. In experiment 2, we tested T4 (1 mg/L) as a thyroid system agonist and PTU (75 mg/L) as an antithyroid substance using the metamorphosis assay under the individual-separated exposure system. In general, when conducting static and semistatic tests, attention should be given to the quality degradation of test solutions during the tests. Since experiment 2 was conducted
Fig. 5. Comparative thyroid gland histology of the S. tropicalis tadpoles in the control (left), T4-treatment (1 mg/L, center), and PTU-treatment (75 mg/L, right) groups. The abbreviations T, F, and Fc indicate thyroid glands, follicles, and follicular cells, respectively. Each scale bar corresponds to 0.1 mm.
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under a semistatic regime with small-sized test vessels, we examined pH, DO, and concentrations of test chemicals of the used test solutions that were allowed to be static for 3 days. The deviations of pH (6.9–7.5) and DO (56.3%–98.8% saturation at 25 1C) fulfilled the criteria determined by OECD (2009) in which the pH and DO should be maintained at 6.5 to 8.5 and 440% air saturation, respectively. As shown in Fig. 2, the T4 concentration clearly decreased as the tadpole development proceeded, and it dropped to nearly 30% of the nominal concentration (1 mg/L) at the test termination. This step-by-step decrease in the T4 concentration may be due to uptake of T4 by the tadpoles. It is likely that the amount of T4 absorbed through the surface of the tadpole body increased as they developed before reaching metamorphic climax stages. Thereafter, alterations in skin permeability occurring with metamorphosis (Nieuwkoop and Faber, 1994) may have relevance to the decrease of T4 in the test solutions. Alternatively, T4 in the test solutions might be absorbed into tadpole feces, because the amount of the tadpole feces seemed to increase since day 7 when the amount of daily food was doubled. On the other hand, the PTU concentration was maintained near the nominal concentration (75 mg/L, exceedingly high compared the nominal concentration of T4) throughout the test. Similar trends have also been reported in the previous studies that tested T4 (1 mg/L) and PTU (75 mg/L) under a semistatic regime with the same renewal interval as in our protocol, although these studies employed different anuran species: X. laevis (Kloas et al., 2003) and R. rugosa (Oka et al., 2009). These facts, coupled with our results, suggest limitations of semistatic tests in general, and therefore, the decrease in the T4 concentration that occurred during experiment 2 does not necessarily invalidate our testing protocol. During experiment 2, T4 was nontoxic to the S. tropicalis tadpoles, however, it consistently induced significant acceleration to metamorphosis, as indicated by significantly further advanced developmental stages, longer hind limb lengths, shorter total body lengths, and smaller wet body mass of the tadpoles exposed to T4 compared with those observed in the control group (Fig. 3). These effects promoting morphological alterations toward metamorphosis rather than tadpole growth (elongation of total body length) reflect the actions of T4. In contrast, PTU, which was also nontoxic to the S. tropicalis tadpoles, caused pronounced inhibitory effects on metamorphosis, as illustrated by the tadpoles showing extremely slow progress in developmental stage and poorly developed hind limbs without fully differentiated digits even on day 28 (Fig. 3). The mean total body length in the PTU group was significantly shorter than that in the control group on days 7–21 (Fig. 3). However, at the test termination, no significant difference was found between the two groups not only in the mean total body length but also in the mean wet body mass. Similar retardation of tadpole growth was also induced by PTU (75 mg/L) in the previous studies employing X. laevis (Kloas et al., 2003) and S. tropicalis (Mitsui et al., 2006). As a potent inhibitor of thyroid peroxidase, PTU blocks T4 synthesis in the thyroid gland in mammals (Cooper et al., 1983; Engler et al., 1982) and anurans (Robertson and Kelley, 1996). Apart from this action, PTU also inhibits extrathyroidal conversion of T4 to triiodothyronine (T3) and augments thyrotropin release in mammals, which is independent of the first effect (Geffner et al., 1975). However, such antithyroidal actions of PTU cannot account for the growth retardation observed in our study because the inhibition of metamorphosis arising from the blockade of T4 synthesis (and T4-to-T3 conversion) does not necessarily lead to tadpole growth inhibition (Opitz et al., 2005). For example, amitrole and ethylenethiourea, both of which act as an antithyroid chemical in X. laevis, do not affect tadpole growth at the concentrations that induce complete blockage of metamorphosis arising from T4 synthesis inhibition (Kloas et al., 2003). The growth retardation
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induced by PTU may therefore suggest that PTU has extrathyroidal and toxic side effects that have not yet been mechanistically ascertained (Kloas et al., 2003; Opitz et al., 2005). Neither total body length data nor snout-vent length data should be solely used to determine thyroid toxicity of test chemicals (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006). Rather, such length data (and also body mass data) should be used in conjunction with developmental stage and histopathology data in order to determine thyroid-disrupting activities (OECD, 2009). In amphibian metamorphosis assays, evaluation of non-thyroidal effects such as growth inhibition is also required to avoid false outcomes because such effects can also induce mild developmental retardation (OECD, 2009). The data of tadpole length and weight can provide useful information to assess possible effects of test chemicals on tadpole growth. Here, we must pay attention to the utility of total body length because tadpoles at stage 60 or further show signs of tail resorption and reshaping of the head, reducing tail length and snout–vent length and consequently reducing total body length (Nieuwkoop and Faber, 1994; Kloas et al., 2003). Under the traditional individualgrouped exposure system, many tadpoles, even in the control, showed development beyond stage 59 at the test termination (Kloas et al., 2003; Opitz et al., 2005; Mitsui et al., 2006). It has therefore been proposed that the use of total body length should be limited to the initial 14 days (Opitz et al., 2005) or 21 days (Kloas et al., 2003; Mitsui et al., 2006) of exposure. Meanwhile, under the individual-separated exposure system, all tadpoles in the control solution did not develop beyond stage 59 both in experiments 1 and 2 due to small interindividual variability in developmental stage (Figs. 1 and 3). We substituted total body length for snout–vent length in order to compare our results with the historical data, and the above-described result suggests the utility of total body length until day 28 under the individualseparated exposure system. In addition, the prolonged metamorphosis assay with the exposure duration of 28 days may find other non-thyroidal effects of chemicals that cannot be detected within 21 days of exposure. In any of the three groups (the control, T4-, and PTU-treatment groups), the median or mean values of the morphological data obtained at 7-day intervals (Fig. 3) were quite similar to those reported by Mitsui et al. (2006), who developed an amphibian metamorphosis assay using S. tropicalis tadpoles under the traditional individual-grouped exposure system. In addition, the variability of the morphological data concerning the three endpoints was greatly reduced compared with that presented by Mitsui et al. (2006). For example, at the test termination (day 28), developmental stage of the tadpoles in the control ranged from stage 51 to stage 64 in the report by Mitsui et al. (2006), whereas from stage 56 to stage 59 in the current work (experiment 2). These comparative evaluations support the validity and superiority of the alternative testing protocol using the individualseparated exposure system. Although Mitsui et al. (2006) did not present any histological observations of thyroid glands, the thyroid gland atrophy observed in the tadpoles exposed to T4 (Fig. 5) is a typical symptom caused by exogenous T4 in X. laevis (OECD, 2007) and R. rugosa (Oka et al., 2009). According to these two previous studies, PTU causes thyroid gland hypertrophy and follicular cell hyperplasia in the above two anuran species. In our study, similar histological lesions were observed in the tadpoles exposed to PTU (Fig. 5). Therefore, our histological observations of the thyroid glands pathologically supported the metamorphosis-promoting and metamorphosis-inhibiting effects of T4 and PTU, respectively. Overall, the amphibian metamorphosis assay using S. tropicalis tadpoles was successfully validated even under the individualseparated exposure system, by testing T4 and PTU. The superiority
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of this alternative testing protocol is mainly due to the fact that it can be successfully performed by only one or two individual researchers. This testing protocol may therefore have the potential to positively contribute to the progress of ecotoxicological studies concerning the detection of chemicals with agonistic and antagonistic activities on the thyroid hormone axis. However, it should be noted that the current study tested only two chemicals that have strong effects on the thyroid system: T4 is a thyroid hormone itself and PTU is a well-known antithyroid substance. In general, effects of thyroid hormone-disrupting chemicals in the environment would not be as strong as those of the two chemicals tested. Further studies using other chemicals will be required to confirm whether our assay can detect slight but significant effects on the thyroid system.
5. Conclusions The current work examined an alternative testing protocol for amphibian (S. tropicalis) metamorphosis assays: an individualseparated exposure system that was expected to reduce interindividual variability in tadpole growth and development and thereby allow the number of test tadpoles to be reduced (n¼30 for each group). Our expectation was verified by the comparative rearing experiment between the individual-separated exposure system and the traditional individual-grouped exposure system. Using the new testing protocol, we tested the well-known thyroid system agonist T4 and antithyroid substance PTU for 28 days of exposure. Morphological data of the tadpoles exposed to T4 and PTU consistently indicated significant metamorphic acceleration and retardation, respectively. The thyroid glands of the tadpoles clearly showed histological alterations caused by T4 and PTU. Our results agree with the historical data generated from previous studies employing the traditional exposure system, thereby indicating the validity of the amphibian metamorphosis assay employing the individual-separated exposure system.
Acknowledgments The frogs S. tropicalis were supplied from the Institute of Amphibian Biology of Hiroshima University as a part of the National BioResource Project by the Japan Ministry of Education, Culture, Sports, Science, and Technology. We thank A. Kashiwagi, M. Takase, and Y. Yaoita for technical advice on the keeping and breeding of S. tropicalis. References ASIH, 2004. Guidelines for Use of Live Amphibians and Reptiles in Field and Laboratory Research. ASIH, Lawrence.
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