LETTER TO THE EDITOR Triclosan Does Not Affect Thyroid-Mediated Metamorphosis in Xenopus laevis—Additional Data

In response to the letter from Helbing et al. (forthcoming) stating that data from our recent paper (Fort et al., 2010) suggested that ‘‘triclosan had an effect on post-embryonic development of Xenopus tadpoles,’’ which was consistent with their observations with the bullfrog (Veldhoen et al., 2006), we offer the following response. In consideration of our data presented in Fort et al. (2010), reevaluation of these data by Helbing et al. (forthcoming), and additional data provided for discussion in this letter, we maintain that there is not sufficient evidence that triclosan accelerates metamorphosis in Xenopus laevis. We agree that developmental stage is an important endpoint in evaluating potential effects on metamorphosis. We also agree that these data are categorical based on specific morphology even though they appear as a numerical data point. Helbing et al. (forthcoming), in their reevaluation of data from Table 3 of Fort et al. (2010), used specific developmental stage sets to establish a contingency table for chi-square testing. Based on their evaluation, statistical significance was found in the lower three test concentrations but not the highest test concentration. However, the stage data sets from the study of Fort et al. (2010) lacked monotonicity, were not normally distributed, possessed missing data where sporadic mortality occurred during the study, and possessed various stages with frequencies < 5 decreasing the practicability of the chi-square test. In order for contingency tables to be established, only stage sets that meet the abundance criteria should be used, which bias the data set. Therefore, we applied the chi-square test over the entire developmental stage data set, which produced the p value cited in Fort et al. (2010). Statistical review of the reevaluated data provided by Helbing et al. (forthcoming) further suggested that the chi-square test was not appropriately applied to the Fort et al. (2010) data set. Chisquare analysis only determines if the distribution of two treatments is different but not whether the distribution is shifted in a given direction. This explains why their reevaluation of this data set produced low p values for the test concentrations. An alternative, more appropriate, approach in reevaluating the data set considering the lack of monotonicity and nonnormality is the Mann-Whitney U-test, which is consistent with the approach specified in EPA 740-C-09-002 (United States Environmental Protection Agency [USEPA], 2009). Results are provided in Table 1. A summary of the endpoint data from Fort et al. (2010) and a second data set from tadpoles exposed to triclosan for 32 days (initiated at Nieuwkoop and Faber NF; Nieuwkoop and Faber, 1994; stage 47) is provided in Table 2. The intent of the second study was to evaluate potential effects of triclosan on metamorphosis with earlier exposure during premetamorphosis and separate effects on growth from metamorphosis. Results from analysis indicated that in the data set of Fort et al. (2010), the median stage obtained at the conclusion of the study in the 0.6 and 7.2 lg triclosan (TCS)/l, but not the 1.5 and 32.3 lg TCS/l treatments, was greater than the control. Because an intermediate concentration (1.5 lg triclosan (TCS/l) and the highest concentration (32.3 lg TCS/l) did not statistically alter developmental stage distribution relative to the control (MannWhitney U-test, see Table 1), biological relevance cannot be inferred from the statistical significance detected in the other two intermediate test concentrations. These findings are supported by results obtained from a recently completed second study indicating that the median stage sets obtained at the conclusion of the supplemental 32-day exposure study were not significantly different from the control (Mann-Whitney U-test, p 1⁄4 0.05). In fact, the median stages in the triclosan treatments were reversed from the study of Fort et al. (2010) with a control median stage of NF 60 and treatment median stage of NF 59. Overall, stage data from neither study indicate that triclosan is accelerating development. In terms of the effects of triclosan on the induction of thyroid hormone receptor beta (TRb) described in Fort et al. (2010), we appreciate the interpretation of relative expression differences