Effects of the anti-microbial contaminant triclocarban on the reproductive function and ovarian transcriptome of the fathead minnow (Pimephales promelas)

Triclocarban (TCC) is a widely used antimicrobial agent that is routinely detected in surface waters. The present study was designed to examine TCC’s efficacy and mode of action as a reproductive toxicant in fish. Reproductively mature Pimephales promelas were continuously exposed to either 1 or 5 μg TCC/L, 0.5 μg 17β-trenbolone (TRB)/L or a mixture (MIX) of 5 μg TCC and 0.5 μg TRB/L for 22 d and a variety of reproductive and endocrine-related endpoints were examined. The data were evaluated to answer several key questions: first, whether exposure to TCC could be linked with an ecologically-relevant adverse outcome, i.e., impaired reproduction; second, whether the present study provided additional support for augmentation of androgen action as an endocrine-disrupting mode of action for TCC; and third, whether there were any novel and/or plausible linkages between changes in the ovarian transcriptome and the apical responses observed in females that could support adverse outcome pathway development and/or further hypothesis-driven testing. Fathead minnows were randomly paired (one male, one female) and placed in tanks at a density of two pairs per tank with six replicate tanks per treatment. Pairs were separated by a water permeable mesh divider and each pair had its own breeding substrate. The fish were held in the test system, receiving UV-treated, filtered Lake Superior (control) water only, for a 21 d acclimation period during which the fecundity and fertility of each pair were assessed daily. After 21 d, exposures were initiated with pairs that had spawned successfully during acclimation. Fish were exposed continuously to target test concentrations of 0 (Lake Superior control), TCC (1 and 5 µg/L), TRB (0.5 μg/L), or a mixture of the two chemicals (5 μg TCC and 0.5 μg TRB/L) for 22 d. Water concentrations of TCC and TRB were determined in stock solutions and all exposure tanks every two to three days. General water quality characteristics (mean ± SD) measured regularly over the course of the Experiment were: temperature, 24.8 ± 0.7 oC; pH, 7.87 ± 0.04; dissolved oxygen, 6.8 ± 0.35 mg/L. Ovary RNA samples from twenty four fish (n=5 for all treatments except TRB alone; n=4) were selected for microarray analysis.

三氯卡班（Triclocarban, TCC）是一种广泛应用的抗菌剂，常被检出于地表水环境中。本研究旨在探究TCC作为鱼类生殖毒物的作用功效与作用模式。
将性成熟的黑头呆鱼（Pimephales promelas）持续暴露于1 μg/L或5 μg/L的TCC、0.5 μg/L的17β-群勃龙（17β-trenbolone, TRB），或5 μg/L TCC与0.5 μg/L TRB的混合液（MIX），时长为22天，并对多项生殖与内分泌相关终点指标进行检测。
本研究通过数据分析旨在解答三个核心问题：其一，TCC暴露是否会引发生态相关的不良结局，即生殖功能受损；其二，本研究是否能为“TCC通过增强雄激素作用发挥内分泌干扰效应”这一作用模式提供额外佐证；其三，雌性个体卵巢转录组的变化与观测到的顶端响应之间是否存在新颖且合理的关联，以支持不良结局途径（adverse outcome pathway, AOP）的构建及后续假说驱动的实验验证。
将黑头呆鱼随机配对（1雄1雌），以每缸2对的密度放置于养殖缸中，每个处理组设置6个重复缸。配对个体间以透水隔板分隔，每对个体配备专属产卵基质。实验前，所有鱼仅使用经紫外线处理、过滤后的苏必利尔湖湖水（对照组）进行21天的驯化，期间每日评估每对的产卵量与受精率。驯化21天后，选取驯化期间成功产卵的配对个体开始暴露处理。
将鱼持续暴露于目标浓度的受试液中22天，目标浓度分别为：0（苏必利尔湖湖水对照组）、1 μg/L和5 μg/L的TCC、0.5 μg/L的TRB，以及5 μg/L TCC与0.5 μg/L TRB的混合液。
每2至3天对储备液及所有暴露缸中的TCC和TRB浓度进行测定。实验期间定期监测的常规水质指标（平均值±标准差）如下：水温24.8±0.7 ℃，pH值7.87±0.04，溶解氧6.8±0.35 mg/L。选取24尾鱼的卵巢RNA样本进行微阵列（microarray）分析，其中除单独TRB处理组样本量为n=4外，其余各组样本量均为n=5。