Article pubs.acs.org/est
Early Life Stage Exposure to BDE-47 Causes Adverse Effects on Reproductive Success and Sexual Differentiation in Fathead Minnows (Pimephales promelas) Leah M. Thornton,† Elise M. Path,† Gunnar S. Nystrom,† Barney J. Venables,‡ and Marlo K. Sellin Jeffries*,† †
Department of Biology, Texas Christian University, Fort Worth, Texas 76129, United States Department of Biological Sciences, University of North Texas, Denton, Texas 76203, United States
‡
S Supporting Information *
ABSTRACT: 2,2′,4,4′-Tetrabromodiphenyl ether (BDE-47), a compound manufactured for use as a flame retardant, is a ubiquitous environmental contaminant and suspected endocrine disruptor. Though several studies have explored the reproductive effects of BDE-47 in adult fish, there is a paucity of data regarding the reproductive effects of early life stage exposure. The goal of this study was to assess the reproductive effects of early life stage BDE-47 exposure in fathead minnows (Pimephales promelas). To achieve this, minnows were exposed to either a low (57.68 μg BDE-47/g Artemia) or high (392.59 μg BDE-47/g Artemia) dose of BDE-47 from fertilization to 34 days postfertilization (dpf) via a combination of maternal transfer and dietary exposure. Larvae were then raised on a clean diet until sexual maturity (∼184 dpf) when reproductive function was evaluated using a 21 day breeding study. Fish exposed to BDE-47 had significantly reduced clutch size and fecundity relative to controls. BDE-47 exposed groups also had female-biased sex ratios and exposed males had fewer tubercles. Overall, this study demonstrates that exposure to BDE-47 during early life stages can alter both sexual differentiation and reproductive function.
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INTRODUCTION
freshwater sites in Virginia, US contained BDE-47 concentrations >1 μg/g lipid weight.3 The presence of BDE-47 in aquatic organisms is of interest not only due to the fact that BDE-47 exhibits thyroid disrupting activity6 but also because it is both weakly estrogenic and antiandrogenic.7−10 Exposure to BDE-47 has been shown to induce adverse reproductive effects in adult fish.6,11,12 For example, fathead minnows (Pimephales promelas) exposed to BDE-47 have been shown to experience alterations in sex steroid-related transcript expression,12 gametogenesis6,11 as well as reproductive output.11 While studying the reproductive effects of BDE-47 in adult organisms is of value, it is also important to consider early life stage organisms for several reasons. First, developing organisms are often more sensitive to contaminants than their adult counterparts.13,14 Second, early life stage exposure to BDE-47 is likely given the ubiquity of PBDEs1 as well as their ability to be maternally transferred.15−17
Polybrominated diphenyl ethers (PBDEs) are a class of compounds applied to a variety of materials (e.g., electronics, textiles, plastics) as flame-retardants.1 The production and use of PBDEs has recently been reduced due to voluntary phaseouts and changes in legislation in response to increased evidence that these compounds may be harmful to various organisms.1 Although these changes will reduce the likelihood of exposures in the future, PBDEs will likely remain problematic given that the products to which they were applied are still widely used and may therefore continue to leach these compounds into the environment.1 Of the 209 possible congeners, 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) is one of the most commonly detected in the environment.1 Like the majority of PBDEs, BDE-47 is both highly stable and lipophilic, allowing it to be both ubiquitous and persistent in the environment.1,2 These characteristics allow for the accumulation of PBDEs by aquatic biota as demonstrated by the results of numerous field studies showing detectable levels of BDE-47 in the tissues of fish collected from waterways throughout the world.1,3−5 In general, total PBDE concentrations in fish are in the ng/g range; however, Hale et al. (2001) found that more than half of the fish analyzed from © XXXX American Chemical Society
Received: April 29, 2016 Revised: June 17, 2016 Accepted: June 21, 2016
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DOI: 10.1021/acs.est.6b02147 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Figure 1. Experimental design for early life stage fathead minnow BDE-47 exposure. Fathead minnows were exposed via a combination of maternal transfer and diet until 34 days postfertilization (dpf). Adult fathead minnow breeding pairs were fed clean, low BDE-47 dose, or high BDE-47 dose bioencapsulated Artemia. At the predicted peak of maternal transfer, embryos were collected from each of the groups and maintained until 6 dpf. From 6 to 34 dpf, larvae were fed Artemia at the same respective doses as the parental generation. At 34 dpf, a subset of larvae was sacrificed for gene expression and PBDE analysis. Remaining minnows were raised until 184 dpf, at which point reproductive success was assessed using a 21 day breeding study. Immediately prior to the breeding study, morphometric endpoints (i.e., body mass (wet weight), total length, and gonadosomatic index) were measured. Secondary sex characteristics (SSCs) were assessed in males included in the breeding study, and sex ratio in each group was assessed in remaining fish following the breeding study. Each group began with 495 embryos collected following maternal transfer of BDE-47. Minnows not used in this study were sampled for experiments not reported here (∼130).
minnows (∼5 mo old) were randomly sorted into 3 groups of 20 breeding pairs: control, low BDE-47 dose, and high BDE-47 dose. During the exposure period, control pairs received 105 mg of clean Artemia. Low dose breeding pairs received 105 mg of bioencapsulated Artemia daily at a measured concentration of 57.68 μg BDE-47/g Artemia (6.06 μg BDE-47/pair/day). High dose breeding pairs received 105 mg of bioencapsulated Artemia daily at a measured concentration of 392.59 μg BDE47/g Artemia (41.22 μg BDE-47/pair/day). Methods for the bioencapsulation of Artemia with BDE-47 and adult fathead minnow husbandry are presented in the Supporting Information. From days 8−12 of the exposure period, 495 embryos were collected from each group. This time period was selected based on the predicted peak of maternal transfer of BDE-47 determined by van de Merwe et al. (2011).17 Upon collection, spawning substrates (PVC pipe halves) were placed in 1 L beakers and incubated in a 26 °C water bath. Ten embryos were sampled from each clutch at approximately 24 h postfertilization (hpf) for BDE-47 analysis. At 6 dpf, 495 larvae were selected from each group for dietary BDE-47 exposure. These embryos were selected from clutches produced by at least five different breeding pairs meeting the following criteria: (1) laid within the selected time period of 8−12 days, (2) more than 50 embryos fertilized, and (3) at least the second clutch for given breeding pair. Additional methods for embryo/larval fathead minnow husbandry are presented in the Supporting Information. Dietary Exposure to BDE-47. From 6 to 34 dpf, larvae were fed live Artemia twice daily based on age. Methods for larval husbandry and feeding regime (Table S1) are found in the Supporting Information. The amount of food per larva was a controlled amount, but in excess of what could be consumed
Exposure to BDE-47 during developmental stages has been shown to negatively alter neural development, cardiac function and gene expression, demonstrating that early life stage fish are sensitive to BDE-47 exposure.18−21 However, to our knowledge, there are no studies that have sought to determine if BDE-47 induces adverse reproductive effects similar to those observed in adults. As such, the goal of this study was to assess the reproductive effects of early life stage BDE-47 exposure in fathead minnows. To achieve this goal, fathead minnows were exposed to BDE-47 via a combination of maternal transfer and diet until 34 days postfertilization (dpf). This exposure period coincides with gonadal differentiation, which occurs by 29 dpf.22 Sex steroid-related gene expression was assessed immediately following BDE-47 exposure, while morphometric measurements (i.e., body mass (wet weight, ww), total length and gonadosomatic index (GSI)), reproductive success (e.g., fecundity, clutch size, etc.), male secondary sex characteristics (SSCs) (i.e., number of nuptial tubercles and fatpad score) and sex ratio were assessed following a depuration period of ∼150 days.
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MATERIALS AND METHODS General Experimental Design. All experimental procedures involving fathead minnows were approved by the Texas Christian University Institutional Animal Care and Use Committee (protocol # 14/06). Figure 1 shows the general experimental design for early life stage BDE-47 exposure. Maternal Transfer of BDE-47. The adult breeding minnows utilized to produce larvae in this study were part of a larger study aimed at investigating the impacts of BDE-47 on adult reproductive endocrinology and reproductive success. Methods related to their care and BDE-47 exposure are found in detail in Thornton et al. (2016).12 Briefly, 120 adult fathead B
DOI: 10.1021/acs.est.6b02147 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
Spawning substrates (PVC pipe halves) were checked for eggs at least twice daily. The total number of eggs was counted and recorded to determine clutch size and fecundity (eggs/ female/day). Fertilization success was determined upon collection by visual inspection. Unfertilized eggs are opaque or clear with a spot where the yolk has precipitated (Ankley et al. 2001).27 If detected, unfertilized eggs were removed from the structure. The number of hatched larvae was recorded for the determination of hatching success, and the mortality of hatched larvae was monitored until 2 dph for the determination of larval survival. Fertilization success, hatching success and larval survival (to 2 dph) were determined for clutches ≥50 eggs. Methods for embryo and larval care are described in the Supporting Information. Sex Ratio. At approximately 9 months old, the sex ratio of the remaining populations of fish not used in the 21 day breeding study was determined. Sex was determined by presence of sex specific phenotypes. When sex could not be determined by phenotype, fish were euthanized and gonads were directly examined for the presence of oocytes using a light microscope. Statistical Analysis. Significant differences between groups for all other endpoints measured were determined using a oneway analysis of variance (ANOVA) followed by a Dunnett’s posthoc multiple comparison test. If variance was unequal between groups, significant differences were determined using a Wilcoxon test. Significant differences in sex ratio between groups were determined using two separate chi square tests comparing the respective treatment group to the control. Statistical significance was set at α < 0.05 for all tests. All analyses were conducted using the statistical software package JMP 11.0.
to ensure adequate rations. Larvae in the BDE-47 groups received BDE-laden Artemia in the morning, but clean Artemia in the evenings, while control larvae were given clean Artemia at both times. From 34 dpf to 184 dpf, larvae were raised on a clean diet. BDE-47 Analysis. Chemical analysis was performed using methods modified from Liu et al. (2009) and previously described by Thornton et al. (2016).12,23 The following sample types were analyzed for BDE-47 using gas chromatography mass spectroscopy (GC/MS): Artemia (n = 3), embryos (n = 5), 34 dpf larval carcass (n = 5), and ∼184 dpf adult carcass (n = 5, immediately prior to breeding study). Details regarding the analytical methods used are presented in the Supporting Information. Gene Expression Analysis. At 34 dpf, whole larvae were sacrificed using a lethal dose (0.3 g/L) of buffered tricaine mesylate (MS-222) and flash frozen in liquid nitrogen for gene expression analysis. Viscera were removed from larvae via dissection in a solution containing RNase inhibitors to prevent RNA degradation. Tissues were homogenized using a QSonica tissue sonicator (QSonica, Farmingdale, NY), and total RNA was extracted from each sample using the Maxwell 16 LEV simplyRNA Purification Kit (Promega, Madison, WI) per manufacturer protocol as outlined in Sellin Jeffries et al. (2014).24 Total RNA was quantified and checked for purity using the NanoDrop 1000 (ThermoScientific, Wilmington, DE). All samples had 260/280 nm ratios ≥2.06. First-strand cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, Hercules, CA) per manufacturer protocol. Target genes analyzed included vitellogenin 1 (vtg), estrogen receptor α (erα), estrogen receptor β1 (erβ), androgen receptor (ar), aromatase (arom), and anti-Mullerian hormone (amh). Target genes were selected due to their utility as biomarkers for exposures to estrogenic compounds and/or their role in sexual differentiation.25,26 Primer sequences for all genes measured are listed in Table S2. The expression of each gene was quantified via the standard curve method using serial diluted cDNA samples, and the expression of each target gene was normalized using ribosomal protein l8 as a reference gene. Statistical analysis revealed that there were no differences between treatment groups for l8 (ANOVA, all p values > 0.05). A list of genes analyzed and their general functions are listed in Table S3. Additional details of gene expression analysis methods are found in the Supporting Information. Morphometric Assessments. Upon sexual maturity (∼184 dpf), fish in each group were identified as male, female, or undifferentiated based on phenotype (e.g., the presence of nuptial tubercles in males or an ovipositor in females). Immediately prior to reproductive assessment, 15 males and 15 females from each group were sacrificed using a lethal dose (0.3 g/L) of buffered MS-222 for the determination of body mass (ww), total length (i.e., snout to the end of the tail), and GSI. GSI was calculated by dividing the mass of gonad tissue by the total body mass and multiplying by 100. In addition, secondary sexual characteristics were assessed in males used in the breeding study by scoring the fatpad according to Ankley et al. (2001) and counting the total number of nuptial tubercles.27 Reproductive Assessment. Of those fish for which sex was identified, 20 males and 20 females were randomly selected from each group to be subjected to a 21 day breeding study modified from Ankley et al. (2001).27 Breeding pairs were fed a clean diet during the 21 day breeding study. General husbandry for breeding pairs is described in the Supporting Information.
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RESULTS BDE-47 Analysis of Artemia, Embryo, and Carcass Tissue. Chemical analysis revealed that adult breeding pairs in the low and high dose treatment groups received 6.06 ± 3.8 and 41.22 ± 22.6 μg BDE-47 daily (mean ± standard deviation, n = 3), respectively. Concentrations of BDE-47 received by larvae during the dietary exposure are presented in Table S1. Concentrations of BDE-47 detected in embryos, 34 dpf larval and 184 dpf carcass tissue are presented in Table 1. In general, Table 1. BDE-47 Concentrations (Mean ± Standard Deviation) Detected in Embryos and 34 dpf (Days Postfertilization) Larval and 184 dpf Carcass Tissue (n = 5)a group
embryo (ng/embryo)
34 dpf larvae (μg/g)
184 dpf female; male (μg/g)
control low high
