Mar 31, 2015 - University of Chinese Academy of Sciences, Beijing 100049, China. § .... phosphate (d15-TMP, 99.9%), CDN Isotopes (Pointe-Claire,. Quebec ...
Article pubs.acs.org/est
Bioconcentration and Transfer of the Organophorous Flame Retardant 1,3-Dichloro-2-propyl Phosphate Causes Thyroid Endocrine Disruption and Developmental Neurotoxicity in Zebrafish Larvae Qiangwei Wang,#,†,‡ Nelson Lok-Shun Lai,#,§,∥,⊥ Xianfeng Wang,†,‡ Yongyong Guo,† Paul Kwan-Sing Lam,§,∥,⊥ James Chung-Wah Lam,*,∥ and Bingsheng Zhou*,† †
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China § State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, China ∥ Research Centre for the Oceans and Human Health, Shenzhen Key Laboratory for Sustainable Use of Marine Biodiversity, City University of Hong Kong Shenzhen Research Institute Building, Shenzhen, Guangdong 518057, China ⊥ Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: Organophosphate flame retardants are emerging environmental contaminants, although knowledge of their health risks is limited. Here, thyroid hormone homeostasis and neuronal development was studied in the progeny of adult zebrafish exposed to tris(1,3-dichloro-2-propyl) phosphate (TDCPP). Adult zebrafish were exposed to TDCPP (0, 4, 20, and 100 μg/L) for 3 months. Increased generation of reactive oxygen species and reduced survival rates was observed in exposed F1 larvae. We also observed a significant decrease in plasma thyroxine and 3,5,3′-triiodothyronine levels in F0 females and F1 eggs/larvae. The mRNA and protein expression of factors associated with neuronal development (e.g., α1tubulin, myelin basic protein, and synapsin IIa) were significantly downregulated in exposed F1 larvae, as was the level of the neurotransmitters dopamine, serotonin, gamma amino butyric acid, and histamine. Larval locomotion was significantly decreased in exposed fish, but there was no effect on acetylcholinesterase activity. Bioconcentration of TDCPP was observed in F0 fish. TDCPP was also detected in F1 eggs following parental exposure, indicating maternal transfer of this compound. This study uniquely shows that TDCPP can be transferred to the offspring of exposed adults, causing thyroid endocrine disruption and developmental neurotoxicity.
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INTRODUCTION Bioaccumulation of environmental contaminants and subsequent maternal transfer to offspring has been recognized as a significant contributing factor in the development of human health and disease.1 The developing fetus (particularly the brain) is especially vulnerable to the negative health effects of toxicant exposure because of its immature organ systems, and because its detoxification enzymes have not fully developed.2Indeed, there is increasing epidemiological evidence showing an association between exposure to toxicants during the perinatal period and neurotoxicity later in life.3 For example, prenatal polybrominated diphenyl ethers (PBDEs) exposures are associated with deficits in intellectual abilities and increased behavior problems in children.4,5 Similarly, an increasing number of wild studies have reported that parental exposure to various organic toxicants, such as polychlorinated biphenyls © 2015 American Chemical Society
(PCBs), PBDEs, organochlorine pesticides (OPs), and Dechlorane Plus flame retardant, can cause toxicant transfer to offspring in fish6−8 and frogs.9 Likewise, the presence of toxicants in these offspring may cause developmental toxicity, particularly in the developing brain. Organophosphorus flame retardants (OPFRs) are mass produced chemicals used as additives in various household and industrial products.10 Brominated flame retardants have been associated with adverse health effects and have been banned in many industrial countries, leading to OPFRs being proposed as replacements.11 Consequently, OPFRs are now Received: Revised: Accepted: Published: 5123
January 30, 2015 March 28, 2015 March 31, 2015 March 31, 2015 DOI: 10.1021/acs.est.5b00558 Environ. Sci. Technol. 2015, 49, 5123−5132
Article
Environmental Science & Technology
Louis, MO USA). TDCPP stock solutions and serial dilutions were prepared in dimethyl sulfoxide (DMSO; purity >99.9%; Amresco, Solon, OH, USA). All other chemicals were of analytical grade. Experimental Design. All experiments were performed in adult zebrafish (4 month old) of the wild type AB strain. Each tank contained 10 fish of a single sex, and there were 3 replicate tanks per sex per concentration of TDCPP (0, 4, 20, and 100 μg/L) (n = 3 replicates/tank/concentration). The lowest exposure concentration (4 μg/L) was based on a previous study28 and is similar to that reported in sewage effluent (3 μg/ L).31 Throughout the experiment, the fish water was aerated, kept at 28 °C and a day-night cycle (14 h light, 10 h dark). The exposure media was prepared with dechlorinated carbon-filtered water, and 50% of the tank volume was replaced with freshly prepared media daily. Control and experimental treatment groups received 0.001% (v/v) DMSO. After 3 months of exposure, survival and growth (weight) were recorded, then the DMSO control fish and TDCPP exposed fish (10 males and 10 females) were paired in clean water (without TDCPP), and the eggs were collected. The embryos were transferred in glass beakers to freshwater to evaluate the parental transfer of TDCPP and to assess transgenerational toxicity. A subset of embryos was collected for TDCPP and thyroid hormone assays. The 5-day postfertilization (dpf) and 10-dpf F1 larvae were sampled at random and frozen immediately in liquid nitrogen, before being stored at −80 °C for subsequent assay of gene/protein expression, thyroid hormones, acetylcholinesterase (AChE) activity, and neurotransmitter levels. A subset of F1 larvae (5 and 10 dpf) was used for locomotor activity measurements. Hatching, malformation, survival, and growth rates were determined for the F1 generation. All studies were conducted in accordance with the guidelines for the care and use of laboratory animals of the National Institute for Food and Drug Control of China. Thyroid Hormone Assays. After 3 months of exposure to TDCPP, adult zebrafish were euthanized in 0.03% MS-222 by prolonged immersion until cessation of opercular movement and blood was collected from the caudal vein (samples from three fish of the same sex were pooled as one sample [approximately 30 μL, n = 3 replicates]), and plasma was stored at −80 °C until further analysis. The method for measuring whole body thyroid hormone contents in eggs (200 eggs; n = 3 replicates) and larvae (200 larvae; n = 3 replicates) were from a previous method29 (Text S1, Supporting Information). Total thyroid hormone levels were measured using a commercial ELISA kit according to the manufacturer’s instructions (EIAab Science, Wuhan, China). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay. Larval samples (5 and 10 dpf; 30 larvae; n = 3 replicates) were collected and preserved with RNAiso Plus according to the manufacturer’s instructions (Takara, Dalian, China). RNA extraction, purification and quantification, and first-strand cDNA synthesis were performed following the protocols described by Chen et al.30 (see Text S2 in the Supporting Information). Oligonucleotide primers specific to each of the selected genes were identified using the online Primer 3 program (http://frodo.wi.mit.edu/; see Supporting Information Table S1). The rpl8 gene was selected as the internal standard. Protein Extraction and Western Blot Analysis. Myelin basic protein (Mbp), α1-tubulin, and synapsin IIa (Syn2a) were selected for analysis of their protein expression level. A Western
ubiquitous contaminants present in many river waters at ng−μg/L levels12−14 (e.g, 294 ng/L-26.4 μg/L in UK river).12 Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is a typical OPFR used as an additive in upholstery foam.15 After the phasing-out of PBDEs in 2005, the use of TDCPP has increased,11,13 and it is now detected across the globe in various environments and biota. In surface water, the concentration of TDCPP is typically at ng/L levels12−14 (e.g, below limit of detection to 200 ng/L in Spain river),12 but TDCPP prone to bioaccumulate in aquatic organisms and up to 251 μg/kg lipid weight has been measured in muscle sample of freshwater fish.16 TDCPP has been detected in human seminal plasma and breast milk,17,18 indicating a potential risk of transmission to infants and leading to concerns about potential toxicity and health effects on progeny. Moreover, the increased presence of TDCPP in house dust has been correlated with decreased levels of circulating thyroid hormone in humans.19 There are numerous in vitro and in vivo studies highlighting the toxic effect of TDCPP. For example, it has been shown to have neurotoxic and cytotoxic effects in cultured neuroendocrine PC12 cells.20,21 It has also been shown to increase the occurrence of developmental abnormalities during zebrafish embryogenesis22,23 and affect swimming activity.24 Exposure to TDCPP also causes thyroid endocrine-disrupting activity and decreased levels of circulating thyroxine (T4) in zebrafish larvae25 and chicken embryos,26 as well as dysregulating thyroid hormone-responsive genes in cultured embryonic chicken hepatocytes.27 Long-term exposure to TDCPP has also been associated with its bioconcentration and neurotoxicity in zebrafish.28 Although much is known about the toxic effects of TDCPP exposure in certain contexts, the effect of long-term exposure to low concentrations of TDCPP in the progeny of exposed adults is unknown. It has been shown that PBDEs bioaccumulate in parental fish, which can be transferred to their offspring;29,30 therefore, we hypothesized that parental exposure to TDCPP (log Kow = 3.76)22 may result in transfer of the compound during the earliest stages of embryogenesis leading to neurotoxicity in the resultant offspring. In accordance with our hypothesis, the objectives of this study were to investigate 1) the chemical accumulation of TDCPP in parental (F0) zebrafish and to investigate its transfer to offspring; 2) the effects of TDCPP on the thyroid endocrine system in the F0 and F1 generation; 3) the effects of TDCPP on neurodevelopment in F1 larvae; and 4) the effects of TDCPP on reactive oxygen species generation in F1 larvae. Taken together, the results of our study further elucidate the environmental risks of OPFRs to aquatic ecosystems and potential health effects on fish.
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MATERIALS AND METHODS Reagents. Reagents were purchased from the following sources: TDCPP (CAS#13674-87-8; >95% purity), TCI Tokyo Chemical Industry Co. (Tokyo, Japan); bis(1,3-dichloro-2propyl) phosphate (BDCPP, >97% purity), Wellington Laboratories (Ontario, Canada); deuterated tri-n-propyl phosphate (d21-TPrP, 99.2%) and deuterated trimethyl phosphate (d15-TMP, 99.9%), CDN Isotopes (Pointe-Claire, Quebec, Canada); and 3,4-dihydroxybenzylamine (DHB, >98.0% purity), dopamine (>98.5% purity), histamine (>97.0% purity), gamma-aminobutyric acid (GABA) (>99.0% purity), and serotonin (>98.0% purity), Sigma−Aldrich (St. 5124
DOI: 10.1021/acs.est.5b00558 Environ. Sci. Technol. 2015, 49, 5123−5132
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Environmental Science & Technology
Table 1. Developmental Parameters of F1 Zebrafish Larvae Derived from Adult Zebrafish Exposed to TDCPP (0, 4, 20, 100 μg/ L) for 3 Monthsa TDCPP (μg/L)
larvae
hatching (%) malformation (%) survival (%)
3-dpf 5-dpf 5-dpf 10-dpf 5-dpf 10-dpf
weight (mg)
0 84.0 1.67 91.5 89.3 0.35 0.38
± ± ± ± ± ±
4 3.2 0.56 1.2 1.6 0.01 0.01
82.8 1.50 91.5 87.8 0.36 0.38
± ± ± ± ± ±
20 1.6 0.62 0.6 1.8 0.01 0.01
69.3 3.17 89.0 77.5 0.35 0.36
± ± ± ± ± ±
100
1.5** 0.95 2.0 2.5** 0.01 0.01
65.8 5.00 85.7 67.0 0.31 0.35
± ± ± ± ± ±
3.0*** 0.82* 1.1* 2.0*** 0.01** 0.02*
a Data are expressed as mean ± SEM of three replicate samples, and each replicate contained 50 embryos/larvae. *Designates significant differences between exposure groups and control group. *P < 0.05; **P < 0.01; *** P < 0.001 (ANOVA followed by Tukey’s test).
Table 2. Total T4 and T3 Levels in F0 Adult Zebrafish after Exposure to TDCPP for 3 Months and in the F1 Eggs/Larvae Derived from Parental Zebrafish Exposed to TDCPPa TDCPP (μg/L) zebrafish F0
TH levels
sex
T4
male female male female
T3 eggs F1 5-dpf F1 10-dpf
T4 T3 T4 T3 T4 T3
0 22.56 26.62 2.19 2.34 12.64 0.89 16.14 1.27 19.58 2.22
± ± ± ± ± ± ± ± ± ±
4 2.36 0.87 0.49 0.21 0.86 0.12 1.33 0.17 0.83 0.25
22.20 25.57 1.94 1.85 12.50 0.87 15.67 1.33 19.07 1.77
± ± ± ± ± ± ± ± ± ±
20 1.41 2.07 0.23 0.17 1.11 0.10 0.66 0.13 1.16 0.14
21.77 20.19 2.04 2.04 12.14 0.75 14.17 1.31 14.26 1.84
± ± ± ± ± ± ± ± ± ±
100 1.21 0.86* 0.46 0.20 1.02 0.15 1.01 0.09 1.20* 0.11
22.16 16.72 2.36 1.29 7.53 0.76 10.52 1.01 12.68 1.27
± ± ± ± ± ± ± ± ± ±
2.11 1.40** 0.53 0.13* 0.87* 0.16 1.47* 0.18 1.70* 0.19*
For the adult zebrafish, plasma samples from three individual fish were pooled and tested with three replicates. For the F1 eggs and larvae, 200 eggs or larvae were measured, also with three replicates. The thyroid hormone levels in the F0 zebrafish are expressed as ng/mL plasma, while levels in F1 eggs and larvae are expressed as ng/g wet weight. All data are expressed as means ± SEM.*P < 0.05 and **P < 0.01 indicate significant differences between exposure groups and the corresponding control group. a
larvae (n = 3) were washed two times with cold PBS (pH 7.4) and then homogenized in cold HEPES-buffered sucrose. The fluorescence intensity was measured using a microplate reader (Molecular Device, M2, Union City, CA) with excitation and emission at 485 and 530 nm, respectively. The ROS concentration was expressed in arbitrary units (DCF mg−1 protein). Results were expressed as the percentage (%) in relation to the value of the control. Measurement of Larval Locomotor Activity. Quantification of larval locomotor activity at 5 and 10 dpf was performed using the Video-Track system (ViewPoint Life Sciences, Montreal, Canada) following a previously described method.30 Larval swimming behavior was monitored under continuous light and in response to dark-to-light transitions. Data (frequency of movements, distance traveled, and total duration of movements) were collected from 30 larvae per treatment every 60 s and further analyzed using custom Open Office.Org 2.4 software. The detailed methodology for monitoring larval swimming is provided in the Supporting Information (Text S5). Quantification of TDCPP and BDCPP. TDCPP concentrations in the exposure solutions (n = 3 replicate tanks) were extracted and analyzed as described previously.28 TDCPP and BDCPP concentrations were determined in F0 adult fish and in F1 eggs (100 eggs, n = 3). The d21-TPrP and d15-TMP were used as internal standards. TDCPP and BDCPP identification and quantification were performed using a liquid chromatography-tandem mass spectrometer system (LC-MS/MS) consisting of an Agilent 1290 Infinity LC (Agilent) coupled to an AB SCIEX QTRAP 5500 LC-MS/MS system. Detailed
blot was performed according to previously described methods30 using approximately 150 larvae (50 μg protein/ sample) for each sample (n = 3 replicates). A detailed procedure is provided in the Supporting Information (Text S3). A quantitative measure of protein expression was obtained by densitometry, with the results normalized to β-actin expression. The rabbit α1-tubulin antibody (Abcam, Cambridge, UK), rabbit mbp antibody (AnaSpec, Fremont, CA), and rabbit syn2a (Synaptic Systems, Göttingen, Germany) antibodies have previously been verified to be reactive and suitable for zebrafish studies32 and previously applied for this purpose.30 Neurotransmitter Measurements. Monoamine extraction from F1 larvae samples (50 larvae, n = 3 replicates) was performed as previously described.33 Monoamine identification and quantification was carried out using an Agilent 1200 liquidchromatograph equipped with a triple quadrupole tandem mass spectrometer (Agilent, Palo Alto, CA, USA). A detailed description of the analytical procedure is provided in the Supporting Information (Text S4). Assay of Larval Acetylcholinesterase Activity. Larvae (50 larvae, n = 3 replicates) were homogenized in Tris-citrate buffer on ice,30 and AChE enzyme activity was measured using a commercial kit following the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China). AChE activity was normalized to the samples protein content, which was measured using the Bradford method. Reactive Oxygen Species Assay. The generation of reactive oxygen species (ROS) in 5 and 10 dpf larvae was measured using 2′,7′-dichlorofluorescein (DCF). Briefly, 20 5125
DOI: 10.1021/acs.est.5b00558 Environ. Sci. Technol. 2015, 49, 5123−5132
Article
Environmental Science & Technology
Table 3. Gene Transcription Levels in F1 Zebrafish Larvae Derived from Parental Zebrafish Exposed to TDCPP (0, 4, 20, and 100 μg/L) for 3 Monthsa zebrafish
5-dpf
TDCPP (μg/L) mbp syn2a α1-tubulin gfap gap43
0 1.0 1.0 1.0 1.0 1.0
± ± ± ± ±
4 0.04 0.08 0.03 0.11 0.04
−1.5 −1.1 −1.1 −1.2 −1.0
± ± ± ± ±
10-dpf 20
0.1 0.1 0.1 0.1 0.1
−1.6 −1.0 −1.4 1.0 −1.1
± ± ± ± ±
100 0.1* 0.1 0.1* 0.2 0.1
−2.3 −1.0 −1.7 −1.1 1.0
± ± ± ± ±
0
0.0** 0.1 0.1* 0.1 0.1
1.0 1.0 1.0 1.0 1.0
± ± ± ± ±
4 0.0 0.1 0.0 0.1 0.1
−1.1 −1.2 1.1 1.0 1.2
± ± ± ± ±
20 0.1 0.1 0.1 0.2 0.1
−1.0 −1.2 −1.4 1.4 1.1
± ± ± ± ±
100 0.1 0.1 0.1* 0.2 0.1
−1.6 −2.1 −1.6 1.5 1.7
± ± ± ± ±
0.1* 0.1* 0.1** 0.0 0.2**
Values represent the mean ± SEM of three replicates (30 larvae per replicate) and are expressed as fold change relative to control. *P < 0.05; **P < 0.01 indicate a significant difference between the exposure groups and the control group. a
Figure 1. Western blot analysis of protein expression in TDCPP-exposed F1 larvae. A representative Western blot of mbp, α1-tubulin, and syn2a expression in 5 dpf F1 larvae is shown (A), with the relative quantification of protein expression shown in (B). A representative Western blot of mbp, α1-tubulin, and syn2a expression in 10 dpf F1 larvae is shown (C), with the relative quantification of protein expression shown in (D). The data represent the means from three replicate samples. All data are expressed as the mean ± SEM fold change relative to the control. * P < 0.05, ** P < 0.01, and *** P < 0.001 indicates significant differences between exposure groups and the corresponding control group.
(11.1%, P = 0.010 and 9.0%, P = 0.034, respectively) in the 100 μg/L TDCPP group (Table 1). An increase in malformation rate was observed in F1 embryos and larvae derived from parents exposed to 100 μg/L (200%; P = 0.025), indicating that parental exposure to TDCPP causes developmental toxicity in their offspring. The survival rate for 5 dpf larvae in the 100 μg/ L TDCPP group was significantly decreased (6.4%, P = 0.028; Table 1); as it was at 10 dpf in the 20 and 100 μg/L TDCPP groups (13.2%, P = 0.002 and 25%, P < 0.001, respectively; Table 1). Thyroid Hormone Contents. Next, the effect of parental TDCPP exposure on thyroid hormone levels was measured in both generations (Table 2). In the F0 adults, long-term exposure to 20 and 100 μg/L TDCPP significantly reduced the total plasma T4 levels (by 24.2%, P = 0.045; by 37.2%; P = 0.004, respectively) in females. A significant decrease in T3 levels (by 44.7%; P = 0.015) was also observed in the group of females exposed to 100 μg/L TDCPP, while no significant effect of TDCPP exposure was observed for males. In F1 eggs and 5 dpf larvae, T4 levels were significantly decreased (by 40.4% and 34.9%, respectively) in the eggs/ larvae derived from adults exposed to 100 μg/L TDCPP (P = 0.049; P = 0.023, respectively), but total T3 content was not
protocols for extraction, cleanup, analysis and quality assurance, and quality control are provided in the Supporting Information (see Text S6). Concentrations of analytes are expressed as μg/ kg wet weight. Statistical Analysis. All data were initially verified for normality and homogeneity of variance using the Kolmogorov−Smirnov and Levene’s tests, respectively. All data are reported as means ± standard error of the mean (SEM). Differences between the control and each exposure group were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test. All analyses were performed with SPSS 16.0 (SPSS, Chicago, IL, USA). A P < 0.05 was considered statistically significant.
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RESULTS
Toxicological Endpoints in the F1 Generation. The effect of parental TDCPP exposure on the hatching, growth, malformation (e.g., spinal curvature), and survival rates of the resultant offspring was measured (Table 1). In the F1 generation derived from TDCPP-exposed adults, the hatching rates were significantly decreased at 3 dpf in the 20 μg/L (17.5%; P = 0.001) and 100 μg/L TDCPP groups (21.6%; P < 0.001), and growth inhibition was observed at 5 and 10 dpf 5126
DOI: 10.1021/acs.est.5b00558 Environ. Sci. Technol. 2015, 49, 5123−5132
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Environmental Science & Technology significantly changed in any group (Table 2). At 10 dpf, total T4 levels were significantly lower in the larvae derived from parents exposed to 20 μg/L (by 27.2%; P = 0.049) and 100 μg/L (by 35.3%; P = 0.01) TDCPP, and the T3 content was significantly reduced in the 100 μg/L TDCPP group (by 42.7%; P = 0.012; Table 2). Gene Transcription in F1 Larvae. The expression levels of several genes associated with the nervous system were assessed in the offspring of TDCPP-exposed zebrafish (Table 3). This included the expression of mbp, growth associated protein 43 (gap-43), synIIa, and glial f ibrillary acidic protein (gfap). As previously described, these genes serve as biomarkers for developmental neurotoxicity and are known to be responsive to neurotoxicant exposure.34 At 5 dpf, mbp expression was significantly downregulated in larvae from the 20 and 100 μg/L TDCPP groups, compared with the control (P = 0.033 and P = 0.003, respectively). There was also a significant downregulation of α1-tubulin (P = 0.03) in the larvae from the 100 μg/L TDCPP group, while expression of syn2a, gfap, and gap-43 was unchanged. At 10 dpf, the expression levels of mbp and syn2a were significantly downregulated in larvae from the 100 μg/L TDCPP group (P = 0.041 and P = 0.013, respectively). Downregulation of α1-tubulin was also observed in the larvae derived from the 20 and 100 μg/L TDCPP groups (P = 0.047 and P = 0.007, respectively), while gap-43 was significantly upregulated in the 100 μg/L TDCPP group (P = 0.002), and gfap gene expression was unchanged (Table 3). Protein Expression in F1 Larvae. To complement our analysis of gene expression, we also assessed the protein expression level of mbp, α1-tubulin, and syn2a by Western blotting (Figure 1). Consistent with our gene expression data, at 5 dpf, mbp protein expression was significantly reduced in larvae from the 100 μg/L TDCPP group (54.7%; P = 0.002), and expression of α1-tubulin was also significantly reduced in the 20 and 100 μg/L TDCPP groups (44.1%, P = 0.005 and 69.9%, P < 0.001, respectively). The expression of syn2a was also significantly reduced in larvae derived from the 20 and 100 μg/L TDCPP groups (40.9%, P = 0.002 and 71.4%, P < 0.001, respectively). At 10 dpf, the expression of mbp (37.6%, P = 0.032 and 60.2%, P = 0.002), α1-tubulin (55.7%, P < 0.001 and 86.4%, P < 0.001), and syn2a (54.9%, P < 0.001 and 73.9%, P < 0.001) were all significantly reduced in the 20 and 100 μg/L TDCPP groups (Figure 1). Neurotransmitter Contents and AChE Activity. The evaluation of neurotransmitters has emerged as an important strategy to assess neurochemical, behavioral, and toxicological phenotypes in zebrafish,35 and larval locomotor behavior can also indicate toxicity.36 Therefore, we examined the concentration of dopamine, serotonin, histamine, and GABA (Figure 2). At 5 dpf, dopamine levels were significantly decreased in F1 larvae from the 20 and 100 μg/L TDCPP groups (31.5%, P = 0.031; 36.4%, P = 0.015, respectively). Serotonin content was also significantly decreased in larvae from the 100 μg/L TDCPP group (45.0%, P = 0.002), while GABA was also significantly reduced in the 20 and 100 μg/L TDCPP groups (19.9%, P = 0.048; 37.2%, P = 0.001, respectively). Histamine content was not significantly changed in any group. At 10 dpf, dopamine levels were significantly decreased in F1 larvae from the 20 and 100 μg/L TDCPP groups (61.4%, P = 0.014; 53.5%, P = 0.028, respectively; Figure 2). Serotonin and GABA content was significantly decreased in the 100 μg/L TDCPP group (50.8%, P = 0.020; 31.9%, P = 0.031,
Figure 2. Neurotransmitter concentration in TDCPP-exposed F1 larvae. The neurotransmitter concentrations of dopamine, serotonin, GABA, and histamine are presented for 5 dpf F1 larvae (A) and 10 dpf F1 larvae (B). All data are expressed as the mean ± SEM of three replicate samples; each replicate contained 50 larvae. * P < 0.05 and ** P < 0.01 indicates significant differences between exposure groups and the corresponding control group.
respectively), whereas histamine levels were significantly reduced in the 20 and 100 μg/L TDCPP groups (32.4%, P = 0.041; 54.3%, P = 0.002, respectively). AChE activity was not significantly changed in 5 and 10 dpf zebrafish larvae derived from TDCPP-exposed parents at any concentration (see Supporting Information, Figure S1). Locomotor Activity. Locomotor activity was monitored in 5 and 10 dpf F1 larvae during continuous light and during a light-dark transition stimulation period. Under continuous light, swimming speed was significantly reduced in 5 dpf (P < 0.001) and 10 dpf (P < 0.001) larvae in the 100 μg/L TDCPP group (see Supporting Information, Figure S2). Locomotor traces of the larvae during light-dark transition stimulation are shown in the Supporting Information (Figure S2). During light-dark transition stimulation of 5 dpf larvae from the 100 μg/L TDCPP group, the average swimming speed was significantly decreased during the first light period (P < 0.001), first dark period (P < 0.001), second light period (P = 0.007), and second dark period (P < 0.001; Figure 3). During light-dark transition stimulation of 10 dpf larvae from the 100 μg/L TDCPP group, both light and dark period average swimming speed was also significantly decreased (both P < 0.001; Figure 3). 5127
DOI: 10.1021/acs.est.5b00558 Environ. Sci. Technol. 2015, 49, 5123−5132
Article
Environmental Science & Technology
Figure 4. Assessment of TDCPP and BDCPP concentration in TDCPP-exposed zebrafish. The concentration of TDCPP and BDCPP was measured in F0 adult zebrafish (A) and the F1 eggs were derived from TDCPP-exposed parents (B). For adult fish, the values represent the mean of three individual fish from each exposure tank. For the eggs, TDCPP and BDCPP were measured in 50 eggs, with three replicate samples. The data are expressed as the mean ± SEM. a, b, c, and d represent the eggs derived from zebrafish exposed to 0, 4, 20, and 100 μg/L TDCPP, respectively.
Figure 3. Locomotor behavior of TDCPP-exposed F1 larvae. The locomotor activity of F1 larvae derived from TDCPP-exposed parents was assessed, and the average swimming speed during the dark-lightdark photoperiod stimulation test is presented for 5 dpf F1 larvae (A) and 10 dpf F1 larvae (B). Data are expressed as the mean ± SEM of three replicates (10 larvae per replicate). * P < 0.05, and *** P < 0.001 indicates significant differences between exposure groups and the corresponding control group.
(Figure 4B), although BDCPP was only detected in F1 eggs derived from parents exposed to 100 μg/L TDCPP. A small amount of TDCPP was detected in the control eggs. The measured TDCPP and BDCPP contents from the F0 adult fish and F1 larvae derived from exposed zebrafish are provided in the Supporting Information, Table S2.
ROS Generation. ROS generation was increased in F1 larvae from the 100 μg/L TDCPP group by 90.1% (P = 0.015) at 5 dpf and by 63.1% (P = 0.013) at 10 dpf (see Supporting Information, Figure S1). Quantification of TDCPP and BDCPP. In order to quantify the degree of TDCPP exposure to zebrafish and its metabolism to BDCPP, we measured the concentration of these compounds under our different experimental conditions. Consistent with previous studies,25,28 the concentration of TDCPP in the exposure water was found to closely match the desired exposure concentration, such that in the 4, 20, and 100 μg/L exposure groups, the actual concentration of TDCPP was 3.7 ± 0.05, 20.4 ± 0.9, and 107.9 ± 5.6 μg/L, respectively. In F0 adult males and females, the total body burden of TDCPP and BDCPP showed a concentration-dependent relationship in the 4, 20, and 100 μg/L exposure groups, (Figure 4A). Based on the data, the bioconcentration factors are 84.1, 43.0, and 10.1 in female fish and 67.5, 28.1, and 15.4 in male fish with treatment of 4, 20, and 100 μg/L TDCPP, respectively. Control males contained detectable amounts of TDCPP and BDCPP content was
