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Environmentally Relevant Concentrations of the Flame Retardant Tris(1,3-dichloro-2-propyl) Phosphate (TDCIPP) Inhibits Growth of Female Zebrafish and Decreases Fecundity Ya Zhu, Xufa Ma, Guanyong Su, Liqin Yu, Robert J. Letcher, Jie Hou, Hongxia Yu, John P. Giesy, and Chunsheng Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03849 • Publication Date (Web): 29 Oct 2015 Downloaded from http://pubs.acs.org on November 3, 2015
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Environmental Science & Technology
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Environmentally Relevant Concentrations of the Flame Retardant
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Tris(1,3-dichloro-2-propyl) Phosphate (TDCIPP) Inhibit Growth of Female Zebrafish
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and Decrease Fecundity
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Ya Zhu‡,O, Xufa Ma‡,O, Guanyong Su†,£,*, Liqin Yu‡, Robert J. Letcher£, Jie Hou‡,
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Hongxia Yu£, John P. Giesy£,ǁ,¶, Chunsheng Liu‡,ζ,*
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‡
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ζ
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
Collaborative Innovation Center for Efficient and Health Production of Fisheries in Hunan
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Province
11
†
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Nanjing University, Nanjing 210089, China
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£
14
ǁ
15
Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada
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¶
17
East Lansing, Michigan 48824, United States
State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,
Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of
Department of Zoology and Centre for Integrative Toxicology, Michigan State University,
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*Authors for correspondence:
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Dr.
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[email protected].
22
Dr.
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[email protected] Chunsheng
Guanyong
Liu,
Su,
Tel:
Tel:
86
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(613)
87282113.
Fax:
660-2390.
Fax:
86
27
(613)
87282114.
Email:
998-0458.
Email:
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The first two authors contributed equally to this work and are indicated with “O”.
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Abstract
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Bioconcentration of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) in brain, gonad and
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liver as well as effects on fecundity and development of zebrafish (Danio rerio) were
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determined. Zebrafish (1-month old) were exposed to environmentally relevant concentrations
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of 29 ± 2.1, 600 ± 21 or 6300 ± 130 ng TDCIPP/L. After 120 days of exposure, TDCIPP
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accumulated in the brain, gonad and liver with bioconcentration factors of 460, 38 and 87 in
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females and 26, 55 and 110 in males, respectively. TDCIPP accumulated to a greater extent in
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brains of females than those of males. Exposure to 6300 ± 130 ng TDCIPP/L resulted in
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significantly (P < 0.05) fewer eggs being produced, but histology of the gonad, plasma
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concentrations of estradiol and 11-ketotestosterone, and expression of genes involved in
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hypothalamic-pituitary-gonadal-liver axis were not significantly (P > 0.05) different between
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individuals exposed to TDCIPP and the unexposed control fish. Exposure to TDCIPP resulted
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in shorter body length, lighter body mass and lesser Gonado-Somatic Index in females. These
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effects were possibly due to down-regulation of expression of genes along the growth
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hormone/insulin-like growth factor (GH/IGF) axis. Correlations between the production of
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eggs and developmental parameters or expression of genes along the GH/IGF axis further
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suggested that environmentally relevant concentrations of TDCIPP could have adverse effects
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on reproduction, possibly due to the inhibition of the growth of females.
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Key Words: reproduction; development; zebrafish; organophosphate flame retardant; growth
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hormone/insulin-like growth factor (GH/IGF) axis
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Introduction
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Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a synthetic organophosphate flame
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retardant (FR) and plasticizer that has been used for decades in various products, such as
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plastics, foams, textiles, varnishes, electronics equipment and furniture [1]. It is estimated that
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annual production of TDCIPP in the United States in the years of 1998, 2002 and 2006 ranged
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from 4,500 to 22,700 tonnes [1]. Recently, because the use of brominated FRs are generally
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being voluntarily reduced and phased out or banned, the consumption of organophosphate FRs
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such as TDCIPP has been increasing [1, 2].
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TDCIPP is an additive FR that is not chemically-bonded to polymeric products, whereas
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reactive FRs are chemically bonded into products. Therefore, TDCIPP is more likely to be
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released into the environment, as evidenced by its frequent detection in aquatic environmental
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compartments [1, 3-14]. For example, 2.5 - 40 ng TDCIPP/L was reported in waters of the
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Songhua River, China [5, 6]. In seawater along the coast of China near the cities of Qingdao
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and Xiamen, concentrations of TDCIPP ranged from 24 to 377 ng/L [7]. In effluents from
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sewage treatment plants in Sweden, concentrations as great as 3 µg TDCIPP/L have been
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reported [13]. TDCIPP has also been detected in various aquatic species of fish.
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perch (Perca fluviatilis) from Djupasjön Lake in Sweden contained concentrations as great as
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140 ng TDCIPP/g lipid weight (lw) [8]. Catfish (Clarius fuscus) and grass carp (Cyprinus
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idellus) from the Pearl River, China were reported to have concentrations of TDCIPP as great
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as 251 ng TDCIPP/g lw [15]. These reported concentrations of TDCIPP in aquatic organisms
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were at least 10- to 100-fold greater than concentrations in water collected from the same
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areas, which indicates the substantial bioconcentration/bioaccumulation potential of TDCIPP
Yellow
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in aquatic systems.
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Limited toxicological information suggests that exposure to TDCIPP has the potential to
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disrupt the endocrine system [16-21], and neurological [22-25], developmental and
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reproductive functions [26-33], depending on species tested. For example, high concentration
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of TDCIPP was shown to delay remethylation in zygotes and inhibit embryonic epiboly in
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embryos of zebrafish, as well as decreased lengths of head and bill, masses and size of
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gallbladder in chicken embryos [29]. Acute exposure to TDCIPP altered expression of
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steroidogenic genes and production of testosterone (T) and 17β-estradiol (E2) in exposed
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H295R cells and zebrafish, and lesser number of eggs produced by zebrafish [32]. Exposure of
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zebrafish to relatively small concentrations (4, 20 or 100 µg TDCIPP/L) for 6 months
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significantly decreased body mass, body length and condition factor (K), affected transcription
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of genes involved in the hypothalamic-pituitary-gonadal-liver (HPGL) axis, increased
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concentrations of T and E2 in blood plasma, and resulted in fewer eggs being produced [17].
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Collectively, these studies suggested that exposure to TDCIPP could cause adverse effects on
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development and reproduction of zebrafish [17, 29, 32]. However, concentrations to which
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fish were exposed in these studies were greater than environmentally relevant concentrations
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ever reported (≤377 ng/L), and thus could not provide reliable thresholds for effects that could
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be used in assessments of risk. Recently, using D. magna as a model, it was found that
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environmentally relevant concentrations of TDCIPP significantly decreased fecundity as well
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as length of F0 and F1 generations, and development of F1 individuals was found to be a more
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sensitive endpoint than was fecundity of F0 individuals [34]. Alterations of expression of genes
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related to the synthesis of proteins and metabolism in endocytosis pathways might be
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responsible for TDCIPP-induced effects on reproduction and development of D. magna [34].
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These data for D. magna suggested a need for further studies to investigate the potential
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toxicological effects due to exposure of other aquatic organisms to environmentally relevant
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concentrations of TDCIPP, such as fish.
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In the present study, bioaccumulation of TDCIPP was evaluated, and effects on
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development and reproduction were examined, after chronic, water-borne exposures of in
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zebrafish to environmentally relevant concentrations of TDCIPP.
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Materials and Methods
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Chemicals and Reagents
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Chemicals and reagents were purchased from the following sources: TDCIPP for
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zebrafish aquatic exposure from Sigma-Aldrich (St. Louis, MO, USA); TRIzol reagent and
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reverse transcription and SYBR Green kits from Takara (Dalian, Liaoning, China); hormone
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detection kits from Cayman Chemical Company (Ann Arbor, MI, USA); MS-222
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(3-aminobenzoic acid ethyl ester, methanesulfonate salt) from Sigma-Aldrich; TDCIPP for use
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as an analytical standard was from Tokyo Chemical Industry America (Portland, OR, USA);
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d15-TDCIPP was purchased from Dr. Vladimir Below via Organic Contaminants Research
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Laboratory (OCRL), NWRC (Ottawa, Canada). All the reagents used in this study were of
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analytical grade.
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Zebrafish Maintenance and TDCIPP Exposure Protocols
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Stock solutions of TDCIPP were prepared in dimethyl sulfoxide (DMSO). Zebrafish were
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maintained in flow-through tanks, according to previously published methods [35].
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One-month old zebrafish were acclimated in 15-L glass tanks for 1 week and then exposed to
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0, 50, 500 or 5000 ng TDCIPP/L for 4 months. Twenty fish were exposed in each of three
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replicate tanks for each concentration. During semi-static exposure, solutions were replaced
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daily with fresh carbon-filtered water containing corresponding concentrations of TDCIPP.
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Exposure solutions were sampled at the last day of exposure, and concentrations of TDCIPP
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and bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) were quantified. Both control and treated
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groups received 0.001% DMSO since previous study demonstrated that no significant effects
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on development and reproduction were observed in zebrafish when DMSO concentrations
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were ≤0.01% [36].
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To examine the effects of TDCIPP on reproduction, fish were spawned in groups during
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the last 21 days of exposure. For each concentration, groups containing four females and four
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males from each replicate (n = 3) were grouped and spawned after lights on in the morning,
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and numbers of eggs spawned were recorded daily. Fecundity was reported as cumulative
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eggs per female in the last 21 days of exposure.
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After exposure, fish were euthanized with MS-222, and body mass (g) and snout-to-vent
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length (mm) were recorded. Blood was collected for quantification of 11-ketotestosterone
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(11-KT) (for males) and E2 (for females) and gonads were sampled and weighed for
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determination of the gonadal-somatic index (GSI), and then fixed in Bouin’s solution for
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subsequent imbedding and histological examination. Brain, gonad and liver samples were
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collected for quantification of TDCIPP or BDCIPP and real-time PCR reactions.
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Quantification of TDCIPP and BDCIPP in Exposure Solutions and Fish Tissues
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Both TDCIPP and BDCIPP were quantified in water and zebrafish samples. Detailed
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protocols used for identification and quantification of TDCIPP in water or tissues have been
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published previously [11, 37, 38] , and are also provided in the supporting information. An
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internal calibration method is used for quantification of TDCIPP and BDCIPP in zebrafish
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tissue samples.
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During quantification of TDCIPP and BDCIPP in water no background contamination
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was detectable, and thus the method limits of quantification were defined as a concentration
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that can generate instrumental response that is 10-fold greater than the signal-to-noise ratio.
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MLOQs of TDCIPP and BDCIPP were 0.01 and 0.015 ng/mL water, respectively. In tissue
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samples, there was unavoidable background contamination with an average of 0.29 ± 0.12 ng
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TDCIPP/sample, and consistently observed in all control fish samples. Thus, the MLOQ for
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TDCIPP was background corrected for fish tissues and was calculated to be 0.36 ng/sample,
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which was three times the standard derivation of measurements in all control fish samples.
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Based on its internal standard, d15-TDCIPP, the mean recovery of TDCIPP for all samples of
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tissues was 99 ± 12%. Like TDCIPP, unavoidable background contamination (0.2±0.05
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ng/sample) was also observed for BDCIPP in analyzed blank samples. The MLOQ of
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BDCIPP in fish tissues was 0.19 ng BDCIPP/sample, and its mean recovery for all analyzed
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samples was 85 ± 17%.
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Histological Examination
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Histological examination of gonads was performed according to previously published
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methods [39]. Firstly, gonadal tissues were dehydrated in 70 % ethanol for 15 min, 80 %
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ethanol for 15 min, 90 % ethanol for 15 min, 95 % ethanol for 30 min, and 100 % ethanol for
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40 min. Secondly, tissues were hyalinized in a mixture of xylene and ethanol (v/v, 1:1) for 8
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min, and 100% xylene for 8 min. Thirdly, tissues were immersed in Paraffix® wax for 60 min
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at 58 0C and then were embedded. Lastly, tissues were sectioned at 5 µm and stained with
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haematoxylin and eosin (H&E). Stages of oogenesis and spermatogenesis were identified and
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quantified using previously reported methods [40]. Six fish for each sex and treatment group
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were included in the assay and a total of three sections were collected from each fish.
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Quantification of Hormone Concentrations
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Quantification of concentrations of 11-KT and E2 in blood plasma of males and females,
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respectively, was performed following previous methods using commercial kits from Cayman
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Chemical Company [35, 39]. In brief, blood from each fish was centrifuged at 5000 × g for 5
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min at 4 oC, and then plasma from 2 fish was collected and pooled to form a sample. Plasma
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from each pooled sample was diluted with ultrapure water and extracted with ethyl ether, and
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then evaporation was performed. EIA buffer provided in the kits was used to re-dissolve
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residues and concentrations of hormones were measured according to the manufacturer’s
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instructions. Limits of quantification for 11-KT and E2 were 1.3 and 19 pg/mL, respectively.
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Three biological replicates were used in this assay. To compare effects of TDCIPP on
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expression of genes and production of hormones, concentrations of 11-KT and E2 in plasma
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were expressed as fold-change relative to control.
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Quantitative Real-time PCR reactions
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Quantitative real-time PCR (qRT-PCR) reactions were performed as previously described
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and met requirements for minimum information for publication of quantitative real-time PCR
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experiment (MIQE) guidelines [35, 41]. In brief, TRIzol reagent was used for the isolation of
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total RNA. Purity and total concentrations of RNA were measured using the EpochTM
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Microplate Spectrophotometer (Bio Tek Instruments, Inc, Vermont, USA). Reverse
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transcription and qRT-PCR were performed using Prime ScriptTM RT reagent kits and SYBR
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Green kits, respectively, from Takara (Dalian, Liaoning, China). Sequences of primers were
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designed by use of Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/) (Table S1)
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(see Supporting Information). Expression of the housekeeping gene rpl8 did not change after
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exposure to various concentrations of TDCIPP, and was thus selected for use as an internal
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control for variations among amplifications. Thermal cycling was done at 95 Co for 10 min,
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followed by 40 cycles of 95 Co for 15 s and 60 Co for 1 min. Relative gene expression to
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DMSO control was calculated using 2-△△CT method, and for down-regulated gene, fold change
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was calculated using formula of “-1/2-△△CT value” according to previous protocol [42]. In this
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study, we selected differentially expressed genes based on fold change >1.5 and P value
