Spatial and temporal variations of trace metal body burdens of live mussels Mytilus galloprovincialis and field validation of the Artificial Mussels in Australian inshore marine environment.

The body burdens of eight trace metals: Cd, Pb, Cu, Zn, Cr, Se, Hg, and As, were measured in live mussels (LMs) Mytilus galloprovincialis at 14 coastal sites in Port Phillip Bay, Victoria, Australia in winter and summer between 2017 and 2018. The spatial and temporal variations of body burdens were evaluated. The results revealed significantly higher body burdens of Cd, Pb, Cu, Zn, Cr, and Hg in summer at the sites where the city centre and industries are located. Elevated levels of most trace metals including 3 toxic, non-essential metals (Cd, Pb, and Hg) were detected in LMs from the site of Geelong. The body burdens of Zn, Cr, Se, and As appeared higher at the sites from the Bellarine Peninsula and the mouth of Port Phillip Bay. Besides, the "Artificial Mussels" (AMs) were deployed at the same sites in summer for 28 d and retrieved when the LMs were collected. The accumulations of the eight metals were compared between AMs and LMs summer results. It indicated significant correlations for Pb, Cu, and Cr, lower correlations in Zn and Hg, and irregular correlations for Se and As. The AM results of Cd were below the detection limit of the analytical method. This study demonstrates that AMs are excellent replacement of LMs for the biomonitoring of multiple kinds of trace metals.

A comparative study on metal contamination in Estero de Urias lagoon, Gulf of California, using oysters, mussels and artificial mussels: Implications on pollution monitoring and public health risk.

The profile of 11 trace metals in two commonly used biomonitors (the native oyster Crassostrea palmula and mussel Mytella strigata) from Estero de Urias lagoon, Gulf of California, were studied for six months, covering both dry and wet seasons. Metal concentrations in these two bivalves were compared with concentrations accumulated by Artificial Mussels (AMs) deployed alongside during the same period. Significant temporal variations in Cd, Cr and Mn were observed in both bivalve species and AMs. Temporal changes were observed for Fe in both bivalve species, Pb in oyster only and Cu in both AMs and oysters, revealing seasonal changes in inputs and/or chemical forms of these metals in the lagoon. Significant correlations for Cd, Cr and Cu were found in mussels and oysters, but their Co, Fe, Mn and Zn profiles were very different, despite these two species being taxonomically closely related and often used as biomonitors for metals. Interestingly, Hg and U were detected in AMs but not in oysters and mussels. The difference in metal profile in oysters, mussels and AMs revealed in the present study clearly showed that different biomonitors and AM take up metals differentially from the same environment, and metal profile in a single biomonitor or AM alone therefore, cannot provide a good estimate on metal concentrations in the ambient environment. As such, different biomonitors and AM should be used in metal monitoring, in order to provide a comprehensive picture on metal levels in aquatic ecosystems. Concentrations of Ni and Pb in oysters, and Cr, Fe and Mn in mussels were among the highest reported in coastal waters worldwide. Concentrations of Pb in oysters exceeded legal limits set for bivalve mollusks in EU. Concentrations of Cr in mussels and oysters exceeded or were very close to, respectively, the legal limit for fish, crab-meat, oysters, prawns, and shrimps in Hong Kong. The results indicate a potential public health risk on human consumption of oysters and mussels commonly harvested from the Estero de Urias lagoon, and corresponding pollution control measures are deemed necessary.

Contamination and risk implications of endocrine disrupting chemicals along the coastline of China: A systematic study using mussels and semipermeable membrane devices.

A systematic study has been carried out to assess the contamination of endocrine disrupting chemicals (EDCs) in five highly urbanized coastal cities spanning from temperate to subtropical environments along the coastline of China. In each of these cities, species of native mussels (Mytilus galloprovincialis, M. coruscus or Perna viridis) were deployed alongside with semipermeable membrane devices (SPMDs) for one month at a reference site and a polluted site. The level of 4-nonylphenol (4-NP), bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in SPMDs and transplanted mussels were determined and compared. The concentration of EDCs in mussels from polluted sites of Qingdao and Shenzhen ranged from 99.4±9.40 to 326.1±3.16ng/g dry wt. for 4-NP, Dalian and Shanghai from 170.3±4.00 to 437.2±36.8ng/g dry wt. for BPA, Dalian and Shenzhen from 82.9±3.03 to 315.6±6.50ng/g dry wt. for E2, and Shenzhen and Shanghai from 124.5±3.25 to 204.5±9.26ng/g dry wt. for EE2, respectively. These results demonstrate that concentrations of EDCs in mussels along the coastline of China are substantially higher than levels reported in mussels and seafood elsewhere. Despite high levels of EDCs and per capita seafood consumption in China, analysis indicated that 4-NP and BPA intake from mussels at polluted sites per se are still below the Tolerable Daily Intake (TDI). In contrast, the daily intake of E2 and EE2 (6.5 and 5.5µg/person/day, respectively) from mussel consumption exceeded the Acceptable Daily Intake (ADI) established by the WHO, USA and Australia by large margins, suggesting significant public health risks. A strong correlation was found between EDC concentrations in SPMDs and transplanted mussels, and the advantages of using mussels and SPMDs for monitoring EDCs in the aquatic environment are discussed.

Juvenile exposure to bisphenol A promotes ovarian differentiation but suppresses its growth - Potential involvement of pituitary follicle-stimulating hormone.

Bisphenol A (BPA), a plastic monomer and plasticizer, is commonly used in plastics industry, and it has been well documented to be an estrogenic endocrine disrupter. In the present study, we investigated the effect of early (juvenile) exposure to BPA on the hypothalamic-pituitary-gonad (HPG) axis in the zebrafish. Estradiol (E2) and testosterone (T) were also included as positive and negative controls respectively. Juvenile zebrafish were exposed to BPA (1 and 10µM), E2 (10nM) and T (10nM) from 20 to 40 dpf (days post-fertilization), the period of sex/gonadal differentiation, followed by histological and expression analyses at 40 dpf. The ovary and hepatic proteomes were also analyzed by mass spectrometry. Our results showed that 20day exposure to BPA and E2 increased the ratio of females; however, they both significantly suppressed ovarian growth. Meanwhile, BPA and E2 significantly suppressed fshb but stimulated lhb expression in the pituitary. These effects did not seem to involve the hypothalamus because neither BPA nor E2 altered the expression of kiss1, kiss2, gnrh2 and gnrh3 in the hypothalamus. At the ovary level, BPA and E2 both decreased lhcgr expression. Interestingly, E2 and BPA displayed different effects in the liver. E2 induced a significant hepatic hypertrophy; however, BPA had no such effect. Analysis of hepatic proteomes revealed distinct protein profiles in the E2 group as compared with the others, especially fructose-bisphospahte aldolase B. These results indicated that BPA has estrogenic effects on female reproduction, but it does not mimic all E2 actions. Our data in the zebrafish suggest that sex differentiation involves estrogens and it is a sensitive window for evaluating estrogenic activities of compounds and their impacts on wildlife reproduction.

A novel approach for estimating the removal efficiencies of endocrine disrupting chemicals and heavy metals in wastewater treatment processes.

The wide occurrence of endocrine disrupting chemicals (EDCs) and heavy metals in coastal waters has drawn global concern, and thus their removal efficiencies in sewage treatment processes should be estimated. However, low concentrations coupled with high temporal fluctuations of these pollutants present a monitoring challenge. Using semi-permeable membrane devices (SPMDs) and Artificial Mussels (AMs), this study investigates a novel approach to evaluating the removal efficiency of five EDCs and six heavy metals in primary treatment, secondary treatment and chemically enhanced primary treatment (CEPT) processes. In general, the small difference between maximum and minimum values of individual EDCs and heavy metals measured from influents/effluents of the same sewage treatment plant suggests that passive sampling devices can smooth and integrate temporal fluctuations, and therefore have the potential to serve as cost-effective monitoring devices for the estimation of the removal efficiencies of EDCs and heavy metals in sewage treatment works.

Heavy metal contamination along the China coastline: A comprehensive study using Artificial Mussels and native mussels.

A comprehensive study was carried out to assess metal contamination in five cities spanning from temperate to tropical environment along the coastal line of China with different hydrographical conditions. At each of the five cities, Artificial Mussels (AM) were deployed together with a native species of mussel at a control site and a polluted site. High levels of Cr, Cu and Hg were found in Qingdao, high level of Cd, Hg and Pb was found in Shanghai, and high level of Zn was found in Dalian. Furthermore, level of Cu contamination in all the five cities was consistently much higher than those reported in similar studies in other countries (e.g., Australia, Portugal, Scotland, Iceland, Korea, South Africa and Bangladesh). Levels of individual metal species in the AM showed a highly significant correlation with that in the native mussels (except for Zn in Mytilus edulis and Cd in Perna viridis), while no significant difference can be found between the regression relationships of metal in the AM and each of the two native mussel species. The results demonstrated that AM can provide a reliable time-integrated estimate of metal concentration in contrasting environments over large biogeographic areas and different hydrographic conditions, and overcome the shortcomings of monitoring metals in water, sediment and the use of biomonitors.

Omics of the marine medaka (Oryzias melastigma) and its relevance to marine environmental research.

In recent years, the marine medaka (Oryzias melastigma), also known as the Indian medaka or brackish medaka, has been recognized as a model fish species for ecotoxicology and environmental research in the Asian region. O. melastigma has several promising features for research, which include a short generation period (3-4 months), daily spawning, small size (3-4 cm), transparent embryos, sexual dimorphism, and ease of mass culture in the laboratory. There have been extensive transcriptome and genome studies on the marine medaka in the past decade. Such omics data can be useful in understanding the signal transduction pathways of small teleosts in response to environmental stressors. An omics-integrated approach in the study of the marine medaka is important for strengthening its role as a small fish model for marine environmental studies. In this review, we present current omics information about the marine medaka and discuss its potential applications in the study of various molecular pathways that can be targets of marine environmental stressors, such as chemical pollutants. We believe that this review will encourage the use of this small fish as a model species in marine environmental research.

Silver nanoparticles disrupt regulation of steroidogenesis in fish ovarian cells.

Despite the influx of silver nanoparticles (nAg) into the marine environment, their effects on fish reproduction remain completely unexplored. Using ovarian primary cells from marine medaka (Oryzias melastigma), in vitro studies were carried out to evaluate the effects of two differently coated nAg particles (Oleic Acid, (OA) nAg and Polyvinylpyrrolidone, (PVP) nAg) on fish ovarian tissues, using AgNO3 as a positive control. Cytotoxicity was evaluated by MTT assay and expression of key genes regulating steroidogenesis (StAR, CYP 19a, CYP 11a, 3ßHSD and 20ßHSD) were determined by Q-RT-PCR. EC50 values for PVP nAg, OA nAg and AgNO3 were 7.25µgL(-1), 924.4µgL(-1), and 42.0µgL(-1) respectively, showing that toxicity of silver was greatly enhanced in the PVP coated nano-form. Down regulation of CYP 19a was observed in both nAg and AgNO3 treatments, while down regulation of 3ßHSD was only found in the OA nAg and AgNO3 treatments. For the first time, our results demonstrated that nAg can affect specific genes regulating steroidogenesis, implicating nAg as a potential endocrine disruptor.

Transcriptome profiling of larvae of the marine medaka Oryzias melastigma by Illumina RNA-seq.

We sequenced the whole transcriptome of the 24h-old larval stage of the marine medaka Oryzias melastigma using Illumina RNA-seq. De novo assembly of 64,914,324 raw reads was performed using Trinity, resulting in 144,953 contigs. TransDecoder found 58,246 candidate coding contigs with homology to other species based on BLAST analysis. Functional gene annotation was performed by GO, KEGG pathway, and COG analyses. We determined an expressed gene catalog for O. melastigma for gene information-based environmental genomic and ecotoxicogenomic research. This information will serve as a resource for elucidating the molecular mechanisms underlying the response of O. melastigma to environmental stresses and chemicals.

Interactive effects of hypoxia and polybrominated diphenyl ethers (PBDEs) on microbial community assembly in surface marine sediments.

Hypoxia alters the oxidation-reduction balance and the biogeochemical processes in sediments, but little is known about its impacts on the microbial community that is responsible for such processes. In this study, we investigated the effects of hypoxia and the ubiquitously dispersed flame-retardant BDE47 on the bacterial communities in marine surface sediments during a 28-days microcosm experiment. Both hypoxia and BDE47 alone significantly altered the bacterial community and reduced the species and genetic diversity. UniFrac analysis revealed that BDE47 selected certain bacterial species and resulted in major community shifts, whereas hypoxia changed the relative abundances of taxa, suggesting slower but nonetheless significant community shifts. These two stressors targeted mostly different taxa, but they both favored Bacteroidetes and suppressed Gammaproteobacteria. Importantly, the impacts of BDE47 on bacterial communities were different under hypoxic and normoxic conditions, highlighting the need to consider risk assessments for BDE47 in a broader context of interaction with hypoxia.

iTRAQ-based proteomic profiling of the marine medaka (Oryzias melastigma) gonad exposed to BDE-47.

A recent study demonstrated that 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) may have an adverse effect on the reproduction in marine medaka (Oryzias melastigma), but the molecular mechanisms remain largely unknown. In this study, we investigated the protein expression profiles of male and female gonads of O. melastigma exposed to dietary BDE-47 at two dosages (0.65 and 1.30 µg/g/day, respectively) for 21 days. Extracted proteins were labeled with iTRAQ and analyzed on a MALDI TOF/TOF analyzer, as results, 133 and 144 unique proteins were identified in testis and ovary, respective, and they exerted dose- and sex-dependent expression patterns. In testis, among the 42 differentially expressed proteins; down-regulation of histone variants and parvalbumins implicated BDE-47 may disrupt the spermatogenesis and induce sterility in fishes. In ovary, 38 proteins were differentially expressed; the elevation of vitellogenins and apolipoprotein A-I expression indicated BDE-47 acts as an estrogen-mimicking compound and led to reproductive impairment in O. melastigma.

Interactive effects of hypoxia and PBDE on larval settlement of a marine benthic polychaete.

Marine benthic polychaete Capitella sp. I is widely known to adapt to polluted habitats; however, its response to xenobiotics under hypoxic conditions has been rarely studied. This research aimed to test the hypothesis that interactive effects of hypoxia and congener BDE-47 of polybrominated diphenyl ethers (PBDE), which is ubiquitous in marine sediments, may alter the settlement of Capitella sp. I. Our results revealed that under hypoxic condition, settlement success and growth in body length of Capitella larvae were significantly reduced compared to those under normoxia of similar BDE-47 concentration. While no significant changes in morphology of settled larvae were noted in both exposure conditions, the presence of BDE-47 could enhance polychaete growth. The present findings demonstrated that the interactive effects of hypoxia and environmentally realistic concentrations of BDE-47 in sediments could affect polychaete settlement, which, in turn, reduce its recruitment and subsequent population size in the marine benthic ecosystem.

Gene expression profile and toxic effects in human bronchial epithelial cells exposed to zearalenone.

Zearalenone (ZEA), a mycoestrogen produced by Fusarium fungal species, is mainly found in cereal crops such as maize, wheat and barley. Although ZEA has been reported to be present in air, little is known about the health risk or the molecular basis of action when lung cells are exposed to ZEA. As ZEA has a similar structure to estrogen, its potential risk as an endocrine disrupting chemical (EDC) has thus aroused both environmental and public health concerns. The purpose of this study is to identify the responses and underlying molecular changes that occur when human bronchial epithelial BEAS-2B cells are exposed to ZEA. Differential gene expression profiles were identified in cells that were treated with 40 µM ZEA for 6 h and 24 h by high-throughput microarray analysis using Affymetrix Human Gene 2.0 GeneChip. The array results showed that after ZEA treatment, 262 genes at 6 h and 1073 genes at 24 h were involved in the differential regulation. Pathway analysis revealed that diverse cellular processes were affected when lung cells were exposed to ZEA resulting in impaired response to DNA damage, cell cycle arrest, down-regulation of inflammatory responses and alterations of epigenetic marks. Results of further experiments indicated that 40 µM ZEA decreased cell viability, induced apoptosis and promoted reactive oxygen species (ROS) generation in a time-dependent manner. Immuno-suppressive effects of ZEA were further revealed through the suppression of lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1ß). Interestingly, the level of global DNA methylation was markedly decreased after 24 h exposure to ZEA. Collectively, these observations suggested that a broad range of toxic effects are elicited by ZEA. Particularly, ROS may play a pivotal role in ZEA-induced cell death. These adverse effects observed in lung cells suggest that exposure to ZEA may increase susceptibility of lung cells to diseases and required further investigations.

Regulation of CYP11B1 and CYP11B2 steroidogenic genes by hypoxia-inducible miR-10b in H295R cells.

Although numerous studies have shown that hypoxia affects cortisol and aldosterone production in vivo, the underlying molecular mechanisms regulating the steroidogenic genes of these steroid hormones are still poorly known. MicroRNAs are post-transcriptional regulators that control diverse biological processes and this study describes the identification and validation of the hypoxia-inducible microRNA, miR-10b, as a negative regulator of the CYP11B1 and CYP11B2 steroidogenic genes in H295R human adrenocortical cells. Using the human TaqMan Low Density miRNA Arrays, we determined the miRNA expression patterns in H295R cells under normoxic and hypoxic conditions, and in cells overexpressing the human HIF-1α. Computer analysis using three in silico algorithms predicted that the hypoxia-inducible miR-10b molecule targets CYP11B1 and CYP11B2 mRNAs. Gene transfection studies of luciferase constructs containing the 3'-untranslated region of CYP11B1 or CYP11B2, combined with miRNA overexpression and knockdown experiments provide compelling evidence that CYP11B1 and CYP11B2 mRNAs are likely targets of miR-10b.

Hypoxia disrupts gene modulation along the brain-pituitary-gonad (BPG)-liver axis.

Hypoxia alters sex hormone concentrations leading to reproductive impairment in fish; however the mechanisms underlying these effects remain largely unknown. Using zebrafish (Danio rerio), this study is the first to demonstrate that hypoxia causes endocrine disruption by simultaneously acting on multiple targets along the brain-pituitary-gonadal (BPG)-liver axis in fish. Alterations in the expression of key genes associated with reproductive endocrine pathways in the brain (sGnRH), pituitary (FSHß and LHß), gonads (FSH-R, LH-R, HMGR, StAR, CYP19A, CYP11A, CYP11ß and 20ß-HSD), and liver were correlated with significant reductions of estradiol in females and testosterone in males. Hypoxia also induced sex-specific and tissue-specific changes in the expression of estrogen, androgen, and membrane progestin receptors along the BPG axis, suggesting disruption of the feedback and synchronization of hormone signals. Furthermore, the hypoxia-induced upregulation of hepatic sex hormone-binding globulin suggests an increase in hormone transport and reduced bioavailability in blood, while upregulation of hepatic CYP3A65 and CYP1A in females suggests an increase in estrogen biotransformation and clearance. Given that the regulation of reproductive hormones and the BPG-liver axis are highly conserved, this study provides new insights into the hypoxia-induced endocrine disrupting mechanisms and reproductive impairment in other vertebrates.

PBDE-47 exposure causes gender specific effects on apoptosis and heat shock protein expression in marine medaka, Oryzias melastigma.

Marine medaka (Oryzias melastigma) was fed with a low and high dose of dietary 2,2',4,4'-tetra-bromodiphenyl ether (PBDE-47), over 21 days. Gender specific changes in caspases 3 and 8 in medaka were found as activities in male medaka were significantly increased in both liver and muscle at both low and high exposure levels whereas caspase activity in female medaka tissue remained unchanged. Results of HSP90 and HSP70 immunoassays also showed gender specific related changes as both HSP families were unchanged in liver and muscle of male medaka but significantly increased in liver and muscle of female medaka, following PBDE-47 exposure. The gender specific effects of PBDE-47 on HSP expression profiles could not be explained by inherent differences in the heat shock response of male and female marine medaka, as the HSP profiles in liver and muscle, induced by acute heat shock, were similar in both sexes. The findings from this study provide evidence that PBDE-47 can cause gender specific modulatory effects on mechanisms critical to the apoptotic cascade as well as HSP regulation and expression.

Label-free detection of endocrine disrupting chemicals by integrating a competitive binding assay with a piezoelectric ceramic resonator.

A piezoelectric biosensor for detection of endocrine disrupting chemicals (EDCs) was developed by incorporating chemical/biochemical recognition elements on the ceramic resonator surface for competitive binding assays. A facile electrodeposition was employed to modify the sensor surface with Au nanoparticles, which increased the surface area and enhanced the binding capacity of the immobilized probes. Thiol-labeled long chain hydrocarbon with bisphenol A (BPA) as head group was synthesized and self-assembled on the Au nanoparticle surface as the sensing probes, which showed a linear response upon the binding of estrogen receptor (ER-α) ranging from 1 to 30 nM. Detection of estrone, 17ß-estradiol and BPA was achieved by integrating a competitive binding assay with the piezoelectric sensor. In this detection scheme, different concentrations of EDCs were incubated with 30 nM of ER-α, and the un-bounded ER-α in the solution was captured by the probes immobilized on the ceramic resonator, which resulted in the frequency changes for different EDCs. The biosensor assay exhibited a linear response to EDCs with a low detection limit of 2.4-2.9 nM (S/N=3), and required only a small volume of sample (1.5 µl) with the assay time of 2h. The performance of the biosensor assay was also evaluated for rapid and facile determination of EDCs of environmental relevant concentrations in drinking water and seawater, and the recovery rate was in the range between 94.7% and 109.8%.