Endocrine Disruption in Marine Mussels: Reproductive Effects and Transcriptomic Applications for Assessing Pollution in Coastal Waters

Endocrine Disrupting Compounds (EDCs) are ubiquitous in marine environments and have been found to cause intersex and developmental impacts across wildlife species including mussels. Innovative approaches are needed to monitor EDCs in coastal environments as these compounds cause effects at low levels (e.g., ng/L), and below analytical detection limits. Additionally, a comprehensive literature search identified at least a hundred compounds that produced an estrogenic response, making monitoring individual contaminants both time and cost intensive. The purpose of this research was to develop a high throughput assay and adverse outcome pathway (AOP) to monitor the exposure and effects of EDCs in coastal environments in marine mussels. Although intersex from EDC exposure has been identified in bivalves, the mechanism linked to this adverse outcome is relatively unknown. A combination of transcriptomic analysis followed by systems biology and computational approaches identified the non-genomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of adverse action. A putative AOP and mechanism-based biomarkers representative of key events (KE) were developed to form the Coastal Biosensor of Endocrine Disruption (C-BED) assay. The C-BED assay was validated in the lab over multiple time-points and was found to respond at the most contaminated field locations in both a native and transplanted mussel study. Adverse effects on reproduction and development were suggested by induction of female specific transcripts in both male and female mussels and delayed development in EDC treatments. In addition to the C-BED assay, this research also showed several promising gene expression applications for biomarker development. First, a novel mtDNA sex identification assay was developed that may be a time and cost-effective alternative to other gender identification methods. Secondly, candidate biomarkers for different contaminants were identified by correlating bioaccumulation in mussel deployed in the Boston Harbor with gene expression data. Finally, the C-BED assay combined with the Integrated Biomarker Response (IBR) also proved to be a reliable method to rank EDC exposure. This study can therefore be used as an example of how transcriptomics and various data analysis approaches can be used to develop novel biomarkers for organisms that lack annotated regulatory and biochemical pathways

Multiple origins of pyrethroid insecticide resistance across the species complex of a nontarget aquatic crustacean, Hyalella azteca

Use of pesticides can have substantial nonlethal impacts on nontarget species, including driving evolutionary change, often with unknown consequences for species, ecosystems, and society. Hyalella azteca, a species complex of North American freshwater amphipods, is widely used for toxicity testing of water and sediment and has frequently shown toxicity due to pyrethroid pesticides. We demonstrate that 10 populations, 3 from laboratory cultures and 7 from California water bodies, differed by at least 550-fold in sensitivity to pyrethroids. The populations sorted into four phylogenetic groups consistent with species-level divergence. By sequencing the primary pyrethroid target site, the voltage-gated sodium channel, we show that point mutations and their spread in natural populations were responsible for differences in pyrethroid sensitivity. At least one population had both mutant and WT alleles, suggesting ongoing evolution of resistance. Although nonresistant H. azteca were susceptible to the typical neurotoxic effects of pyrethroids, gene expression analysis suggests the mode of action in resistant H. azteca was not neurotoxicity but was oxidative stress sustained only at considerably higher pyrethroid concentrations. The finding that a nontarget aquatic species has acquired resistance to pesticides used only on terrestrial pests is troubling evidence of the impact of chronic pesticide transport from land-based applications into aquatic systems. Our findings have far-reaching implications for continued uncritical use of H. azteca as a principal species for monitoring and environmental policy decisions

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, <i>Mytilus edulis</i>

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed <i>Mytilus edulis</i> to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 μg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an <i>M. edulis</i> microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field

Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, <i>Mytilus edulis</i>

Transcriptomics, high-throughput assays, and adverse outcome pathways (AOP) are promising approaches applied to toxicity monitoring in the 21st century, but development of these methods is challenging for nonmodel organisms and emerging contaminants. For example, Endocrine Disrupting Compounds (EDCs) may cause reproductive impairments and feminization of male bivalves; however, the mechanism linked to this adverse outcome is unknown. To develop mechanism-based biomarkers that may be linked through an AOP, we exposed <i>Mytilus edulis</i> to 17-alpha-ethinylestradiol (5 and 50 ng/L) and 4-nonylphenol (1 and 100 μg/L) for 32 and 39 days. When mussels were exposed to these EDCs, we found elevated female specific transcripts and significant female-skewed sex ratios using a RT-qPCR assay. We performed gene expression analysis on digestive gland tissue using an <i>M. edulis</i> microarray and through network and targeted analyses identified the nongenomic estrogen signaling pathway and steroidogenesis pathway as the likely mechanisms of action for a putative AOP. We also identified several homologues to genes within the vertebrate steroidogenesis pathway including the cholesterol side chain cleavage complex. From this AOP, we designed the Coastal Biosensor for Endocrine Disruption (C-BED) assay which was confirmed in the laboratory and tested in the field

Toxicogenomic Responses of Nanotoxicity in <i>Daphnia magna</i> Exposed to Silver Nitrate and Coated Silver Nanoparticles

Applications for silver nanomaterials in consumer products are rapidly expanding, creating an urgent need for toxicological examination of the exposure potential and ecological effects of silver nanoparticles (AgNPs). The integration of genomic techniques into environmental toxicology has presented new avenues to develop exposure biomarkers and investigate the mode of toxicity of novel chemicals. In the present study we used a 15k oligonucleotide microarray for <i>Daphnia magna</i>, a freshwater crustacean and common indicator species for toxicity, to differentiate between particle specific and ionic silver toxicity and to develop exposure biomarkers for citrate-coated and PVP-coated AgNPs. Gene expression profiles revealed that AgNO₃ and AgNPs have distinct expression profiles suggesting different modes of toxicity. Major biological processes disrupted by the AgNPs include protein metabolism and signal transduction. In contrast, AgNO₃ caused a downregulation of developmental processes, particularly in sensory development. Metal responsive and DNA damage repair genes were induced by the PVP AgNPs, but not the other treatments. In addition, two specific biomarkers were developed for the environmental detection of PVP AgNPs; although further verification under different environmental conditions is needed

Toward Sustainable Environmental Quality: Priority Research Questions for North America.

Anticipating, identifying, and prioritizing strategic needs represent essential activities by research organizations. Decided benefits emerge when these pursuits engage globally important environment and health goals, including the United Nations Sustainable Development Goals. To this end, horizon scanning efforts can facilitate identification of specific research needs to address grand challenges. We report and discuss 40 priority research questions following engagement of scientists and engineers in North America. These timely questions identify the importance of stimulating innovation and developing new methods, tools, and concepts in environmental chemistry and toxicology to improve assessment and management of chemical contaminants and other diverse environmental stressors. Grand challenges to achieving sustainable management of the environment are becoming increasingly complex and structured by global megatrends, which collectively challenge existing sustainable environmental quality efforts. Transdisciplinary, systems-based approaches will be required to define and avoid adverse biological effects across temporal and spatial gradients. Similarly, coordinated research activities among organizations within and among countries are necessary to address the priority research needs reported here. Acquiring answers to these 40 research questions will not be trivial, but doing so promises to advance sustainable environmental quality in the 21st century. Environ Toxicol Chem 2019;38:1606-1624. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC

The toxicogenome of <i>Hyalella azteca</i>:a model for sediment ecotoxicology and evolutionary toxicology

<i>Hyalella azteca</i> is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the <i>H. azteca</i> U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of <i>H. azteca</i> exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The <i>H. azteca</i> genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health