Using eRNA/eDNA metabarcoding to detect community-level impacts of nanoplastic exposure to benthic estuarine ecosystems.

Plastic particles are ubiquitous in marine systems and fragment into smaller pieces, such as nanoplastics (NPs). The effects of NPs on marine organisms are of growing concern but are not well understood. Marine sediments act as a sink for many contaminants, like microplastics, and are rich habitats for benthic micro- and meiofauna which are ecologically-important components of marine food webs; however, little is known about the sensitivities of specific organisms to NPs or the effects on community diversity and composition. Utilizing molecular methods, such as metabarcoding of environmental DNA/RNA, allows for the rapid and comprehensive detection of microscopic organisms via high-throughput sequencing to assess adverse effects at the community level. The objective of this study was to use a metabarcoding approach to investigate the effects of NPs on benthic micro- and meiofaunal community diversity. Mesocosms were created with sediment cores collected from the Narrow River estuary (Rhode Island, USA) and exposed to 900 nm diameter weathered polystyrene beads at concentrations of 0.1, 1, 10, or 100 mg/kg dry weight in sediment for two weeks. Following exposure, RNA and DNA were co-extracted from the sediment, RNA was reverse-transcribed, 18S and COI markers were PCR-amplified, and amplicons were sequenced on an Illumina MiSeq. Using the 18S marker and eRNA template, increases to α-diversity and significant differences to ß-diversity were observed in the highest NP exposures relative to the control. Observed differences in community composition were driven by the differential abundance of several types of protists and arthropods. Significant dose-dependent shifts in composition were observed in ß-diversity Jaccard and Unweighted-Unifrac metrics with the 18S marker using the RNA template. To our knowledge, this is the first demonstration of a dose-response relationship for NPs at a community level, and it highlights the value of using community-level endpoints to assess environmental impacts of nanoparticles.

Building an inclusive wave in marine science: Sense of belonging and Society for Women in Marine Science symposia.

Achieving gender equity is a long-standing and ubiquitous challenge in marine science. Creating equitable experiences for all genders in marine science requires recognizing scientists' intersectional identities, and how this leads to unique lived experiences of privilege and marginalization. One approach to increase equitable experiences for women in marine science is to create affinity groups where women can learn from each other, share their experiences, and provide support and mentorship. The Society for Women in Marine Science (SWMS) is one such organization, founded to amplify the work of early career women in marine science and create community, through events such as full-day symposium events. This study investigates the experiences of symposium attendees for four events held from 2018 through 2020, as reported in pre- and post-symposium surveys. We used quantitative analysis of the open-ended survey questions to examine the demographics of attendees and their fields of study. Qualitative thematic analysis identified the most effective aspects of the symposia, areas of logistical and content improvement for future symposia, and emphasized the unique challenges women in marine science experience. The majority of symposium attendees were white graduate students. Nearly all attendees identified as women, with a small number of men and non-binary individuals. Symposia attendees enjoyed opportunities for professional development and interactions with colleagues across career stages. We present recommendations for continuing to foster a sense of belonging in marine science and STEM more broadly, both specific to SWMS and transferable actions that can be applied for other affinity groups. These suggestions include empathetic event logistics, continual democratic evaluation, identity reflexivity among group leaders, and professional development activities targeted towards the unique needs of the affinity group. The positive responses received from SWMS's adaptive integration of survey results into symposia demonstrate that incorporating these recommendations and findings will help create an inclusive wave in marine science.

Environmental RNA as a Tool for Marine Community Biodiversity Assessments.

Microscopic organisms are often overlooked in traditional diversity assessments due to the difficulty of identifying them based on morphology. Metabarcoding is a method for rapidly identifying organisms where Environmental DNA (eDNA) is used as a template. However, legacy DNA is problematically detected from organisms no longer in the environment during sampling. Environmental RNA (eRNA), which is only produced by living organisms, can also be collected from environmental samples and used for metabarcoding. The aim of this study was to determine differences in community composition and diversity between eRNA and eDNA templates for metabarcoding. Using mesocosms containing field-collected communities from an estuary, RNA and DNA were co-extracted from sediment, libraries were prepared for two loci (18S and COI), and sequenced using an Illumina MiSeq. Results show a higher number of unique sequences detected from eRNA in both markers and higher α-diversity compared to eDNA. Significant differences between eRNA and eDNA for all ß-diversity metrics were also detected. This study is the first to demonstrate community differences detected with eRNA compared to eDNA from an estuarine system and illustrates the broad applications of eRNA as a tool for assessing benthic community diversity, particularly for environmental conservation and management applications.

Assessing the Environmental Effects Related to Quantum Dot Structure, Function, Synthesis and Exposure.

Quantum dots (QDs) are engineered semiconductor nanocrystals with unique fluorescent, quantum confinement, and quantum yield properties, making them valuable in a range of commercial and consumer imaging, display, and lighting technologies. Production and usage of QDs are increasing, which increases the probability of these nanoparticles entering the environment at various phases of their life cycle. This review discusses the major types and applications of QDs, their potential environmental exposures, fates, and adverse effects on organisms. For most applications, release to the environment is mainly expected to occur during QD synthesis and end-product manufacturing since encapsulation of QDs in these devices prevents release during normal use or landfilling. In natural waters, the fate of QDs is controlled by water chemistry, light intensity, and the physicochemical properties of QDs. Research on the adverse effects of QDs primarily focuses on sublethal endpoints rather than acute toxicity, and the differences in toxicity between pristine and weathered nanoparticles are highlighted. A proposed oxidative stress adverse outcome pathway framework demonstrates the similarities among metallic and carbon-based QDs that induce reactive oxygen species formation leading to DNA damage, reduced growth, and impaired reproduction in several organisms. To accurately evaluate environmental risk, this review identifies critical data gaps in QD exposure and ecological effects, and provides recommendations for future research. Future QD regulation should emphasize exposure and sublethal effects of metal ions released as the nanoparticles weather under environmental conditions. To date, human exposure to QDs from the environment and resulting adverse effects has not been reported.

The effects of temperature and salinity on the endocrinology in two life stages of juvenile rainbow/steelhead trout (Oncorhynchus mykiss).

The San Francisco Bay Delta is experiencing seasonally warmer waters and saltwater intrusion into historically freshwater ecosystems due to climate change. Steelhead/rainbow trout (Oncorhynchus mykiss) are resident in the Bay-Delta from juvenile development through the smoltification process. Due to increases in sea level, premature seawater (SW) acclimation may co-occur with increased temperatures on pre-smolt juveniles. To evaluate the interactive effects of salinity and temperature on juvenile life stages of salmonids, rainbow trout alevin (3 days post-hatching) were exposed to 13, 16.4 and 19°C for 10 days and then challenged for 24 h to 18 parts per thousand SW. Similarly, fry (4 weeks post-hatching) were exposed to 13, 16.4 and 19°C for 2 weeks (14 days) and then challenged to SW. Estradiol-17ß (E2 ), cortisol, triiodothyronine (T3 ) and thyroxine (T4 ) were measured in whole animal homogenates and muscle tissue using enzyme-linked immunosorbent assays. Transcripts of gill Na+ /K+ ATPase ß (NKAα1b), brain growth hormone I (gh1) and brain gonadotropin-releasing hormone receptor 2 (gnrh2) were also measured. Alevin exhibited a significant temperature-dependent decrease in survival, and fry showed a temperature-dependent decrease in condition factor. The gene expression of NKAα1b, gh1 and gnrh2 significantly decreased in all SW-challenged alevin, and a significant decrease in gnrh2 expression was observed in fry with temperature. Alevin T3 and T4 concentrations significantly increased with increasing temperature. There was a temperature-dependent increase in E2 of fry but not of alevin. The results of this study demonstrate that increasing temperature and SW exposure may adversely affect the survival and SW acclimation of alevin and fry stages of salmonids and that the tolerances of younger juvenile stages should be considered when assessing the response of salmonid populations to climate change stressors.

Alteration of neuro-dopamine and steroid hormone homeostasis in wild Bank voles in relation to tissue concentrations of PFAS at a Nordic skiing area.

Perfluoroalkyl substances (PFAS) are contaminants that are applied in a wide range of consumer products, including ski products. The present study investigated the neuro-dopamine (DA) and cellular steroid hormone homeostasis of wild Bank voles (Myodes glareolus) from a skiing area in Norway (Trondheim), in relation to tissue concentrations of PFAS. We found a positive association between brain DA concentrations and the concentration of several PFAS, while there was a negative association between PFAS and dopamine receptor 1 (dr1) mRNA. The ratio between DA and its metabolites (3,4-dihydroxyphenylacetic acid: DOPAC and homovanillic acid: HVA) showed a negative association between DOPAC/DA and several PFAS, suggesting that PFAS altered the metabolism of DA via monoamine oxidase (Mao). This assumption is supported by an observed negative association between mao mRNA and PFAS. Previous studies have shown that DA homeostasis can indirectly regulate cellular estrogen (E2) and testosterone (T) biosynthesis. We found no association between DA and steroid hormone levels, while there was a negative association between some PFAS and T concentrations, suggesting that PFAS might affect T through other mechanisms. The results from the current study indicate that PFAS may alter neuro-DA and steroid hormone homeostasis in Bank voles, with potential consequences on reproduction and general health.

Recessivity of pyrethroid resistance and limited interspecies hybridization across Hyalella clades supports rapid and independent origins of resistance.

Several populations of the amphipod, Hyalella azteca, have developed resistance to pyrethroid insecticides due to non-target exposure, but the dominance of the resistance trait is unknown. The current study investigated the dominance level of point mutations in natural populations of insecticide-resistant H. azteca and determined whether H. azteca from different clades with and without resistant alleles can hybridize and produce viable offspring. A parent generation (P0) of non-resistant homozygous wild type H. azteca was crossbred with pyrethroid-resistant homozygous mutant animals and the tolerance of the filial 1 (F1) generation to the pyrethroid insecticide, permethrin, was measured. Then the genotypes of the F1 generation was examined to assure heterozygosity. The resistant parents had permethrin LC50 values that ranged from 52 to 82 times higher than the non-resistant animals and both crossbreeding experiments produced heterozygous hybrid offspring that had LC50 values similar to the non-resistant H. azteca parent. Dominance levels calculated for each of the crosses showed values close to 0, confirming that the L925I and L925V mutations were completely recessive. The lack of reproduction by hybrids of the C x D breeding confirmed that these clades are reproductively isolated and therefore introgression of adaptive alleles across these clades is unlikely. Potential evolutionary consequences of this selection include development of population bottlenecks, which may arise leading to fitness costs and reduced genetic diversity of H. azteca.

The use of non-targeted metabolomics to assess the toxicity of bifenthrin to juvenile Chinook salmon (Oncorhynchus tshawytscha).

An increase in urban and agricultural application of pyrethroid insecticides in the San Francisco Bay Estuary and Sacramento San Joaquin Delta has raised concern for the populations of several salmonids, including Chinook salmon (Oncorhynchus tshawytscha). Bifenthrin, a type I pyrethroid, is among the most frequently detected pyrethroids in the Bay-Delta watershed, with surface water concentrations often exceeding chronic toxicity thresholds for several invertebrate and fish species. To better understand the mechanisms of bifenthrin-induced neurotoxicity, juvenile Chinook salmon were exposed to concentrations of bifenthrin previously measured in the Delta. Non-targeted metabolomic profiles were used to identify transcriptomic changes in the brains of bifenthrin-exposed fish. Pathway analysis software predicted increased apoptotic, inflammatory, and reactive oxygen species (ROS) responses in Chinook following exposure to 0.15 and 1.50 µg/L bifenthrin for 96 h. These responses were largely driven by reduced levels of inosine, hypoxanthine, and guanosine. Subsequently, in the brain, the expression of caspase 3, a predominant effector for apoptosis, was significantly upregulated following exposure to 1.50 µg/L bifenthrin. This data suggests that metabolites involved in inflammatory and apoptotic responses, as well as those involved in maintaining proper neuronal function may be disrupted following sublethal exposure to bifenthrin and further suggests that additional population studies should focus on behavioral responses associated with impaired brain function.

Mechanisms behind interactive effects of temperature and bifenthrin on the predator avoidance behaviors in parr of chinook salmon (Oncorhynchus tshawytscha).

Many coastal systems have been experiencing the effects of non-chemical and chemical anthropological stressors through respective increases in surface water temperatures and rainstorm-derived runoff events of pyrethroid pesticide movement into waterways such as the San Francisco Bay-Delta. Salmonid populations in the Bay-Delta have been dramatically declining in recent decades. Therefore, the aim of this study was to investigate the interactive effects of bifenthrin, a pyrethroid insecticide, and increasing water temperatures on targeted neuroendocrine and behavioral responses in Chinook salmon (Oncorhynchus tshawytscha) parr (10- month post-hatch). Parr were reared at 11 °C, 16.4 °C, or 19 °C for 14 days and, in the final 96 h of rearing, exposed to nominal concentrations of 0, 0.15, or 1.5 µg/L bifenthrin. A predatory avoidance Y-Maze behavioral assay was conducted immediately following exposures. Parr were presented a choice of clean or odorant zones, and locomotive behavior was recorded. Thyroid hormones (T3 and T4), estradiol, and testosterone were quantified within plasma using ELISAs, and the expression of brain hormone and dopamine receptor genes were also evaluated by qPCR. Brain dopamine levels were analyzed by LC/MS. No significant changes were observed in brain transcripts or plasma hormone concentrations with bifenthrin or increasing temperature. However, temperature did significantly lower brain dopamine levels in fish reared at 19 °C compared to 11 °C controls, but was unaltered by bifenthrin treatment. In contrast, parr reared at 11 °C and exposed to 1.5 µg/L bifenthrin spent significantly less time avoiding a predatory odorant compared to vehicle controls reared at 11 °C. The 16.4 °C and 1.5 µg/L-treated fish spent significantly more time in the neutral arm compared to the odorant and clean arms, as well as spending significantly less time in the clean arm compared to the 11 °C control fish. These results suggest that the interaction of temperature and bifenthrin may be adversely impacting predator-avoidance behavior, which may not be related to dopaminergic responses.

Effects of bifenthrin on sex differentiation in Japanese Medaka (Oryzias latipes).

Bifenthrin (BF) is a pyrethroid insecticide used in urban and agricultural applications. Previous studies in early life stages of fish have indicated anti-estrogenic activity; however, estrogenic activity has been observed in adults. To test the hypothesis that BF impairs sex differentiation, larval Japanese Medaka were exposed to BF during a critical developmental window for phenotypic sexual differentiation. Fish were exposed to environmentally relevant concentrations of BF (0.15 µg/L and 1.5 µg/L), a single concentration (0.3 mg/L) of an estrogen receptor (ER) antagonist (ICI 182,780), and an ER agonist (0.2 ug/L) (17ß-estradiol). Fish were exposed at 8 days post hatch (dph) larvae for 30 days. Phenotypic sex, secondary sexual characteristics (SSC) and genotypic sex were investigated at sexual maturity (8 weeks). A trend towards masculinization (p = 0.06) based on the presence of papillary processes in anal fin rays of Japanese Medaka was observed in fish exposed to the lowest concentration of BF. However, genotypic gender ratios were not altered. These results show sex differentiation was not significantly altered by larval exposure to BF in Japanese medaka.

Effects of Chlorpyrifos on Cholinesterase and Serine Lipase Activities and Lipid Metabolism in Brains of Rainbow Trout (Oncorhynchus mykiss).

Chlorpyrifos is an organophosphorus insecticide that elicits acute toxicity through inhibition of acetylcholinesterase (AChE), leading to acetylcholine accumulation and prolonged stimulation of cholinergic receptors throughout the central and peripheral nervous systems. Previous studies have indicated that neurodevelopment may also be impaired through alternative pathways, including reduction of cyclic adenosine monophosphate (cAMP)-catalyzed downstream events. The upstream initiating events that underlie noncholinergic neurological actions of chlorpyrifos and other organophosphorus compounds remain unclear. To investigate the potential role of fatty acid signaling disruption as a mechanism of toxicity, lipid metabolism and fatty acid profiles were examined to identify alterations that may play a critical role in upstream signaling in the central nervous system (CNS). Juvenile rainbow trout were treated for 7 days with nominal chlorpyrifos concentrations previously reported to diminish olfactory responses (10, 20, and 40 µg/l). Although lethality was noted higher in doses, measured chlorpyrifos concentrations of 1.38 µg/l (nominal concentration 10 µg/l) significantly reduced the activity of AChE and two serine lipases, monoacylglycerol lipase, and fatty acid amide hydrolase in the brain. Reductions in lysophosphatidylethanolamines (16:0, 18:0, 18:1, and 22:6) derived from the phosphatidylethanolamines and free fatty acids (palmitic acid 16:0, linolenic acid 18:3, eicosadienoic acid 20:2, arachidonic acid 20:4, and docosahexaenoic acid 22:6) were also noted, suggesting that chlorpyrifos inhibited the metabolism of select phospholipid signaling precursors at sublethal concentrations. These results indicate that in addition to AChE inhibition, environmentally relevant chlorpyrifos exposure alters serine lipase activity and lipid metabolites in the trout brain, which may compromise neuronal signaling and impact neurobehavioral responses in aquatic animals.

The effects of bifenthrin and temperature on the endocrinology of juvenile Chinook salmon.

The San Francisco Bay delta (USA) is experiencing seasonally warmer waters attributable to climate change and receives rainstorm runoff containing pyrethroid pesticides. Chinook salmon (Oncorhynchus tshawytscha) inhabit the affected waterways from hatch through smoltification, and thus juvenile fish may experience both pyrethroid and warmer water exposures. The effects of higher temperatures and pesticide exposure on presmolt Chinook are unknown. To improve understanding of the potential interaction between temperature and pesticide exposure on salmonid development, juvenile alevin and fry were reared in 11, 16.4, and 19 °C freshwater for 11 d and 2 wk, respectively, and exposed to nominal concentrations of 0, 0.15, and 1.5 µg/L bifenthrin for the final 96 h of rearing. Estradiol-17ß (E2), testosterone, triiodothyronine, and thyroxine levels were measured in whole-body homogenates using hormone-specific enzyme-linked immunosorbent assays. Brain gonadotropin-releasing hormone receptor (GnRH2), dopamine receptor 2A, and growth hormone 1 (GH1) mRNA levels were measured using quantitative PCR. Results showed significantly decreased survival and condition factors observed with increasing temperature in alevin. Alevin thyroid hormones increased significantly with temperature, but fry thyroid hormones trended toward a decrease at lower temperatures with increasing bifenthrin exposure. There were significant reductions in fry testosterone and E2 at 11 °C with increasing bifenthrin treatments and significant changes in GnRH2 and GH1 gene expression in both alevin and fry, indicating potential disruption of hormonal and signaling pathways. Environ Toxicol Chem 2019;38:852-861. © 2019 SETAC.

Changes in thyroid status of Menidia beryllina exposed to the antifouling booster irgarol: Impacts of temperature and salinity.

The triazine-based herbicide irgarol is widely used in antifouling systems as an algicide and has been detected recently in multiple coastal environments. Studies evaluating sub-lethal responses of fish following exposure to irgarol are limited. Moreover, impacts of climate change on fish endocrinology may also contribute to the sublethal toxicity of irgarol. We assessed the effects of irgarol on thyroid endpoints in juveniles of Menidia beryllina under two different treatments of salinity (10 and 20 ‰) and two temperatures (10 and 20°C). Condition factor coefficients (K) of animals were significantly affected by 0.1 to 10 µg/L of irgarol at the higher temperature. Levels of T3 were changed in whole body homogenates from both temperatures at 10‰ following exposure to 1 to 10 µg/L. T4 levels were altered only at 10°C when animals were treated with 1 to 10 µg/L (10 ‰), and in 0.1 and 10 µg/L (20 ‰). Increased transcripts of deiodinase enzymes at 10 °C may be impacted by salinity and alter thyroid hormone homeostasis. Impact on gene expression of thyroid (α and ß) and growth hormone receptors were also determined. Our results highlight the relevance of environmental variable that may impact the ecological risk of irgarol in estuarine systems.

Impacts of Salinity and Temperature on the Thyroidogenic Effects of the Biocide Diuron in Menidia beryllina.

Diuron is a herbicide used in agricultural and urban settings and also as an antifouling agent. Recent studies have indicated sublethal responses of diuron in the endocrine system of fish and amphibians. Given the potential of climate change to also alter fish endocrinology, the combination of environmental stressors with diuron may contribute to its sublethal toxicity. In this study, the effects of temperature and salinity on thyroid targets of diuron were assessed in juveniles of the estuarine fish Menidia beryllina under different conditions of salinity (10 and 20‰) and temperature (10 and 20 °C). Environmentally relevant concentrations of diuron affected the growth, and the higher temperature reduced the condition factor of animals. Increased levels of T3 were observed in fish from all treatments, and at 10 °C, T4 levels were augmented at 10‰ but reduced at 20‰. Increased gene expression of deiodinases at 20‰ in both temperatures suggests the influence of salinity on the regulation of hormone imbalance via deiodination pathway activation. Decreased transcripts of thyroid and growth hormone receptors were also observed following diuron treatment. These results indicate that changes in environmental stressors may have significant impacts on the ecological risk of diuron in estuarine fish.

The effect of chlorpyrifos on salinity acclimation of juvenile rainbow trout (Oncorhynchus mykiss).

As a part of their unique life cycle, most salmonids undergo a transition from fresh water to salt water requiring various adjustments in metabolism, osmoregulation and ion regulation. Exposure to pesticides may affect the acclimation of juvenile salmonids to salt water during downstream migration to estuaries. Using the Caspian Sea as a model waterbody, the present study aimed to determine how the toxicity of the organophosphate pesticide chlorpyrifos (CPF) impacts saline acclimation of rainbow trout (Oncorhynchus mykiss). We pre-exposed 4-month-old fish to nominal concentrations of 0, 20, 40, 80, 160 µg/L of CPF for seven days, and then gradually to salinity (12 ppt) for another seven days. Mortality, levels of cortisol, T3 and T4 in serum, and expression of genes involved in gill ion transport (Na+/K+ATPase α1a and α1b) and liver xenobiotic detoxification (Glutathione-S-Transferase pi, GST) were measured at day fourteen. Cortisol concentrations in serum were not changed by CPF exposure in freshwater, but serum T3 increased up to three fold relative to controls in freshwater. Following salinity acclimation, T3 and T4 concentrations in the serum were both increased up to 2.5 and 8.8 fold in animals treated with CPF followed by saltwater. Na+/K + ATPase α1a and α1b mRNA in gill were unchanged by CPF treatment in freshwater but trended higher in CPF-treated animals after salinity acclimation. Hepatic mRNA of GST was significantly increased following exposure to CPF but was unchanged after saltwater exposure. Although saltwater treatment reduced the acute lethality of CPF, changes in T3/T4 suggest sublethal impacts may occur in CPF-treated fish after they acclimate to Caspian seawater.