The effects of plastic additives on swimming activity and startle response in marine amphipod Echinogammarus marinus.

Plastic additives are widely used in plastic production and are found in the environment owing to their widespread applications. Among these additives, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP) are under international watchlist for evaluation, with limited studies on amphipods. Di-ethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are banned in some countries and categorised as substances of very high concern. This study aimed to investigate the effects of NBBS, TPHP, DEHP and DBP on the swimming activity of a coastal intertidal marine amphipod, Echinogammarus marinus. Furthermore, this study is the first to quantify startle response in E. marinus in response to light stimuli. Amphipods were exposed to 0, 0.5, 5, 50 and 500 µg/l concentrations of all test compounds. Swimming activity and startle responses were assessed by video tracking and analysis using an 8-min alternating dark and light protocol after exposure on days 7 and 14. We observed an overall compound and light effect on the swimming activity of E. marinus. A significant decrease in swimming distance was found in 500 µg/l NBBS and TPHP. We observed that the startle response in E. marinus had a latency period of >2 s and animals were assessed at 1 s and the sum of the first 5 s. There was a clear startle response in E. marinus during dark to light transition, evident with increased swimming distance. NBBS exposure significantly increased startle response at environmental concentrations, while significant effects were only seen in 500 µg/l TPHP at 5 s. We found no significant effects of DEHP and DBP on swimming behaviour at the concentrations assessed. The findings of this study affirm the necessity for a continuous review of plastic additives to combat adverse behavioural effects that may be transferable to the population levels.

Evaluation of precopulatory pairing behaviour and male fertility in a marine amphipod exposed to plastic additives.

Plastics contain a mixture of chemical additives that can leach into the environment and potentially cause harmful effects on reproduction and the endocrine system. Two of these chemicals, N-butyl benzenesulfonamide (NBBS) and triphenyl phosphate (TPHP), are among the top 30 organic chemicals detected in surface and groundwater and are currently placed on international watchlist for evaluation. Although bans have been placed on legacy pollutants such as diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP), their persistence remains a concern. This study aimed to examine the impact of plastic additives, including NBBS, TPHP, DBP, and DEHP, on the reproductive behaviour and male fertility of the marine amphipod Echinogammarus marinus. Twenty precopulatory pairs of E. marinus were exposed to varying concentrations of the four test chemicals to assess their pairing behaviour. A high-throughput methodology was developed and optimised to record the contact time and re-pair time within 15 min and additional point observations for 96 h. The study found that low levels of NBBS, TPHP, and DEHP prolonged the contact and re-pairing time of amphipods and the proportion of pairs reduced drastically with re-pairing success ranging from 75% to 100% in the control group and 0%-85% in the exposed groups at 96 h. Sperm count declined by 40% and 60% in the 50 µg/l and 500 µg/l DBP groups, respectively, whereas TPHP resulted in significantly lower sperms in 50 µg/l exposed group. Animals exposed to NBBS and DEHP showed high interindividual variability in all exposed groups. Overall, this study provides evidence that plastic additives can disrupt the reproductive mechanisms and sperm counts of amphipods at environmentally relevant concentrations. Our research also demonstrated the usefulness of the precopulatory pairing mechanism as a sensitive endpoint in ecotoxicity assessments to proactively mitigate population-level effects in the aquatic environment.

Conflicts of Interest in the Assessment of Chemicals, Waste, and Pollution.

Pollution by chemicals and waste impacts human and ecosystem health on regional, national, and global scales, resulting, together with climate change and biodiversity loss, in a triple planetary crisis. Consequently, in 2022, countries agreed to establish an intergovernmental science-policy panel (SPP) on chemicals, waste, and pollution prevention, complementary to the existing intergovernmental science-policy bodies on climate change and biodiversity. To ensure the SPP's success, it is imperative to protect it from conflicts of interest (COI). Here, we (i) define and review the implications of COI, and its relevance for the management of chemicals, waste, and pollution; (ii) summarize established tactics to manufacture doubt in favor of vested interests, i.e., to counter scientific evidence and/or to promote misleading narratives favorable to financial interests; and (iii) illustrate these with selected examples. This analysis leads to a review of arguments for and against chemical industry representation in the SPP's work. We further (iv) rebut an assertion voiced by some that the chemical industry should be directly involved in the panel's work because it possesses data on chemicals essential for the panel's activities. Finally, (v) we present steps that should be taken to prevent the detrimental impacts of COI in the work of the SPP. In particular, we propose to include an independent auditor's role in the SPP to ensure that participation and processes follow clear COI rules. Among others, the auditor should evaluate the content of the assessments produced to ensure unbiased representation of information that underpins the SPP's activities.

Biomimetic generation of the strongest known biomaterial found in limpet tooth.

The biomaterial with the highest known tensile strength is a unique composite of chitin and goethite (α-FeO(OH)) present in teeth from the Common Limpet (Patella vulgata). A biomimetic based on limpet tooth, with corresponding high-performance mechanical properties is highly desirable. Here we report on the replication of limpet tooth developmental processes ex vivo, where isolated limpet tissue and cells in culture generate new biomimetic structures. Transcriptomic analysis of each developmental stage of the radula, the organ from which limpet teeth originate, identifies sequential changes in expression of genes related to chitin and iron processing. We quantify iron and chitin metabolic processes in the radula and grow isolated radula cells in vitro. Bioinspired material can be developed with electrospun chitin mineralised by conditioned media from cultured radula cells. Our results inform molecular processes behind the generation of limpet tooth and establish a platform for development of a novel biomimetic with comparable properties.

Priorities to inform research on marine plastic pollution in Southeast Asia.

Southeast Asia is considered to have some of the highest levels of marine plastic pollution in the world. It is therefore vitally important to increase our understanding of the impacts and risks of plastic pollution to marine ecosystems and the essential services they provide to support the development of mitigation measures in the region. An interdisciplinary, international network of experts (Australia, Indonesia, Ireland, Malaysia, the Philippines, Singapore, Thailand, the United Kingdom, and Vietnam) set a research agenda for marine plastic pollution in the region, synthesizing current knowledge and highlighting areas for further research in Southeast Asia. Using an inductive method, 21 research questions emerged under five non-predefined key themes, grouping them according to which: (1) characterise marine plastic pollution in Southeast Asia; (2) explore its movement and fate across the region; (3) describe the biological and chemical modifications marine plastic pollution undergoes; (4) detail its environmental, social, and economic impacts; and, finally, (5) target regional policies and possible solutions. Questions relating to these research priority areas highlight the importance of better understanding the fate of marine plastic pollution, its degradation, and the impacts and risks it can generate across communities and different ecosystem services. Knowledge of these aspects will help support actions which currently suffer from transboundary problems, lack of responsibility, and inaction to tackle the issue from its point source in the region. Being profoundly affected by marine plastic pollution, Southeast Asian countries provide an opportunity to test the effectiveness of innovative and socially inclusive changes in marine plastic governance, as well as both high and low-tech solutions, which can offer insights and actionable models to the rest of the world.

Shipping in the north-east Atlantic: Identifying spatial and temporal patterns of change.

Maritime traffic is increasing globally, with a four-fold increase in commercial vessel movements between 1992 and 2012. Vessels contribute to noise and air pollution, provide pathways for non-native species, and collide with marine wildlife. While knowledge of shipping trends and potential environmental impacts exists at both local and global levels, key information on vessel density for regional-scale management is lacking. This study presents the first in-depth spatio-temporal analysis of shipping in the north-east Atlantic region, over three years in a five-year period. Densities increased by 34%, including in 73% of Marine Protected Areas. Western Scotland and the Bay of Biscay experienced the largest increases in vessel density, predominantly from small and slow vessels. Given well-documented impacts that shipping can have on the marine environment, it is crucial that this situation continues to be monitored - particularly in areas designated to protect vulnerable species and ecosystems which may already be under pressure.

Frontiers in quantifying wildlife behavioural responses to chemical pollution.

Animal behaviour is remarkably sensitive to disruption by chemical pollution, with widespread implications for ecological and evolutionary processes in contaminated wildlife populations. However, conventional approaches applied to study the impacts of chemical pollutants on wildlife behaviour seldom address the complexity of natural environments in which contamination occurs. The aim of this review is to guide the rapidly developing field of behavioural ecotoxicology towards increased environmental realism, ecological complexity, and mechanistic understanding. We identify research areas in ecology that to date have been largely overlooked within behavioural ecotoxicology but which promise to yield valuable insights, including within- and among-individual variation, social networks and collective behaviour, and multi-stressor interactions. Further, we feature methodological and technological innovations that enable the collection of data on pollutant-induced behavioural changes at an unprecedented resolution and scale in the laboratory and the field. In an era of rapid environmental change, there is an urgent need to advance our understanding of the real-world impacts of chemical pollution on wildlife behaviour. This review therefore provides a roadmap of the major outstanding questions in behavioural ecotoxicology and highlights the need for increased cross-talk with other disciplines in order to find the answers.

Radiocaesium accumulation and fluctuating asymmetry in the Japanese mitten crab, Eriocheir japonica, along a gradient of radionuclide contamination at Fukushima.

The 2011 Tohoku earthquake-tsunami and the subsequent nuclear accident at the Fukushima Dai-ichi Nuclear Power Station (FDNPS) led to large-scale radionuclide contamination of the marine and freshwater environment. Monitoring studies of marine food products in the Fukushima region have generally demonstrated a declining trend in radiocaesium concentrations. However, the accumulation and elimination of radiocaesium and potential biological effects remain poorly understood for freshwater biota inhabiting highly contaminated areas at Fukushima. Consequently, the present study aimed to assess radiocaesium accumulation and developmental effects on the commercially important catadromous Japanese mitten crab, Eriocheir japonica. E. japonica were collected from four sites along a gradient of radionuclide contamination 4-44 km in distance from the FDNPS in 2017. To determine potential developmental effects, fluctuating asymmetry (FA) was used as a measure of developmental stability. Combined 134Cs and 137Cs values for whole E. japonica from highly contaminated sites 4 and 16 km in distance from the FDNPS were 3040 ± 521 and 2250 ± 908 Bq kg-1 wet weight respectively, 30 and 22 times greater than the Japanese standard limit of 100 Bq kg-1. Estimated total dose rates based on radiocaesium concentrations in whole crabs and sediment ranged from 0.016 to 37.7 µGy h-1. No significant relationship between radiocaesium accumulation and FA was recorded, suggesting that chronic radiation exposure at Fukushima is not inducing developmental effects in E. japonica as measured using fluctuating asymmetry. Furthermore, estimated dose rates were below proposed regulatory limits where significant deleterious effects are expected. The present study will aid in the understanding of the long-term consequences of radiation exposure for non-human biota and the management of radioactively contaminated environments.

High-Throughput Screening of Psychotropic Compounds: Impacts on Swimming Behaviours in Artemia franciscana.

Animal behaviour is becoming increasingly popular as an endpoint in ecotoxicology due to its increased sensitivity and speed compared to traditional endpoints. However, the widespread use of animal behaviours in environmental risk assessment is currently hindered by a lack of optimisation and standardisation of behavioural assays for model species. In this study, assays to assess swimming speed were developed for a model crustacean species, the brine shrimp Artemia franciscana. Preliminary works were performed to determine optimal arena size for this species, and weather lux used in the experiments had an impact on the animals phototactic response. Swimming speed was significantly lower in the smallest arena, whilst no difference was observed between the two larger arenas, suggesting that the small arena was limiting swimming ability. No significant difference was observed in attraction to light between high and low light intensities. Arena size had a significant impact on phototaxis behaviours. Large arenas resulted in animals spending more time in the light side of the arena compared to medium and small, irrespective of light intensity. The swimming speed assay was then used to expose specimens to a range of psychotropic compounds with varying modes of action. Results indicate that swimming speed provides a valid measure of the impacts of behaviour modulating compounds on A. franciscana. The psychotropic compounds tested varied in their impacts on animal behaviour. Fluoxetine resulted in increased swimming speed as has been found in other crustacean species, whilst oxazepam, venlafaxine and amitriptyline had no significant impacts on the behaviours measured. The results from this study suggest a simple, fast, high throughput assay for A. franciscana and gains insight on the impacts of a range of psychotropic compounds on the swimming behaviours of a model crustacean species used in ecotoxicology studies.

The Role of Behavioral Ecotoxicology in Environmental Protection.

For decades, we have known that chemicals affect human and wildlife behavior. Moreover, due to recent technological and computational advances, scientists are now increasingly aware that a wide variety of contaminants and other environmental stressors adversely affect organismal behavior and subsequent ecological outcomes in terrestrial and aquatic ecosystems. There is also a groundswell of concern that regulatory ecotoxicology does not adequately consider behavior, primarily due to a lack of standardized toxicity methods. This has, in turn, led to the exclusion of many behavioral ecotoxicology studies from chemical risk assessments. To improve understanding of the challenges and opportunities for behavioral ecotoxicology within regulatory toxicology/risk assessment, a unique workshop with international representatives from the fields of behavioral ecology, ecotoxicology, regulatory (eco)toxicology, neurotoxicology, test standardization, and risk assessment resulted in the formation of consensus perspectives and recommendations, which promise to serve as a roadmap to advance interfaces among the basic and translational sciences, and regulatory practices.

Unusual male size vs sperm count relationships in a coastal marine amphipod indicate reproductive impairment by unknown toxicants.

Sperm quantity/quality are significant reproductive endpoints with clear links to population level dynamics. Amphipods are important model organisms in environmental toxicology. Despite this, field monitoring of male fertility in invertebrates has rarely been used in monitoring programs. The aim of this study was to compare sperm quality/quantity in an amphipod collected at six UK locations with differing water quality. Due to low sperm counts and an observed lack of relationship between sperm count and weight in amphipods collected from a nationally protected conservation area (Langstone Harbour, England), we also compared datasets from this site over a decade to determine the temporal significance of this finding. One collection to evaluate a female reproductive endpoint was also performed at this site. Interestingly, this harbour consistently presented some of the lowest sperm counts comparable to highly industrial sites and low eggs number from females. Amphipods collected from all the sites, except from Langstone Harbour, presented strong positive correlations between sperm count and weight. Given Langstone Harbour has several international and national protected statutes primarily for marine life and birds, our results indicate that E. marinus, one important food component for wading birds, might be impacted by unknown reproductive stressors. These unknown stressors maybe related to agricultural runoff, leachate from historical landfills and effluent from storm water overflows. This study highlights the importance of exploring new reproductive endpoints such as sperm quantity/quality in marine monitoring programs.

A Crab Is Not a Fish: Unique Aspects of the Crustacean Endocrine System and Considerations for Endocrine Toxicology.

Crustaceans-and arthropods in general-exhibit many unique aspects to their physiology. These include the requirement to moult (ecdysis) in order to grow and reproduce, the ability to change color, and multiple strategies for sexual differentiation. Accordingly, the endocrine regulation of these processes involves hormones, receptors, and enzymes that differ from those utilized by vertebrates and other non-arthropod invertebrates. As a result, environmental chemicals known to disrupt endocrine processes in vertebrates are often not endocrine disruptors in crustaceans; while, chemicals that disrupt endocrine processes in crustaceans are often not endocrine disruptors in vertebrates. In this review, we present an overview of the evolution of the endocrine system of crustaceans, highlight endocrine endpoints known to be a target of disruption by chemicals, and identify other components of endocrine signaling that may prove to be targets of disruption. This review highlights that crustaceans need to be evaluated for endocrine disruption with consideration of their unique endocrine system and not with consideration of the endocrine system of vertebrates.

Feeding Behavioural Studies with Freshwater Gammarus spp.: The Importance of a Standardised Methodology.

Freshwater Gammarids are common leaf-shredding detritivores, and they usually feed on naturally conditioned organic material, in other words leaf litter that is characterised by an increased palatability, due to the action and presence of microorganisms (Chaumot et al. 2015; Cummins 1974: Maltby et al. 2002). Gammarus spp. are biologically omnivorous organisms, so they are involved in shredding leaf litter and are also prone to cannibalism, predation behaviour (Kelly et al. 2002) and coprophagy when juveniles (McCahon and Pascoe 1988). Gammarus spp. is a keystone species (Woodward et al. 2008), and it plays an important role in the decomposition of organic matter (Alonso et al. 2009; Bundschuh et al. 2013) and is also a noteworthy prey for fish and birds (Andrén and Eriksson Wiklund 2013; Blarer and Burkhardt-Holm 2016). Gammarids are considered to be fairly sensitive to different contaminants (Ashauer et al. 2010; Bloor et al. 2005; Felten et al. 2008a; Lahive et al. 2015; Kunz et al. 2010); in fact Amphipods have been reported to be one of the most sensitive orders to metals and organic compounds (Wogram and Liess 2001), which makes them representative test organisms for ecotoxicological studies and valid sentinel species for assessing water quality status (Garcia-Galan et al. 2017).

The effects of wastewater effluent on multiple behaviours in the amphipod, Gammarus pulex.

The prevalence of pharmaceuticals and personal care products (PPCPs) in lotic habitats is increasing, with the main source of these contaminants being effluent from waste water treatment works (WwTW). There is still much uncertainty about the impacts of these PPCPs at environmentally relevant concentrations and their potential effects on aquatic ecology. Behaviour is a sensitive endpoint which can help evaluate possible population level effects from changes in physiology. This paper evaluates the effects of WwTW effluent on a range of behaviours in the freshwater invertebrate, Gammarus pulex. Effluent taken from the outflow of two WwTW in southern England was used in the study. Behavioural analyses, namely feeding rate, phototaxis, activity, velocity and precopula pairing, were measured in G. pulex following a period of one and three weeks after exposure to a 50% or 100% effluent and a control. Mortality remained very low throughout the 3 week experiment (0-10%, n = 20) and no significant changes in moulting frequency were observed (p > 0.05). No significant effects on feeding or velocity or phototaxis following 3 weeks of effluent exposures were observed (p > 0.05). However, significant reductions were observed in the overall activity over 3 weeks across which appeared to be exacerbated by exposure to effluents. Interestingly, males exposed for 3 weeks to WwTW effluent re-paired with unexposed females significantly faster (4-6x) than control animals. This result was consistent between the effluents taken from the two WwTW. The implications of these behavioural changes are currently unknown but highlight the need for a varied set of tools to study the behavioural changes in wildlife.

Endocrine Disruption in Invertebrates: A Survey of Research Progress.

Manmade chemicals can interfere with endocrine processes and have permeated many ecosystems. Arguably, the most devastating example of endocrine disruption occurred in gastropod molluscs which led to the banning of tributyltin. The invertebrates consist of ∼95% of all known animals and possess endocrine systems that can significantly differ from that of vertebrates. An expert group in the late 1990s highlighted considerable paucity in our knowledge of these endocrine systems and the limited ability to ascertain risks of endocrine-disrupting chemicals (EDCs) to invertebrates. Twenty years later, we surveyed experts in this field on the current state of the science. Respondents agreed that endocrine disruption is still a significant issue and noted that there was key evidence that EDCs were impacting invertebrates groups. Respondents noted a variety of impediments to advancing the science, including inadequate funding, insufficient knowledge to develop appropriate assays, and generally low support for invertebrate studies. Several scientists highlighted that resources were being misdirected with studies that address impacts of vertebrate EDCs or using biomarkers specific to vertebrate endocrine disruption. Sadly, many of the recommendations proposed by respondents matched those made over two decades ago. Accordingly, the field has not advanced as much as one might have expected.

Effects of the antidepressant fluoxetine on pigment dispersion in chromatophores of the common sand shrimp, Crangon crangon: repeated experiments paint an inconclusive picture.

The effects of antidepressants in the environment are starting to generate considerable interest due to the fact that neurotransmitters influence a range of biological processes. Crypsis is an important behavioural and physiological response in many crustaceans modulated by monoamine and pigment dispersing/concentrating hormones. This study aimed to develop a test methodology and investigate the effects of the selective serotonin reuptake inhibitor (SSRI), fluoxetine, on a chromatophore index and overall carapace 'darkness' in the common sand shrimp Crangon crangon. Adult shrimp were exposed for either 1 h, 1 day or 1 week across a range of nominal fluoxetine concentrations (10 ng/L, 100 ng/L and 1000 ng/L) and the chromatophore index or carapace percentage 'darkness' was recorded following 30 min on white and black substrates. These experiments were repeated three times using different specimens. Animals became significantly darker (~20%) on darker background and lighter on light backgrounds as one might expect. However, time periods over which the animals were recorded had a significant impact on the colouration suggesting habituation to laboratory conditions. Fluoxetine exposure came up as a significant factor in two of the three trials for the chromatophore index but the results was inconsistent between trials. There was a high degree of correlation between the chromatophore index and the percentage darkness analyses however, there was no significant effects for fluoxetine exposure with the percentage darkness data. We conclude that the effects on antidepressants on colour change remain inconclusive from these experiments and we discuss potential areas to improve the repeatability of the experiments.

Complete transcriptome assembly and annotation of a critically important amphipod species in freshwater ecotoxicological risk assessment: Gammarus fossarum.

Because of their crucial role in ecotoxicological risk assessment, amphipods (Crustacea) are commonly employed as model species in a wide range of studies. However, despite their ecological importance, their genome has not yet been completely annotated and molecular mechanisms underlying key pathways, such as the serotonin pathway, in development of ecotoxicological biomarkers of exposure to neuroactive pharmaceuticals are still poorly understood. Furthermore, genetic similarities and discrepancies with other model arthropods (e.g., Drosophila melanogaster) have not been completely clarified. In this report, we present a new transcriptome assembly of Gammarus fossarum, an important amphipod species, widespread in Central Europe. RNA-Seq with Illumina HiSeq technology was used to analyse samples extracted from total internal tissues. We used the Trinity and Trinotate software suites for transcriptome assembly and annotation, respectively. The quality of this assembly and the affiliated targeted homology searches greatly enrich the molecular knowledge on this species. Because of the lack of publicly available molecular information on the serotonin pathway, we also highlighted sequence homologies and divergences of the genes encoding the serotonin pathway components of the well-annotated arthropod D. melanogaster, and Crustacea with the corresponding genes of our assembly. An inferior number of hits was found when running a BLAST analysis of both D. melanogaster and Crustacea mRNA sequences encoding serotonin receptors available in GenBank against the total assembly, compared to other serotonin pathway components. A lack of information on important components for serotonin biosynthesis and vesicle endocytosis (i.e., tryptophan hydroxylase and vesicular monoamine transporter) in Crustacea was also brought to light. Our results will provide an extensive transcriptional resource for this important species in ecotoxicological risk assessment and highlight the need for a more detailed categorization of neuronal pathways components in invertebrates.

Emerging investigator series: use of behavioural endpoints in the regulation of chemicals.

Interest in behavioural ecotoxicology is growing, partly due to technological and computational advances in recording behaviours but also because of improvements of detection capacity facilitating reporting effects at environmentally relevant concentrations. The peer-reviewed literature now contains studies investigating the effects of chemicals, including pesticides and pharmaceuticals, on migration, dispersal, aggression, sociability, reproduction, feeding and anti-predator behaviours in vertebrates and invertebrates. To understand how behavioural studies could be used in regulatory decision-making we: (1) assessed the legal obstacles to using behavioural endpoints in EU chemicals regulation; (2) analysed the known cases of use of behavioural endpoints in EU chemicals regulation; and (3) provided examples of behavioural endpoints of relevance for population level effects. We conclude that the only legal obstacle to the use of behavioural endpoints in EU chemicals regulation is whether an endpoint is considered to be relevant at the population level or not. We also conclude that ecotoxicity studies investigating behavioural endpoints are occasionally used in the EU chemicals regulation, and underscore that behavioural endpoints can be relevant at the population level. To improve the current use of behavioural studies in regulatory decision-making contribution from all relevant stakeholders is required. We have the following recommendations: (1) researchers should conduct robust, well-designed and transparent studies that emphasize the relevance of the study for regulation of chemicals; (2) editors and scientific journals should promote detailed, reliable and clearly reported studies; (3) regulatory agencies and the chemical industry need to embrace new behavioural endpoints of relevance at the population level.