Towards reliable data: Validation of a machine learning-based approach for microplastics analysis in marine organisms using Nile red staining.

Microplastic (MP) research faces challenges due to costly, time-consuming, and error-prone analysis techniques. Additionally, the variability in data quality across studies limits their comparability. This study addresses the critical need for reliable and cost-effective MP analysis methods through validation of a semi-automated workflow, where environmentally relevant MP were spiked into and recovered from marine fish gastrointestinal tracts (GITs) and blue mussel tissue, using Nile red staining and machine learning automated analysis of different polymers. Parameters validated include trueness, precision, uncertainty, limit of quantification, specificity, sensitivity, selectivity, and method robustness. For fish GITs a 95 ± 9 % recovery rate was achieved, and 87 ± 11 % for mussels. Polymer identification accuracies were 76 ± 8 % for fish GITs and 80 ± 13 % for mussels. Polyethylene terephthalate fragments showed more variability with lower accuracies. The proposed validation parameters offer a step towards quality management guidelines, as such aiding future researchers and fostering cross-study comparability.

Health risks associated with the consumption of sea turtles: A review of chelonitoxism incidents and the presumed responsible phycotoxins.

Consuming the meat of some marine turtles can lead to a specific type of seafood poisoning known as chelonitoxism. A recent poisoning event (March 2024) on the Tanzanian island Pemba, resulting in the death of 9 people and hospitalization of 78 others, underscores the need to obtain an up to date overview and understanding of chelonitoxism. Here, we document a global overview of poisoning incidents resulting from the consumption of sea turtle flesh worldwide. All events combined involved over 2400 victims and 420 fatalities. Incidents were predominantly reported in remote regions (often islands) across the Indo-Pacific region. Reported health effects of consuming poisonous sea turtles include epigastric pain, diarrhea, vomiting, a burning mouth and throat sensation, and dehydration. In addition, ulcerative oeso-gastro-duodenal lesions, which occasionally have resulted in hospitalization and death, have been reported. Lyngbyatoxins have been suggested as (one of) the causative agents, originating from the cyanobacterium Moorena producens, growing epiphytically on the seagrass and seaweed consumed by green turtles. However, due to the limited evidence of their involvement, the actual etiology of chelonitoxism remains unresolved and other compounds may be responsible. The data outlined in this review offer valuable insights to both regulatory bodies and the general public regarding the potential risks linked to consuming sea turtles.

Temperature dependent sensitivity of the harpacticoid copepod Nitokra spinipes to marine algal toxins.

Harmful algal blooms (HABs) - proliferated algae densities, often producing toxins - have increasingly been found in ocean and coastal areas. Recent studies show that rising temperatures contribute to HAB occurrence, but the broader influence of climate change on these outbreaks is less quantified. Of particular concern is the limited research on HAB toxin effects under varying temperatures, especially regarding primary consumers such as copepods, a crucial component of aquatic ecosystems. Therefore, we examined the impact of marine toxins on the harpacticoid copepod Nitokra spinipes, a model organism for marine ecotoxicology, in the context of climate change. We evaluated the toxicity of four purified, commonly occurring algal toxins, at three different temperatures in the laboratory. First, adult females were exposed to a concentration series of toxins at 15, 20, and 25 °C for 48 h. EC50 values of domoic acid ranged from 8.79 ± 1.93 µg L-1 to 25.97 ± 11.96 µg L-1. Nauplii, aged 48-72 h, were exposed at 18, 20 and 22 °C for the same duration. Less sensitive compared to adults, the EC50 of domoic acid in this case varied from 57.26 ± 6.82 µg L-1 to 97.24 ± 6.45 µg L-1. Both results indicated a temperature-dependent EC50. For the chronic toxicity tests, larval development ratio (LDR), brood size and inter-brood time of domoic acid (DA), yessotoxin (YTX), saxitoxin (STX), and microcystin-LR (MC-LR) were examined at 18, 20 and 22 °C. We observed that with increasing temperatures, LDR increased, whereas brood size significantly decreased as DA, YTX or STX concentrations rose. No interaction between temperature and algal toxins was found but a temperature dependent sensitivity of copepods towards DA, YTX and STX was revealed. Our research provides insights into the effects of long-term exposure to algal toxins on marine copepods and the potential impacts of climate warming.

Aerosolization of micro- and nanoplastics via sea spray: Investigating the role of polymer type, size, and concentration, and potential implications for human exposure.

Micro- and nanoplastics (MNPs) can enter the atmosphere via sea spray aerosols (SSAs), but the effects of plastic characteristics on the aerosolization process are unclear. Furthermore, the importance of the transport of MNPs via these SSAs as a possible new exposure route for human health remains unknown. The aim of this study was two-fold: (1) to examine if a selection of factors affects aerosolization processes of MNPs, and (2) to estimate human exposure to MNPs via aerosols inhalation. A laboratory-based bubble bursting mechanism, simulating the aerosolization process at sea, was used to investigate the influence of MNP as well as seawater characteristics. To determine the potential human exposure to microplastics via inhalation of SSAs, the results of the laboratory experiments were extrapolated to the field based on sea surface microplastic concentrations and the volume of inhaled aerosols. Enrichment seemed to be influenced by MNP size, concentration and polymer type. With higher enrichment for smaller particles and denser polymers. Experiments with different concentrations showed a larger range of variability but nonetheless lower concentrations seemed to result in higher enrichment, presumably due to lower aggregation. In addition to the MNP characteristics, the type of seawater used seemed to influence the aerosolization process. Our human exposure estimate to microplastic via inhalation of sea spray aerosols shows that in comparison with reported inhaled concentrations in urban and indoor environments, this exposure route seems negligible for microplastics. Following the business-as-usual scenario on plastic production, the daily plastic inhalation in coastal areas in 2100 is estimated to increase but remain far below 1 particle per day. This study shows that aerosolization of MNPs is a new plastic transport pathway to be considered, but in terms of human exposure it seems negligible compared to other more important sources of MNPs, based on current reported environmental concentrations.

Contribution of combined stressors on density and gene expression dynamics of the copepod Temora longicornis in the North Sea.

The impact of multiple environmental and anthropogenic stressors on the marine environment remains poorly understood. Therefore, we studied the contribution of environmental variables to the densities and gene expression of the dominant zooplankton species in the Belgian part of the North Sea, the calanoid copepod Temora longicornis. We observed a reduced density of copepods, which were also smaller in size, in samples taken from nearshore locations when compared to those obtained from offshore stations. To assess the factors influencing the population dynamics of this species, we applied generalised additive models. These models allowed us to quantify the relative contribution of temperature, nutrient levels, salinity, turbidity, concentrations of photosynthetic pigments, as well as chemical pollutants such as polychlorinated biphenyls and polycyclic aromatic hydrocarbons (PAHs), on copepod density. Temperature and Secchi depth, a proxy for turbidity, were the most important environmental variables predicting the densities of T. longicornis, followed by summed PAH and chlorophyll concentrations. Analysing gene expression in field-collected adults, we observed significant variation in metabolic and stress-response genes. Temperature correlated significantly with genes involved in proteolytic activities, and encoding heat shock proteins. Yet, concentrations of anthropogenic chemicals did not induce significant differences in the gene expression of genes involved in the copepod's fatty acid metabolism or well-known stress-related genes, such as glutathione transferases or cytochrome P450. Our study highlights the potential of gene expression biomonitoring and underscores the significance of a changing environment in future studies.

Accelerated fragmentation of two thermoplastics (polylactic acid and polypropylene) into microplastics after UV radiation and seawater immersion.

To better understand the fate and assess the ingestible fraction of microplastics (by aquatic organisms), it is essential to quantify and characterize of their released from larger items under environmental realistic conditions. However, the current information on the fragmentation and size-based characteristics of released microplastics, for example from bio-based thermoplastics, is largely unknown. The goal of our work was to assess the fragmentation and release of microplastics, under ultraviolet (UV) radiation and in seawater, from polylactic acid (PLA) items, a bio-based polymer, and from polypropylene (PP) items, a petroleum-based polymer. To do so, we exposed pristine items of PLA and PP, immersed in filtered natural seawater, to accelerated UV radiation for 57 and 76 days, simulating 18 and 24 months of mean natural solar irradiance in Europe. Our results indicated that 76-day UV radiation induced the fragmentation of parent plastic items and the microplastics (50 - 5000 µm) formation from both PP and PLA items. The PP samples (48 ± 26 microplastics / cm2) released up to nine times more microplastics than PLA samples (5 ± 2 microplastics / cm2) after a 76-day UV exposure, implying that the PLA tested items had a lower fragmentation rate than PP. The particles' length of released microplastics was parameterized using a power law exponent (α), to assess their size distribution. The obtained α values were 3.04 ± 0.11 and 2.54 ± 0.06 (-) for 76-day UV weathered PP and PLA, respectively, meaning that PLA microplastics had a larger sized microplastics fraction than PP particles. With respect to their two-dimensional shape, PLA microplastics also had lower width-to-length ratio (0.51 ± 0.17) and greater fiber-shaped fractions (16%) than PP microplastics (0.57 ± 0.17% and 11%, respectively). Overall, the bio-based PLA items under study were more resistant to fragmentation and release of microplastics than the petroleum-based PP tested items, and the parameterized characteristics of released microplastics were polymer-dependent. Our work indicates that even though bio-based plastics may have a slower release of fragmented particles under UV radiation compared to conventional polymer types, they still have the potential to act as a source of microplastics in the marine environment, with particles being available to biota within ingestible size fractions, if not removed before major fragmentation processes.

Assessment of road run-off and domestic wastewater contribution to microplastic pollution in a densely populated area (Flanders, Belgium).

Plastics are omnipresent in our daily life. Unfortunately, the produced plastics will partly end up in the environment including aquatic ecosystems. People often refer to littering or illegal waste dumping as sources of plastic emission to the environment. However, daily-life sources could also, unknowingly, contribute considerably to the total microplastic pollution in the ecosystem. Hence, there is an urgent need to study these potential sources. In this research, two common sources, i.e. domestic wastewater and road run-off from tire and road wear particles, were studied in detail to quantify the relative contribution of both domestic sources towards microplastic pollution in freshwater ecosystems in Flanders, Belgium. This assessment shows that every person (in studied area) emits on average 1145 microplastics (25-1000 µm) daily through domestic wastewater, resulting in a yearly discharge of 418,000 microplastic particles per person. The road run-off samples contained between 0.02 and 9.2 mg tire wear particles per litre per day, which corresponds to an emission of 10.8 mg tire wear particles per driven vehicle km. The gross and net emissions of both above mentioned microplastic sources were extrapolated to the whole Flanders region using an emission model. From the yearly gross microplastic pollution in the domestic wastewater, 623 kg (20%) will be discharged in the freshwater. The highest losses originated from the households that have a private drain or are not (yet) connected to an active wastewater treatment plant. In Flanders, the yearly net microplastic emission into the aquatic environment of tire wear particles is estimated to be 246 tonnes (38%), mainly from the direct run-off from the road surface. Based on the results, specific mitigation measures can be installed to reduce the emission of microplastics towards the freshwater ecosystem. Other sources should be quantified in a similar way for a more holistic strategy to counteract plastic pollution.

Decadal decline of dominant copepod species in the North Sea is associated with ocean warming: Importance of marine heatwaves.

Time-series are crucial to understand the status of zooplankton communities and to anticipate changes that might affect the entire food web. Long-term time series allow us to understand impacts of multiple environmental and anthropogenic stressors, such as chemical pollution and ocean warming, on the marine ecosystems. Here, a recent time series (2018-2022) of abundance data of four dominant calanoid and one harpacticoid copepod species from the Belgian Part of the North Sea was combined with previously collected (2009-2010, 2015-2016) datasets for the same study area. The time series reveals a significant decrease (up to two orders of magnitude) in calanoid copepod abundance (Temora longicornis, Acartia clausi, Centropages spp., Calanus helgolandicus), while this was not the case for the harpacticoid Euterpina acutifrons. We applied generalized additive models to quantify the relative contribution of temperature, nutrients, salinity, primary production, turbidity and pollution (anthropogenic chemicals, i.e., polychlorinated biphenyls and polycyclic aromatic hydrocarbons) to the population dynamics of these species. Temperature, turbidity and chlorophyll a concentrations were the only variables consistently showing a relative high contribution in all models predicting the abundances of the selected species. The observed heat waves which occurred during the summer periods of the investigated years coincided with population collapses (versus population densities in non-heatwave years) and are considered the most likely cause for the observed copepod abundance decreases. Moreover, the recorded water temperatures during these heatwaves correspond to the physiological thermal limit of some of the studied species. As far as we know, this is the first study to observe ocean warming and marine heat waves having such a dramatic impact (population collapse) on the dominant zooplankton species in shallow coastal areas.

Micro- and nanoplastics transfer from seawater to the atmosphere through aerosolization under controlled laboratory conditions.

Sea spray has been suggested to enable the transfer of micro- and nanoplastics (MNPs) from the ocean to the atmosphere, but only a few studies support the role of sea spray aerosols (SSAs) as a source of airborne particles. We demonstrated that MNPs are aerosolized during wave action, via SSAs, under controlled laboratory conditions. We used a mini-Marine-Aerosol-Reference-Tank (miniMART), a device that mimics naturally occurring physical mechanisms producing SSAs, and assessed the aerosolization of fluorescent polystyrene beads (0.5-10 µm), in artificial seawater. The SSAs contained up to 18,809 particles/mL of aerosols for 0.5 µm beads, with an enrichment factor of 19-fold, and 1977 particles/mL of aerosols for 10 µm beads with a 2-fold enrichment factor. Our study demonstrates that the use of the miniMART is essential to assess MNPs aerosolization in a standardized way, supporting the hypothesis which states that MNPs in the surface of the ocean may be transferred to the atmosphere.

Synthesizing the impact of sea-dumped munition and related chemicals on humans and the environment.

Marine environments are globally impacted by vast quantities of munition disposed following both World Wars. Dumped munitions contain conventional explosives, chemicals warfare agents as well as a variety of metals. Field monitoring studies around marine dumpsites report the presence of munition constituents in water and sediment samples. The growing interest and developments in the ocean as a new economic frontier underline the need to remediate existing dumpsites. Here, we provide a comprehensive assessment of the magnitude and potential risks associated with marine munition dumpsites. An overview of the global distribution of dumpsites identifying the most impacted areas is provided, followed by the currently available data on the detection of munition constituents in environmental samples and evidence of their toxic potential to human and environmental health. Finally, existing data gaps are identified and future research needs promoting better understanding of the impact of the dumped material on the marine environment suggested.

Toward a Better Understanding of the Contribution of Wastewater Treatment Plants to Microplastic Pollution in Receiving Waterways.

Microplastics (1 µm-5 mm), are ubiquitous in daily-use products and regularly end up in the wastewater. The main part of the wastewater is treated in wastewater treatment plants (WWTPs), which allow for at least partial removal of microplastics. The present study aimed to understand the contribution of domestic wastewater to microplastic pollution in Flanders (Belgium) via two main discharge routes of microplastics: (1) the effluent, and (2) removed fractions. Furthermore the effect of effluent discharge on the microplastic contamination in the waterway was studied in both surface water and sediment samples of upstream and downstream locations of the discharge from three WWTPs. On average, 12.64 ± 20.20 microplastic/L entered a WWTP (10 µm-5 mm). The effluent contained on average 0.41 ± 0.91 microplastic/L, resulting in an average removal efficiency of 97.46% ± 2.33%, which is comparable with various (non-)European countries. Removal efficiencies are both polymer- and size-specific, and data suggest that smaller particles are less efficiently removed from the wastewater, which also causes an increased input of smaller particles to the environment. The sludge is the most efficient treatment process to remove microplastics. Despite the high removal efficiencies, still 1.11 × 107 ± 3.07 × 107 microplastics end up in the nearby waterway daily. Nonetheless, based on the results gathered in the present study, this does not seem to impact the microplastic concentration in the waterway significantly. In summary, the present study offers a holistic approach in the research on the impact of wastewater on microplastic pollution in the ecosystem, integrating different discharge routes and measuring the impact on environmental microplastic pollution. Environ Toxicol Chem 2023;42:642-654. © 2022 SETAC.

Indole decreases the virulence of the bivalve model pathogens Vibrio tasmaniensis LGP32 and Vibrio crassostreae J2-9.

Indole signaling plays an important role in bacterial pathogenesis. In this study, the impact of indole on biofilm formation, swimming and swarming motility were explored in Vibrio tasmaniensis LGP32 and Vibrio crassostreae J2-9, two model pathogens of bivalves. The results showed that indole decreased swimming and swarming motility in both strains, and decreased biofilm formation in V. crassostreae J2-9. Furthermore, indole affected a large number of genes at RNA level, including genes related to metabolism, ABC transporters, flagellar assembly, chemotaxis, and response regulators. Finally, the bacterial virulence towards mussel larvae was decreased by pretreatment with indole in both V. tasmaniensis LGP32 and V. crassostreae J2-9. After 5 days, the survival rate of mussel larvae increased 2.4-fold and 2.8-fold in mussel larvae challenged with V. tasmaniensis LGP32 pretreated with 200 µM and 500 µM indole, respectively. The survival rate of mussel larvae increased 1.5-fold and 1.9-fold in mussel larvae challenged with V. crassostreae J2-9 pretreated with 200 µM and 500 µM indole, respectively. These data indicate that indole has a significant impact on the virulence of V. tasmaniensis LGP32 and V. crassostreae J2-9, and indole signaling could be a promising target for antivirulence therapy.

Effects of Microplastic on the Population Dynamics of a Marine Copepod: Insights from a Laboratory Experiment and a Mechanistic Model.

Microplastic is ubiquitously and persistently present in the marine environment, but knowledge of its population-level effects is limited. In the present study, to quantify the potential theoretical population effect of microplastic, a two-step approach was followed. First, the impact of microplastic (polyethylene, 0.995 g cm-3 , diameter 10-45 µm) on the filtration rate of the pelagic copepod Temora longicornis was investigated under laboratory conditions. It was found that the filtration rate decreased at increasing microplastic concentrations and followed a concentration-response relationship but that at microplastic concentrations <100 particles L-1 the filtration rate was not affected. From the concentration-response relationship between the microplastic concentrations and the individual filtration rate a median effect concentration of the individual filtration rate (48 h) of 1956 ± 311 particles L-1 was found. In a second step, the dynamics of a T. longicornis population were simulated for realistic environmental conditions, and the effects of microplastics on the population density equilibrium were assessed. The empirical filtration rate data were incorporated in an individual-based model implementation of the dynamic energy budget theory to deduct potential theoretical population-level effects. The yearly averaged concentration at which the population equilibrium density would decrease by 50% was 593 ± 376 particles L-1 . The theoretical effect concentrations at the population level were 4-fold lower than effect concentrations at the individual level. However, the theoretical effect concentrations at the population level remain 3-5 orders of magnitude higher than ambient microplastic concentrations. Because the present experiment was short-term laboratory-based and the results were only indirectly validated with field data, the in situ implications of microplastic pollution for the dynamics of zooplankton field populations remain to be further investigated. Environ Toxicol Chem 2022;41:1663-1674. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Sea Spray Aerosols Contain the Major Component of Human Lung Surfactant.

Marine phytoplankton influence the composition of sea spray aerosols (SSAs) by releasing various compounds. The biogenic surfactant dipalmitoylphosphatidylcholine (DPPC) is known to accumulate in the sea surface microlayer, but its aerosolization has never been confirmed. We conducted a 1 year SSA sampling campaign at the Belgian coast and analyzed the SSA composition. We quantified DPPC at a median and maximum air concentration of 7.1 and 33 pg m-3, respectively. This discovery may be of great importance for the field linking ocean processes to human health as DPPC is the major component of human lung surfactant and is used as excipient in medical aerosol therapy. The natural airborne exposure to DPPC seems too low to induce direct human health effects but may facilitate the effects of other marine bioactive compounds. By analyzing various environmental variables in relation to the DPPC air concentration, using a generalized linear model, we established that wave height is a key environmental predictor and that it has an inverse relationship. We also demonstrated that DPPC content in SSAs is positively correlated with enriched aerosolization of Mg2+ and Ca2+. In conclusion, our findings are not only important from a human health perspective but they also advance our understanding of the production and composition of SSAs.

Phycotoxin-Enriched Sea Spray Aerosols: Methods, Mechanisms, and Human Exposure.

To date, few studies have examined the role of sea spray aerosols (SSAs) in human exposure to harmful and beneficial marine compounds. Two groups of phycotoxins (brevetoxins and ovatoxins) have been reported to induce respiratory syndromes during harmful algal blooms. The aerosolization and coastal air concentrations of other common marine phycotoxins have, however, never been examined. This study provides the first (experimental) evidence and characterization of the aerosolization of okadaic acid (OA), homoyessotoxin, and dinophysistoxin-1 using seawater spiked with toxic algae combined with the realistic SSA production in a marine aerosol reference tank (MART). The potential for aerosolization of these phycotoxins was highlighted by their 78- to 1769-fold enrichment in SSAs relative to the subsurface water. To obtain and support these results, we first developed an analytical method for the determination of phycotoxin concentrations in SSAs, which showed good linearity (R2 > 0.99), recovery (85.3-101.8%), and precision (RSDs ≤ 17.2%). We also investigated natural phycotoxin air concentrations by means of in situ SSA sampling with concurrent aerosolization experiments using natural seawater in the MART. This approach allowed us to indirectly quantify the (harmless) magnitude of OA concentrations (0.6-51 pg m-3) in Belgium's coastal air. Overall, this study provides new insights into the enriched aerosolization of marine compounds and proposes a framework to assess their airborne exposure and effects on human health.

Spatio-temporal patterns in the gene expression of the calanoid copepod Temora longicornis in the Belgian part of the North Sea.

Marine zooplankton are increasingly being affected by recent environmental changes, such as climate change, and respond with profound spatial relocations and shifts in phenology and physiology. In order to predict whether populations are able to persist or adapt to such new conditions, it is essential to understand the molecular basis of such adaptations, which ultimately get translated into these physiological responses. To explore variation in population gene expression across time and space, we investigated transcriptome-level profiles of the calanoid copepod Temora longicornis, that were collected at four different locations in the Belgian Part of the North Sea (BPNS) on three different time points (April, June, October) in 2018. RNA-seq analysis of field collected adults identified large seasonal differences in gene expression, mainly between spring-summer and autumn samples. The largest log-fold changes occurred in a set of genes encoding for ribosomal and myosin (heavy chain) transcripts. Enrichment analysis revealed a strong seasonal pattern in vitellogenin, cuticle and glycolytic gene expression as well. We also found a positive correlation between vitellogenin expression and densities of T. longicornis. No clear spatial variation in expression patterns was found in the BPNS. This study underlines the potential of field gene expression studies for biomonitoring purposes and the significance of considering seasonal variation in future studies.

Aerosolizable Marine Phycotoxins and Human Health Effects: In Vitro Support for the Biogenics Hypothesis.

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L-1 - mg·L-1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.

Acute and Chronic Toxicity of Cobalt to Freshwater Organisms: Using a Species Sensitivity Distribution Approach to Establish International Water Quality Standards.

Water quality standards for cobalt (Co) have not been developed for the European Union or United States. The objective of the present study was to produce freshwater Co toxicity data that could be used by both the European Union and the United States to develop appropriate regulatory standards (i.e., environmental quality standards or predicted-no-effect concentrations in Europe and ambient water quality criteria or state water quality standards in the United States). Eleven species, including algae, an aquatic plant, and several invertebrate and fish species, were used in the performance of acute and chronic Co toxicity tests. Acute median lethal or median effective concentration (LC50 or EC50) values ranged from 90.1 µg Co/L for duckweed (Lemna minor) to 157 000 µg Co/L for midges (Chironomus tentans). Chronic 10% effect concentration (EC10) values ranged from 4.9 µg Co/L for duckweed to 2170 µg Co/L for rainbow trout (Oncorhynchus mykiss). Chronic 20% effect concentration (EC20) values ranged from 11.1 µg Co/L for water flea (Ceriodaphnia dubia) to 2495 µg Co/L for O. mykiss. Results indicated that invertebrate and algae/plant species are more sensitive to chronic Co exposures than fish. Acute-to-chronic ratios (derived as acute LC50s divided by chronic EC20s) were lowest for juvenile O. mykiss (0.6) and highest for the snail Lymnaea stagnalis (2670). Following the European-based approach and using EC10 values, species sensitivity distributions (SSDs) were developed and a median hazardous concentration for 5% of the organisms of 1.80 µg Co/L was derived. Chronic EC20 values were used, also in an SSD approach, to derive a US Environmental Protection Agency-style final chronic value of 7.13 µg Co/L. Environ Toxicol Chem 2020;39:799-811. © 2020 SETAC.

Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community.

The implementation of cost-effective monitoring programs for zooplankton remains challenging due to the requirements of taxonomical expertise and the high costs of sampling and species identification. To reduce costs, molecular methods have been proposed as alternatives to morphology-based monitoring. Metatranscriptomics can contribute to promote both cost-effectiveness and accuracy of biological assessments of aquatic ecosystems. Here, we describe and evaluate the construction of a metatranscriptome dataset from a pelagic crustacean zooplankton community. We sampled zooplankton in one marine station, named LW02, in the North Sea, in both winter and summer, and generated transcripts using Oxford Nanopore Technology (ONT), a third-generation nanopore-based sequencing technology. ONT is, uniquely, capable of sequencing RNA directly, rather than depending on reverse transcription and PCR, and applicable to be used directly in the field. We found that metatranscriptomics is capable of species detection, including screening for the presence of endoparasites, hence competing with morphological identification. Taxonomic analysis based on ribosomal 18S transcripts identified calanoid copepods, particularly Temora longicornis and Acartia clausi, as the most abundant community members. Moreover, up to 40.4% and 50.5% of all sequences could be assigned to predicted genes in the winter and summer sample, respectively. The most abundant mRNA transcripts with known function coded for essential metabolic processes. GO term annotation revealed that genes involved in glycolytic and translation-related processes were most expressed in the community. Although small in scale, our study provides the basis for future efforts to characterize the metatranscriptome of marine zooplankton communities and its application in biomonitoring programs.