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.

Influence of the Belgian Coast on Well-Being During the COVID-19 Pandemic.

There is increasing evidence that blue spaces, particularly coastal environments, are beneficial for well-being. During the first-wave lockdown of the COVID-19 pandemic in Belgium, access to the coast was restricted due to restraint in circulation. Making use of this unique opportunity, this study investigated whether access and visits to the coast were positively associated with well-being by using a quasi-experimental design. The emotions of awe and nostalgia were studied as potential mediators between coastal visits and well-being. A total of 687 Flemish adults took part in an online survey that was launched end of April until beginning of June 2020. After controlling for covariates, results showed that access to the coast, but not visit frequency, was positively associated with well-being. More specifically, coastal residents reported less boredom and worry, and more happiness than inland residents. Awe and nostalgia were not significantly associated with coastal visits, but awe was negatively correlated with boredom. The study suggests a potential buffer effect of residential proximity to the coast against negative psychological consequences of the COVID-19 pandemic, supporting the notion that the coast has a positive impact on well-being.

Environmentally relevant concentrations and sizes of microplastic do not impede marine diatom growth.

The current knowledge about the ecological effects of microplastic (MP) remains limited, and to-date ecotoxicity tests often utilize standard microplastic with one or two distinct size classes and expose the organisms to unrealistically high MP concentrations. We exposed the marine diatom Phaeodactylum tricornutum to microplastic particles of a mimicked realistic size frequency distribution complemented with serial experiments with distinct size classes. To do so, we exposed this diatom to a concentration series of different sized polyethylene (PE) microbeads (sizes: 10-106 µm; 1.25 ×102-1.25 ×107 particles/L) in a 72-h growth inhibition test. No effect on the growth of P. tricornutum by virgin PE microbeads up to 1.25 × 107 particles/L (or 499 mg/L), indicating environmentally relevant concentrations and sizes of MP does not alter the growth of marine diatoms. Results of smaller sized MPs (10-20 µm) did not differ from those obtained with larger MPs (90-106 µm) and mix sized MPs (10-106 µm), i.e. no impact on the microalgae growth. As a pioneer work, our results contribute with high quality dose-response data to an improved risk assessment of microplastic under realistic present and future marine MP pollution.

Salinity, dissolved organic carbon, and interpopulation variability hardly influence the accumulation and effect of copper in Mytilus edulis.

To improve the ecological relevance of environmental risk assessment, an improved understanding is needed of 1) the influence of environmental conditions on the toxicity of pollutants, and 2) the effect of these factors in combination with possible interpopulation variability. The influences of salinity and dissolved organic carbon (DOC) on the accumulation and effect of copper (Cu) to settled mussels were investigated with mussels from a North Sea and a Baltic Sea population. We found that both populations were equally Cu-sensitive, even though the Baltic Sea population lives in suboptimal conditions. Baltic Sea mussels, however, accumulated more Cu. This suggests that these populations may have different ways of coping with excess Cu. The influence of salinity on Cu toxicity to settled mussels was limited for both populations. An increase in DOC did not decrease the Cu accumulation or effect in either population. This suggests that DOC-Cu complexes are bioavailable for settled mussels. These findings are in contrast with previous research which indicated that DOC decreased the toxicity and accumulation of Cu in the D-larvae life stage. As a consequence, the mussel larval stage is not the most Cu-sensitive life stage at high DOC concentrations. Furthermore, a DOC correction factor for Cu toxicity cannot be used for settled mussels. This should be accounted for in future marine Cu environmental risk assessment. Environ Toxicol Chem 2017;36:2074-2082. © 2017 SETAC.

Non-lethal heat shock increases tolerance to metal exposure in brine shrimp.

Pollution and temperature increase are two of the most important stressors that aquatic organisms are facing. Exposure to elevated temperatures and metal contamination both induce heat shock proteins (HSPs), which may thus be involved in the induced cross-tolerance in various organisms. This study aimed to test the hypothesis that exposure to a non-lethal heat shock (NLHS) causes an increased tolerance to subsequent metal exposure. Using gnotobiotic cultures of the brine shrimp Artemia franciscana, the tolerance to Cd and Zn acute exposures was tested after a prior NLHS treatment (30min exposure to 37°C). The effects of NLHS and metal exposure were also assessed by measuring 70kDa-HSPs production, along with the analysis of epigenetic markers such as DNA methylation and histone H3 and histone H4 acetylation. Our results showed that heat-shocked Artemia had increased acute tolerance to Cd and Zn. However, different patterns of HSPs were observed between the two metal compounds and no epigenetic alterations were observed in response to heat shock or metal exposure. These results suggest that HSP production is a phenotypically plastic trait with a potential role in temperature-induced tolerance to metal exposure.

Salinity and dissolved organic carbon both affect copper toxicity in mussel larvae: Copper speciation or competition cannot explain everything.

Predicting copper (Cu) toxicity in marine and estuarine environments is challenging because of the influence of anions on Cu speciation, competition between Cu(2+) and other cations at the biotic ligand and the effect of salinity on the physiology of the organism. In the present study the combined effect of salinity and dissolved organic carbon (DOC) on Cu toxicity to larvae of Mytilus galloprovincialis was assessed. Two statistical models were developed and used to elucidate the relationship between Cu toxicity, salinity, and DOC. All models based on dissolved Cu indicate a decrease in Cu toxicity with increasing DOC concentrations, which can partly be explained by complexation of Cu(2+) ions with DOC. These models also indicate an increase in Cu toxicity (modeled with dissolved Cu or Cu(2+) activity) with increasing salinity, suggesting a salinity-induced alteration in the physiology of the mussel larvae. When based on Cu body burdens, neither of the models indicates an effect of salinity or DOC. This shows that the Cu body burden is a more constant predictor of Cu toxicity, regardless of the water chemistry influencing Cu speciation or competition and possible physiological alterations or changes in Cu speciation or competition.

Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype × environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia.

Microplastics are taken up by mussels (Mytilus edulis) and lugworms (Arenicola marina) living in natural habitats.

We studied the uptake of microplastics under field conditions. At six locations along the French-Belgian-Dutch coastline we collected two species of marine invertebrates representing different feeding strategies: the blue mussel Mytilus edulis (filter feeder) and the lugworm Arenicola marina (deposit feeder). Additional laboratory experiments were performed to assess possible (adverse) effects of ingestion and translocation of microplastics on the energy metabolism (cellular energy allocation) of these species. Microplastics were present in all organisms collected in the field: on average 0.2 ± 0.3 microplastics g(-1) (M. edulis) and 1.2 ± 2.8 particles g(-1) (A. marina). In a proof of principle laboratory experiment, mussels and lugworms exposed to high concentrations of polystyrene microspheres (110 particles mL(-1) seawater and 110 particles g(-1) sediment, respectively) showed no significant adverse effect on the organisms' overall energy budget. The results are discussed in the context of possible risks as a result of the possible transfer of adsorbed contaminants.

The combined effect of dissolved organic carbon and salinity on the bioaccumulation of copper in marine mussel larvae.

Larvae of Mytilus spp. are among the most Cu sensitive marine species. In this study we assessed the combined effect of salinity and dissolved organic carbon (DOC) on Cu accumulation on mussel larvae. Larvae were exposed for 48 h to three Cu concentrations in each of nine salinity/DOC treatments. Synchrotron radiation X-ray fluorescence was used to determine the Cu concentration in 36 individual larvae with a spatial resolution of 10 × 10 µm. Cu body burden concentrations varied between 1.1 and 27.6 µg/g DW larvae across all treatments and Cu was homogeneously distributed at this spatial resolution level. Our results indicate decreasing Cu accumulation with increasing DOC concentrations which can be explained by an increase in Cu complexation. In contrast, salinity had a nonlinear effect on Cu. This cannot be explained by copper speciation or competition processes and suggests a salinity-induced alteration in physiology.

Epigenetics in an ecotoxicological context.

Epigenetics can play a role in interactions between chemicals and exposed species, between species and abiotic ecosystem components or between species of the same or another population in a community. Technological progress and advanced insights into epigenetic processes have led to the description of epigenetic features (mainly DNA methylation) in many ecologically relevant species: algae, plants, several invertebrates and fish. Epigenetic changes in plants, insects and cladocerans have been reported to be induced by various environmental stress factors including nutrition or water deficiency, grazing, light or temperature alterations, social environment, and dissolved organic matter concentrations. As regards chemicals, studies in rats and mice exposed to specific pesticides, hydrocarbons, dioxins, and endocrine disrupting chemicals demonstrated the induction of epigenetic changes, suggesting the need for further research with these substances in an ecotoxicological context. In fish and plants, exposure to polyaromatic hydrocarbons, metals, and soluble fractions of solid waste affected the epigenetic status. A novel concept in ecotoxicological epigenetics is the induction of transgenerational stress resistance upon chemical exposure, as demonstrated in rice exposed to metals. Evaluating epigenetics in ecotoxicological field studies is a second relatively new approach. A cryptic lineage of earthworms had developed arsenic tolerance in the field, concurrent with specific DNA methylation patterns. Flatfish caught in the framework of environmental monitoring had developed tumours, exhibiting specific DNA methylation patterns. Two main potential implications of epigenetics in an ecotoxicological context are (1) the possibility of transgenerationally inherited, chemical stress-induced epigenetic changes with associated phenotypes and (2) epigenetically induced adaptation to stress upon long-term chemical exposure. Key knowledge gaps are concerned with the causality of the relation between epigenetic and phenotypic changes, the persistence of transgenerational effects, the implications at population level and the costs of tolerance. Epigenetic changes following exposure to multiple stressors constitute another promising area of further research.

Assessment of marine debris on the Belgian Continental Shelf.

A comprehensive assessment of marine litter in three environmental compartments of Belgian coastal waters was performed. Abundance, weight and composition of marine debris, including microplastics, was assessed by performing beach, sea surface and seafloor monitoring campaigns during two consecutive years. Plastic items were the dominant type of macrodebris recorded: over 95% of debris present in the three sampled marine compartments were plastic. In general, concentrations of macrodebris were quite high. Especially the number of beached debris reached very high levels: on average 6429±6767 items per 100 m were recorded. Microplastic concentrations were determined to assess overall abundance in the different marine compartments of the Belgian Continental Shelf. In terms of weight, macrodebris still dominates the pollution of beaches, but in the water column and in the seafloor microplastics appear to be of higher importance: here, microplastic weight is approximately 100 times and 400 times higher, respectively, than macrodebris weight.

New techniques for the detection of microplastics in sediments and field collected organisms.

Microplastics have been reported in marine environments worldwide. Accurate assessment of quantity and type is therefore needed. Here, we propose new techniques for extracting microplastics from sediment and invertebrate tissue. The method developed for sediments involves a volume reduction of the sample by elutriation, followed by density separation using a high density NaI solution. Comparison of this methods' efficiency to that of a widely used technique indicated that the new method has a considerably higher extraction efficiency. For fibres and granules an increase of 23% and 39% was noted, extraction efficiency of PVC increased by 100%. The second method aimed at extracting microplastics from animal tissues based on chemical digestion. Extraction of microspheres yielded high efficiencies (94-98%). For fibres, efficiencies were highly variable (0-98%), depending on polymer type. The use of these two techniques will result in a more complete assessment of marine microplastic concentrations.

Whole sediment toxicity tests for metal risk assessments: on the importance of equilibration and test design to increase ecological relevance.

Current laboratory-based approaches for predicting metal toxicity in sediments exhibit a number of limitations. The most important are (1) a lack of sufficient equilibration resulting in unrealistically low pH values or unnaturally high porewater metal concentrations and (2) an inadequate test design regarding the metal concentrations selected for spiking. The present study illustrates that by explicitly accounting for these limitations, one obtains reliable and environmentally realistic toxicity data, thus advancing the metal risk assessments of sediments. To this end, a toxicity test design with natural sediments was developed in which the administered metal concentrations were selected to comprise a range of the difference between the molar concentration of simultaneously extracted metals and acid volatile sulfides (SEM-AVS) closely surrounding zero. In addition, the test design presented includes a 35- or 40-d equilibration period with overlying water renewal during which conductivity, pH, and metal concentrations in the overlying water are monitored. This allows toxicity testing to start after equilibrium for these parameters has been reached. This test design was applied to Ephoron virgo (Olivier, 1791), Gammarus pulex (Linnaeus, 1758), and Lumbriculus variegatus (Mueller, 1774) exposed to Zn and Pb. These tests indicated that the general concept of absence of toxicity when SEM-AVS<0 could not be rejected. However, the onset of Zn toxicity occurred at lower concentrations than generally assumed.

Locust phase polyphenism: Does epigenetic precede endocrine regulation?

The morphological, physiological and behavioural differences between solitarious and gregarious desert locusts are so pronounced that one could easily mistake the two phases as belonging to different species, if one has no knowledge of the phenomenon of phenotypic plasticity. A number of phase-specific features are hormonally controlled. Juvenile hormone promotes several solitarious features, the green cuticular colour being the most obvious one. The neuropeptide corazonin elicits the dark cuticular colour that is typical for the gregarious phase, as well as particular gregarious behavioural characteristics. However, it had to be concluded, for multiple reasons, that the endocrine system is not the primary phase-determining system. Our observation that longevity gets imprinted in very early life by crowding of the young hatchlings, and that it cannot be changed thereafter, made us consider the possibility that, perhaps, epigenetic control of gene expression might be, if not the missing, a primary phase-determining mechanism. Imprinting is likely to involve DNA methylation and histone modification. Analysis of a Schistocerca EST database of nervous tissue identified the presence of several candidate genes that may be involved in epigenetic control, including two DNA methyltransferases (Dnmts). Dnmt1 and Dnmt2 are phase-specifically expressed in certain tissues. In the metathoracic ganglion, important in the serotonin pathway for sensing mechanostimulation, their expression is clearly affected by crowding. Our data urge for reconsidering the role of the endocrine system as being sandwiched in between genetics and epigenetics, involving complementary modes of action.

Epigenetics and its implications for ecotoxicology.

Epigenetics is the study of mitotically or meiotically heritable changes in gene function that occur without a change in the DNA sequence. Interestingly, epigenetic changes can be triggered by environmental factors. Environmental exposure to e.g. metals, persistent organic pollutants or endocrine disrupting chemicals has been shown to modulate epigenetic marks, not only in mammalian cells or rodents, but also in environmentally relevant species such as fish or water fleas. The associated changes in gene expression often lead to modifications in the affected organism's phenotype. Epigenetic changes can in some cases be transferred to subsequent generations, even when these generations are no longer exposed to the external factor which induced the epigenetic change, as observed in a study with fungicide exposed rats. The possibility of this phenomenon in other species was demonstrated in water fleas exposed to the epigenetic drug 5-azacytidine. This way, populations can experience the effects of their ancestors' exposure to chemicals, which has implications for environmental risk assessment. More basic research is needed to assess the potential phenotypic and population-level effects of epigenetic modifications in different species and to evaluate the persistence of chemical exposure-induced epigenetic effects in multiple subsequent generations.

Gene transcription profiles, global DNA methylation and potential transgenerational epigenetic effects related to Zn exposure history in Daphnia magna.

A reduced level of DNA methylation has recently been described in both Zn-exposed and non-exposed offspring of Daphnia magna exposed to Zn. The hypothesis examined in this study is that DNA hypomethylation has an effect on gene transcription. A second hypothesis is that accumulative epigenetic effects can affect gene transcription in non-exposed offspring from parents with an exposure history of more than one generation. Transcriptional gene regulation was studied with a cDNA microarray. In the exposed and non-exposed hypomethylated daphnids, a large proportion of common genes were similarly up- or down-regulated, indicating a possible effect of the DNA hypomethylation. Two of these genes can be mechanistically involved in DNA methylation reduction. The similar transcriptional regulation of two and three genes in the F0 and F1 exposed daphnids on one hand and their non-exposed offspring on the other hand, could be the result of a one-generation temporary transgenerational epigenetic effect, which was not accumulative.

Gene transcription and higher-level effects of multigenerational Zn exposure in Daphnia magna.

Zn exposure of Daphnia magna during one generation has been shown to modulate gene transcription differently in Zn exposed organisms compared to their non-exposed offspring. Here we studied the transcriptional gene regulation with a cDNA microarray in D.magna exposed to Zn for three generations (F0-F2). For the first time molecular effects of multigeneration toxicant exposure in D. magna are described. Out of 73 differentially transcribed genes in the F1Zn exposed generation (compared to the F1 control), only seven genes were also differentially transcribed in the same direction in the F0Zn exposed daphnids (up or down, compared to the F0 control). The majority of the differentially transcribed unigenes in F1Zn exposed daphnids (78%) were not differentially transcribed in the F0Zn exposed organisms. This indicates that Zn exposure affected other molecular pathways in the second exposed generation, although a reduced reproduction and a reduction in juvenile growth were observed in both Zn exposed generations, compared to the respective controls. In the third Zn exposed generation (F2), no reduction in growth or reproduction compared to the control was observed. This acclimation was reflected in a significantly lower number of differentially transcribed genes, compared to the Zn exposed F0 and F1 generations.

Direct and transgenerational impact on Daphnia magna of chemicals with a known effect on DNA methylation.

The purpose of this study is to investigate (1) the induction of epigenetic effects in the crustacean Daphnia magna using DNA methylation as an epigenetic mark and (2) the potential stable transfer of such an epigenetic effect to non-exposed subsequent generations. Daphnids were exposed to chemical substances known to affect DNA methylation in mammals: vinclozolin, 5-azacytidine, 2'-deoxy-5-azacytidine, genistein and biochanin A. Effects on overall DNA cytosine methylation, body length and reproduction were evaluated in 21day experiments. Using a multi-generational experimental design these endpoints were also evaluated in the F(1) and F(2) generation of both exposed and non-exposed offspring from F(0) daphnids exposed to 5-azacytidine, genistein or vinclozolin. A reduction in DNA methylation was consistently observed in daphnids exposed to vinclozolin and 5-azacytidine. Only in organisms exposed to 5-azacytidine was this effect transferred to the two subsequent non-exposed generations. A concurrent reduction in body length at day 7 was observed in these treatments. For the first time, exposure to environmental chemicals was shown to affect DNA methylation in the parental generation of D. magna. We also demonstrated a transgenerational alteration in an epigenetic system in D. magna, which indicates the possibility of transgenerational inheritance of environment-induced epigenetic changes in non-exposed subsequent generations.

Can metal stress induce transferable changes in gene transcription in Daphnia magna?

DNA methylation has recently been reported in Daphnia magna, which indicates the possible presence of epigenetic mechanisms regulating gene expression in this species. As such, effects of transient chemical exposure could be transferred through epigenetic inheritance to non-exposed generations. In this study, in the Zn-exposed daphnids, a large number of genes were found to be differentially transcribed, amongst which transcription and translation related genes (downregulated), genes associated with oxidative stress (upregulated) and different types of metabolism-related genes (mostly upregulated). In the two subsequent generations of non-exposed daphnids, a considerable number of differentially regulated genes were observed, indicating an effect of Zn-exposure in the non-exposed progeny. However, none of the differentially transcribed genes observed in the Zn-exposed generation were regulated in the same direction in both non-exposed subsequent generations. The exposure of D. magna to a sublethal Zn concentration for one generation did not result in a stable transgenerational epigenetic effect with consequences for reproductive output nor was a stably epigenetically inheritable effect observed on the transcription of any of the studied genes. An important observation was the large number of genes that were differentially transcribed between different control generations with no pre-exposure history. These genes were not considered in the analysis of the effect of Zn exposure on gene transcription. This differential regulation between subsequent control generations was attributed to possible differences in synchronization of the molting and reproductive cycle of the daphnids in the different generations. This finding is of major importance for the interpretation and design of future microarray experiments with adult Daphnia.

Occurrence of DNA methylation in Daphnia magna and influence of multigeneration Cd exposure.

Most of the research on the epigenetic phenomenon of DNA methylation has been performed with vertebrates and plants. Knowledge on DNA methylation in Daphnia magna, a key test organism in aquatic toxicology, is completely lacking. Through epigenetic inheritance, effects of transient chemical exposure could be transferred to non-exposed generations, which could have a major impact on ecological risk assessment procedures. In this study, we determined if CpG methylation occurs in D. magna and if this can be influenced by exposure to toxic substances. Homologs of human DNA methyltransferases DNMT1, DNMT2 and DNMT3A were found in the partially available D. magna genome. Using an optimized "Amplification of Intermethylated Sites (AIMS)" technique, two methylated fragments were discovered in D. magna DNA. No homology was found for these sequences. The methylation and the D. magna origin of the fragments were confirmed with Southern analysis. This optimized AIMS technique was then applied to DNA of D. magna which were exposed to 180 microg/L Cd for two generations. Exposure resulted in a significant decrease in reproduction. The same methylated fragments with the same band intensity were observed in DNA of both non-exposed and exposed daphnids. As such, it could not be demonstrated that Cd exposure altered DNA methylation. However, the presence of DNA methylation in D. magna shows that potentially epigenetic effects may occur in this species.