General health and residential proximity to the coast in Belgium: Results from a cross-sectional health survey.

The health risks of coastal areas have long been researched, but the potential benefits for health are only recently being explored. The present study compared the general health of Belgian citizens a) according to the EU's definition of coastal (<50 km) vs. inland (>50 km), and b) between eight more refined categories of residential proximity to the coast (<5 km to >250 km). Data was drawn from the Belgian Health Interview Survey (n = 60,939) and investigated using linear regression models and mediation analyses on several hypothesized mechanisms. Results indicated that populations living <5 km of the coast reported better general health than populations living at >50-100 km. Four commonly hypothesized mechanisms were considered but no indirect associations were found: scores for mental health, physical activity levels and social contacts were not higher at 0-5 km from the coast, and air pollution (PM10 concentrations) was lower at 0-5 km from the coast but not statistically associated with better health. Results are controlled for typical variables such as age, sex, income, neighbourhood levels of green and freshwater blue space, etc. The spatial urban-rural-nature mosaic at the Belgian coast and alternative explanations are discussed. The positive associations between the ocean and human health observed in this study encourage policy makers to manage coastal areas sustainably to maintain associated public health benefits into the future.

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.

Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model.

The notion that phenotypic traits emerging from environmental experiences are heritable remains under debate. However, the recent report of nonmendelian transgenerational epigenetic inheritance, i.e., the inheritance of traits not determined by the DNA sequence, might make such a phenomenon plausible. In our study, by carrying out common garden experiments, we could provide clear evidences that, on exposure to nonlethal heat shocks, a parental population of parthenogenetic (all female) Artemia (originating from one single female) experiences an increase in levels of Hsp70 production, tolerance toward lethal heat stress, and resistance against pathogenic Vibrio campbellii. Interestingly, these acquired phenotypic traits were transmitted to three successive generations, none of which were exposed to the parental stressor. This transgenerational inheritance of the acquired traits was associated with altered levels of global DNA methylation and acetylated histones H3 and H4 in the heat-shocked group compared to the control group, where both the parental and successive generations were reared at standard temperature. These results indicated that epigenetic mechanisms, such as global DNA methylation and histones H3 and H4 acetylation, have particular dynamics that are crucial in the heritability of the acquired adaptive phenotypic traits across generations.

Quantification and profiling of lipophilic marine toxins in microalgae by UHPLC coupled to high-resolution orbitrap mass spectrometry.

During the last decade, a significant increase in the occurrence of harmful algal blooms (HABs), linked to repetitive cases of shellfish contamination has become a public health concern and therefore, accurate methods to detect marine toxins in different matrices are required. In this study, we developed a method for profiling lipophilic marine microalgal toxins based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS). Extraction of selected toxins (okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX) and 13-desmethyl spirolide C (SPX-1)) was optimized using a Plackett-Burman design. Three key algal species, i.e., Prorocentrum lima, Protoceratium reticulatum and Alexandrium ostenfeldii were used to test the extraction efficiency of OA, YTXs and SPXs, respectively. Prorocentrum micans, fortified with certified reference solutions, was used for recovery studies. The quantitative and confirmatory performance of the method was evaluated according to CD 2002/657/EC. Limits of detection and quantification ranged between 0.006 and 0.050 ng mL(-1) and 0.018 to 0.227 ng mL(-1), respectively. The intra-laboratory reproducibility ranged from 6.8 to 11.7 %, repeatability from 6.41 to 11.5 % and mean corrected recoveries from 81.9 to 119.6 %. In addition, algae cultures were retrospectively screened for analogues and metabolites through a homemade database. Using the ToxID software programme, 18 toxin derivates were detected in the extract of three toxin producing microalgae species. In conclusion, the generic extraction and full-scan HRMS approach offers an excellent quantitative performance and simultaneously allows to profile analogues and metabolites of marine toxins in microalgae. Graphical Abstract Optimization of extraction, detection and quantification of lipophilic marine toxins in microalgae by UHPLC-HR Orbitrap MS.

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.

Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS.

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion ((13)C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software.

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.

Microplastic detection and identification by Nile red staining: Towards a semi-automated, cost- and time-effective technique.

Microplastic pollution is an issue of concern due to the accumulation rates in the marine environment combined with the limited knowledge about their abundance, distribution and associated environmental impacts. However, surveying and monitoring microplastics in the environment can be time consuming and costly. The development of cost- and time-effective methods is imperative to overcome some of the current critical bottlenecks in microplastic detection and identification, and to advance microplastics research. Here, an innovative approach for microplastic analysis is presented that combines the advantages of high-throughput screening with those of automation. The proposed approach used Red Green Blue (RGB) data extracted from photos of Nile red-fluorescently stained microplastics (50-1200 µm) to train and validate a 'Plastic Detection Model' (PDM) and a 'Polymer Identification Model' (PIM). These two supervised machine learning models predicted with high accuracy the plastic or natural origin of particles (95.8%), and the polymer types of the microplastics (88.1%). The applicability of the PDM and the PIM was demonstrated by successfully using the models to detect (92.7%) and identify (80%) plastic particles in spiked environmental samples that underwent laboratorial processing. The classification models represent a semi-automated, high-throughput and reproducible method to characterize microplastics in a straightforward, cost- and time-effective yet reliable way.

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.

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.

Environmental stability of porcine respiratory coronavirus in aquatic environments.

Coronaviruses (CoVs) are a family of viruses that are best known as the causative agents of human diseases like the common cold, Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS) and COVID-19. CoVs spread by human-to-human transmission via droplets or direct contact. There is, however, concern about potential waterborne transmission of SARS-CoV-2, the virus responsible for COVID-19, as it has been found in wastewater facilities and rivers. To date, little is known about the stability of SARS-CoV-2 or any other free coronavirus in aquatic environments. The inactivation of terrestrial CoVs in seawater is rarely studied. Here, we use a porcine respiratory coronavirus (PRCV) that is commonly found in animal husbandry as a surrogate to study the stability of CoVs in natural water. A series of experiments were conducted in which PRCV (strain 91V44) was added to filtered and unfiltered fresh- and saltwater taken from the river Scheldt and the North Sea. Virus titres were then measured by TCID50-assays using swine testicle cell cultures after various incubation times. The results show that viral inactivation of PRCV in filtered seawater can be rapid, with an observed 99% decline in the viral load after just two days, which may depend on temperature and the total suspended matter concentration. PRCV degraded much slower in filtered water from the river Scheldt, taking over 15 days to decline by 99%, which was somewhat faster than the PBS control treatment (T99 = 19.2 days). Overall, the results suggest that terrestrial CoVs are not likely to accumulate in marine environments. Studies into potential interactions with exudates (proteases, nucleases) from the microbial food web are, however, recommended.

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.

Risk assessment of microplastics in the ocean: Modelling approach and first conclusions.

We performed an environmental risk assessment for microplastics (<5 mm) in the marine environment by estimating the order of magnitude of the past, present and future concentrations based on global plastic production data. In 2100, from 9.6 to 48.8 particles m-3 are predicted to float around in the ocean, which is a 50-fold increase compared to the present-day concentrations. From a meta-analysis with effect data available in literature, we derived a safe concentration of 6650 buoyant particles m-3 below which adverse effects are not likely to occur. Our risk assessment (excluding the potential role of microplastics as chemical vectors) suggests that on average, no direct effects of free-floating microplastics in the marine environment are to be expected up to the year 2100. Yet, even today, the safe concentration can be exceeded in sites that are heavily polluted with buoyant microplastics. In the marine benthic compartment between 32 and 144 particles kg-1 dry sediment are predicted to be present in the beach deposition zone. Despite the scarcity of effect data, we expect adverse ecological effects along the coast as of the second half of the 21st century. From then ambient concentrations will start to outrange the safe concentration of sedimented microplastics (i.e. 540 particles kg-1 sediment). Additional ecotoxicological research in which marine species are chronically exposed to realistic environmental microplastic concentration series are urgently needed to verify our findings.

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.

Soil-solution speciation of Cd as affected by soil characteristics in unpolluted and polluted soils.

Total metal content by itself is insufficient as a measure to indicate actual environmental risk. Understanding the mobility of heavy metals in the soil and their speciation in the soil solution is of great importance for accurately assessing environmental risks posed by these metals. In a first explorative study, the effects of general soil characteristics on Cd mobility were evaluated and expressed in the form of empirical formulations. The most important factors influencing mobility of Cd proved to be pH and total soil content. This may indicate that current legislation expressing the requirement for soil sanitation in Flanders (Belgium) as a function of total soil content, organic matter, and clay does not successfully reflect actual risks. Current legal frameworks focusing on total content, therefore, should be amended with criteria that are indicative of metal mobility and availability and are based on physicochemical soil properties. In addition, soil-solution speciation was performed using two independent software packages (Visual Minteq 2.23 and Windermere Humic Aqueous model VI [WHAM VI]). Both programs largely were in agreement in concern to Cd speciation in all 29 soils under study. Depending on soil type, free ion and the organically complexed forms were the most abundant species. Additional inorganic soluble species were sulfates and chlorides. Minor species in solution were in the form of nitrates, hydroxides, and carbonates, the relative importance of which was deemed insignificant in comparison to the four major species.

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.

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.

In vivo X-ray elemental imaging of single cell model organisms manipulated by laser-based optical tweezers.

We report on a radically new elemental imaging approach for the analysis of biological model organisms and single cells in their natural, in vivo state. The methodology combines optical tweezers (OT) technology for non-contact, laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time. The main objective of this work is to establish a new method for in vivo elemental imaging in a two-dimensional (2D) projection mode in free-standing biological microorganisms or single cells, present in their aqueous environment. Using the model organism Scrippsiella trochoidea, a first proof of principle experiment at beamline ID13 of the European Synchrotron Radiation Facility (ESRF) demonstrates the feasibility of the OT XRF methodology, which is applied to study mixture toxicity of Cu-Ni and Cu-Zn as a result of elevated exposure. We expect that the new OT XRF methodology will significantly contribute to the new trend of investigating microorganisms at the cellular level with added in vivo capability.

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.