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.

Probing Long-Term Impacts: Low-Dose Polystyrene Nanoplastics Exacerbate Mitochondrial Health and Evoke Secondary Glycolysis via Repeated and Single Dosing.

Nanoplastics (NPs) are omnipresent in the environment and contribute to human exposure. However, little is known regarding the long-term effects of NPs on human health. In this study, human intestinal Caco-2 cells were exposed to polystyrene nanoplastics (nanoPS) in an environmentally relevant concentration range (102-109 particles/mL) under two realistic exposure scenarios. In the first scenario, cells were repeatedly exposed to nanoPS every 2 days for 12 days to study the long-term effects. In the second scenario, only nanoPS was added once and Caco-2 cells were cultured for 12 days to study the duration of the initial effects of NPs. Under repeated dosing, initial subtle effects on mitochondria induced by low concentrations would accrue over consistent exposure to nanoPS and finally lead to significant impairment of mitochondrial respiration, mitochondrial mass, and cell differentiation process at the end of prolonged exposure, accompanied by significantly increased glycolysis over the whole exposure period. Single dosing of nanoPS elicited transient effects on mitochondrial and glycolytic functions, as well as increased reactive oxygen species (ROS) production in the early phase of exposure, but the self-recovery capacity of cells mitigated these effects at intermediate culture times. Notably, secondary effects on glycolysis and ROS production were observed during the late culture period, while the cell differentiation process and mitochondrial mass were not affected at the end. These long-term effects are of crucial importance for comprehensively evaluating the health hazards arising from lifetime exposure to NPs, complementing the extensively observed acute effects associated with prevalent short-term exposure to high concentrations. Our study underlines the need to study the toxicity of NPs in realistic long-term exposure scenarios such as repeated dosing.

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.

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.

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.

Characteristics and Sinking Behavior of Typical Microplastics Including the Potential Effect of Biofouling: Implications for Remediation.

Microplastics are ubiquitous pollutants within the marine environment, predominantly (>90%) accumulating in sediments worldwide. Despite the increasing global concern regarding these anthropogenic pollutants, research into the remediation of microplastics is lacking. Here, we examine those characteristics of microplastics that are essential to adequately evaluate potential remediation techniques such as sedimentation and (air) flotation techniques. We analyzed the sinking behavior of typical microplastics originating from real plastic waste samples and identified the best-available drag model to quantitatively describe their sinking behavior. Particle shape is confirmed to be an important parameter strongly affecting the sinking behavior of microplastics. Various common shape descriptors were experimentally evaluated on their ability to appropriately characterize frequently occurring particle shapes of typical microplastics such as spheres, films, and fibers. This study is the first in this field to include film particles in its experimental design, which were found to make up a considerable fraction of marine pollution and are shown to significantly affect the evaluation of shape-dependent drag models. Circularity χ and sphericity Φ are found to be appropriate shape descriptors in this context. We also investigated the effect of biofouling on the polarity of marine plastics and estimated its potential contribution to the settling motion of initially floating microplastics based on density-modification. It is found that biofouling alters the polarity of plastics significantly; this is from (near) hydrophobic (i.e., water contact angles from 70 to 100°) to strong hydrophilic (i.e., water contact angles from 30 to 40°) surfaces, rendering them more difficult to separate from sediment based on polarity as a primary separation factor. Thus, besides providing a better understanding of the fate and behavior of typical marine microplastics, these findings serve as a fundamental stepping-stone to the development of the first large-scale sediment remediation technique for microplastics to address the global microplastic accumulation issue.

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.

Biodiversity effects on ecosystem functioning respond unimodally to environmental stress.

Understanding how biodiversity (B) affects ecosystem functioning (EF) is essential for assessing the consequences of ongoing biodiversity changes. An increasing number of studies, however, show that environmental conditions affect the shape of BEF relationships. Here, we first use a game-theoretic community model to reveal that a unimodal response of the BEF slope can be expected along environmental stress gradients, but also how the ecological mechanisms underlying this response may vary depending on how stress affects species interactions. Next, we analysed a global dataset of 44 experiments that crossed biodiversity with environmental conditions. Confirming our main model prediction, the effect of biodiversity on ecosystem functioning tends to be greater at intermediate levels of environmental stress, but varies among studies corresponding to differences in stress-effects on species interactions. Together, these results suggest that increases in stress from ongoing global environmental changes may amplify the consequences of biodiversity changes.

Selective and context-dependent effects of chemical stress across trophic levels at the basis of marine food webs.

Human activities increasingly impact the functioning of marine food webs, but anthropogenic stressors are seldom included in ecological study designs. Diet quality, as distinct from just diet quantity, has moreover rarely been highlighted in food web studies in a stress context. We measured the effects of metal and pesticide stress (copper and atrazine) on the contribution of a benthic intertidal diatom community to two processes that are key to the functioning of intertidal systems: biomass (diet quantity) and lipid (diet quality) production. We then examined if stressors affected diatom functioning by selectively targeting the species contributing most to functioning (selective stress effects) or by changing the species' functional contribution (context-dependent effects). Finally, we tested if stress-induced changes in diet quality altered the energy flow to the diatoms' main grazers (harpacticoid copepods). Diatom diet quantity was reduced by metal stress but not by low pesticide levels due to the presence of an atrazine-tolerant, mixotrophic species. Selective effects of the pesticide reduced diatom diet quality by 60% and 75% at low and high pesticide levels respectively, by shifting diatom community structure from dominance by lipid-rich species toward dominance by an atrazine-tolerant, but lipid-poor, species. Context-dependent effects did not affect individual diatom lipid content at low levels of both stressors, but caused diatoms to lose 40% of their lipids at high copper stress. Stress-induced changes in diet quality predicted the energy flow from the diatoms to their copepod consumers, which lost half of their lipids when feeding on diatoms grown under low and high pesticide and high metal stress. Selective pesticide effects were a more important threat for trophic energy transfer than context-dependent effects of both stressors, with shifts in diatom community structure affecting the energy flow to their copepod grazers at stress levels where no changes in diatom lipid content were detected.

Integrated Three-Dimensional Microanalysis Combining X-Ray Microtomography and X-Ray Fluorescence Methodologies.

A novel 3D elemental and morphological analysis approach is presented combining X-ray computed tomography (µCT), X-ray fluorescence (XRF) tomography, and confocal XRF analysis in a single laboratory instrument (Herakles). Each end station of Herakles (µCT, XRF-CT, and confocal XRF) represents the state-of-the-art of currently available laboratory techniques. The integration of these techniques enables linking the (quantitative) spatial distribution of chemical elements within the investigated materials to their three-dimensional (3D) internal morphology/structure down to 1-10 µm resolution level, which has not been achieved so-far using laboratory X-ray techniques. The concept of Herakles relies strongly on its high precision (around 100 nm) air-bearing motor system that connects the different end-stations, allowing combined measurements based on the above X-ray techniques while retaining the coordinate system. In-house developed control and analysis software further ensures a smooth integration of the techniques. Case studies on a Cu test pattern, a Daphnia magna model organism and a perlite biocatalyst support material demonstrate the attainable resolution, elemental sensitivity of the instrument, and the strength of combining these three complementary methodologies.

The Consequences of Nonrandomness in Species-Sensitivity in Relation to Functional Traits for Ecosystem-Level Effects of Chemicals.

Estimating ecosystem-level effects from single-species bioassays is a major challenge in environmental risk assessment. Most extrapolation procedures are based on the implicit assumption that species sensitivities are random with regard to their functional traits. Here, we explore how nonrandomness in species sensitivities affects how species-level and ecosystem level effects of chemical exposure correspond. The effect of a correlation between the trait value under control conditions and the sensitivity of the trait to chemical stress is studied for two traits (per capita growth rate and monoculture yield) under constant and temporary exposure. Theoretical model predictions are thereby validated against a 3-week microcosm experiment, in which eight marine diatoms systems with different correlations between trait values and sensitivities were temporary (1 week) or constantly (3 weeks) exposed to two concentrations of the herbicide atrazine (100 and 250 µg L-1). Negative correlations increased the reduction in ecosystem functioning (productivity) by atrazine for both traits. However, correlations in the per capita growth rate affected productivity only shortly following changes in environmental conditions, whereas correlations in the monoculture yield affected productivity throughout exposure. Correlations between species sensitivities and functional trait values can thus help to identify when ecosystem-level effects are likely to exceed species-level effects.

Bisulfite Sequencing with Daphnia Highlights a Role for Epigenetics in Regulating Stress Response to Microcystis through Preferential Differential Methylation of Serine and Threonine Amino Acids.

Little is known about the influence that environmental stressors may have on genome-wide methylation patterns, and to what extent epigenetics may be involved in environmental stress response. Yet, studies of methylation patterns under stress could provide crucial insights on stress response and toxicity pathways. Here, we focus on genome-wide methylation patterns in the microcrustacean Daphnia magna, a model organism in ecotoxicology and risk assessment, exposed to the toxic cyanobacterium Microcystis aeruginosa. Bisulfite sequencing of exposed and control animals highlighted differential methylation patterns in Daphnia upon exposure to Microcystis primarily in exonic regions. These patterns are enriched for serine/threonine amino acid codons and genes related to protein synthesis, transport and degradation. Furthermore, we observed that genes with differential methylation corresponded well with genes susceptible to alternative splicing in response to Microcystis stress. Overall, our results suggest a complex mechanistic response in Daphnia characterized by interactions between DNA methylation and gene regulation mechanisms. These results underscore that DNA methylation is modulated by environmental stress and can also be an integral part of the toxicity response in our study species.

Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 µg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions.

Laboratory Scale X-ray Fluorescence Tomography: Instrument Characterization and Application in Earth and Environmental Science.

A new laboratory scale X-ray fluorescence (XRF) imaging instrument, based on an X-ray microfocus tube equipped with a monocapillary optic, has been developed to perform XRF computed tomography experiments with both higher spatial resolution (20 µm) and a better energy resolution (130 eV @Mn-K(α)) than has been achieved up-to-now. This instrument opens a new range of possible applications for XRF-CT. Next to the analytical characterization of the setup by using well-defined model/reference samples, demonstrating its capabilities for tomographic imaging, the XRF-CT microprobe has been used to image the interior of an ecotoxicological model organism, Americamysis bahia. This had been exposed to elevated metal (Cu and Ni) concentrations. The technique allowed the visualization of the accumulation sites of copper, clearly indicating the affected organs, i.e. either the gastric system or the hepatopancreas. As another illustrative application, the scanner has been employed to investigate goethite spherules from the Cretaceous-Paleogene boundary, revealing the internal elemental distribution of these valuable distal ejecta layer particles.

Biodiversity increases functional and compositional resistance, but decreases resilience in phytoplankton communities.

There is now ample evidence that biodiversity stabilizes aggregated ecosystem functions, such as primary production, in changing environments. In primary producer systems, this stabilizing effect is found to be driven by higher functional resistance (i.e., reduced changes in functions by environmental changes) rather than through higher functional resilience (i.e., rapid recovery following environmental changes) in more diverse systems. The stability of aggregated ecosystem functions directly depends on changes in species composition and by consequence their functional contributions to ecosystem functions. Still, it remains only theoretically explored how biodiversity can stabilize ecosystem functions by affecting compositional stability. Here, we demonstrate how biodiversity effects on compositional stability drive biodiversity effects on functional stability in diatom communities. In a microcosm experiment, we exposed 39 communities of five different levels of species richness (1, 2, 4, 6, and 8 species) to three concentrations of a chemical stressor (0, 25, and 250 µg/L atrazine) for four weeks, after which all communities were transferred to atrazine-free medium for three more weeks. Biodiversity simultaneously increased, increasing functional and compositional resistance, but decreased functional and compositional resilience. These results confirm the theoretically proposed link between biodiversity effects on functional and compositional stability in primary producer systems, and provide a mechanistic underpinning for observed biodiversity-stability relationships. Finally, we discuss how higher compositional stability can be expected to become increasingly important in stabilizing ecosystem functions under field conditions when multiple environmental stressors fluctuate simultaneously.

Microplastic as a Vector for Chemicals in the Aquatic Environment: Critical Review and Model-Supported Reinterpretation of Empirical Studies.

The hypothesis that 'microplastic will transfer hazardous hydrophobic organic chemicals (HOC) to marine animals' has been central to the perceived hazard and risk of plastic in the marine environment. The hypothesis is often cited and has gained momentum, turning it into paradigm status. We provide a critical evaluation of the scientific literature regarding this hypothesis. Using new calculations based on published studies, we explain the sometimes contrasting views and unify them in one interpretive framework. One explanation for the contrasting views among studies is that they test different hypotheses. When reframed in the context of the above hypothesis, the available data become consistent. We show that HOC microplastic-water partitioning can be assumed to be at equilibrium for most microplastic residing in the oceans. We calculate the fraction of total HOC sorbed by plastics to be small compared to that sorbed by other media in the ocean. We further demonstrate consistency among (a) measured HOC transfer from microplastic to organisms in the laboratory, (b) measured HOC desorption rates for polymers in artificial gut fluids (c) simulations by plastic-inclusive bioaccumulation models and (d) HOC desorption rates for polymers inferred from first principles. We conclude that overall the flux of HOCs bioaccumulated from natural prey overwhelms the flux from ingested microplastic for most habitats, which implies that microplastic ingestion is not likely to increase the exposure to and thus risks of HOCs in the marine environment.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. 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 at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

Conserved transcriptional responses to cyanobacterial stressors are mediated by alternate regulation of paralogous genes in Daphnia.

Despite a significant increase in genomic data, our knowledge of gene functions and their transcriptional responses to environmental stimuli remains limited. Here, we use the model keystone species Daphnia pulex to study environmental responses of genes in the context of their gene family history to better understand the relationship between genome structure and gene function in response to environmental stimuli. Daphnia were exposed to five different treatments, each consisting of a diet supplemented with one of five cyanobacterial species, and a control treatment consisting of a diet of only green algae. Differential gene expression profiles of Daphnia exposed to each of these five cyanobacterial species showed that genes with known functions are more likely to be shared by different expression profiles, whereas genes specific to the lineage of Daphnia are more likely to be unique to a given expression profile. Furthermore, while only a small number of nonlineage-specific genes were conserved across treatment type, there was a high degree of overlap in expression profiles at the functional level. The conservation of functional responses across the different cyanobacterial treatments can be attributed to the treatment-specific expression of different paralogous genes within the same gene family. Comparison with available gene expression data in the literature suggests differences in nutritional composition in diets with cyanobacterial species compared to diets of green algae as a primary driver for cyanobacterial effects on Daphnia. We conclude that conserved functional responses in Daphnia across different cyanobacterial treatments are mediated through alternate regulation of paralogous gene families.

Multidecadal Field Data Support Intimate Links between Phytoplankton Dynamics and PCB Concentrations in Marine Sediments and Biota.

We analyzed three decades of field observations in the North Sea with additive models to infer spatiotemporal trends of chlorophyll a concentration, sediment organic carbon content, and polychlorinated biphenyls (PCBs) concentrations in mussels and sediments. By doing so, we separated long-term changes in PCB concentrations from seasonal variability. Using the inferred seasonal variability, we demonstrated that phytoplankton blooms in spring and autumn correspond to the annual maxima of the organic carbon content (r = 0.56; p = 0.004) and the PCB concentrations in sediments (r = 0.57; p = 0.004). Furthermore, we found a negative correlation between the PCB concentrations in sediments and in blue mussels (Mytilus edulis; r = -0.33, p = 0.012), which is probably related to the cleansing of the dissolved PCB phase driven by sinking organic matter during phytoplankton blooms and the filter-feeding behavior of the blue mussel. The present research demonstrates the role of seasonal phytoplankton dynamics in the environmental fate of PCBs at large spatiotemporal scales.

Rapid Adaptation of a Daphnia magna Population to Metal Stress Is Associated with Heterozygote Excess.

Although natural populations can harbor evolutionary potential to adapt genetically to chemical stress, it is often thought that natural selection leads to a general reduction of genetic diversity and involves costs. Here, a 10 week microevolution experiment was conducted with a genetically diverse and representative sample of one natural Daphnia magna population that was exposed to copper and zinc. Both Cu- and Zn-selected populations developed a significantly higher metal tolerance (i.e., genetic adaptation), indicated by higher reproduction probabilities of clonal lines in Cu and Zn exposures than observed for the original and control populations. The complete recovery of the population densities after 10 weeks of Zn selection (following an initial decrease of 74%) illustrates an example of evolutionary rescue. Microsatellite genotyping revealed a decrease in clonal diversity but no change in allelic richness, and showed an excess in heterozygosity in the Cu- and Zn-selected populations compared to the control and original populations. The excess heterozygosity in metal-selected populations that we observed has important consequences for risk assessment, as it contributes to the maintenance of a higher allelic diversity under multigenerational chemical exposure. This study is, to our knowledge, the first report of an increase in heterozygosity following multigenerational exposure to metal stress, despite a decline in clonal diversity. In a follow-up study with the Zn-selected populations, we observed no effect of Zn selection on the tolerance to heat and cyanobacteria. However, we observed higher tolerance to Cd in the Zn-selected than in the original and control populations if the 20% effective concentration of Cd was considered (cross-tolerance). Our results suggest only limited costs of adaptation but future research is needed to evaluate the adaptive potential of metal-selected populations to novel stressors and to determine to what extent increased heterozygosity is preserved after genetic recombination following periods of sexual reproduction.