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.

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

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

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

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

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.

Genome-Wide Stress Responses to Copper and Arsenic in a Field Population of Daphnia.

Over the past decade, significant advances have been made to unravel molecular mechanisms of stress response in different ecotoxicological model species. Within this study, we focus on population level transcriptomic responses of a natural population of Daphnia magna Straus, (1820), to heavy metals. We aim to characterize the population level transcriptomic responses, which include standing genetic variation, and improve our understanding on how populations respond to environmental stress at a molecular level. We studied population level responses to two heavy metals, copper and arsenic, and their binary mixture across time. Transcriptomic patterns identified significantly regulated gene families and genes at the population level including cuticle proteins and resilins. Furthermore, some of these differentially regulated gene families, such as cuticle proteins, were also significantly enriched for genetic variations including SNPs and MNPs. In general, genetic variation was observed in specific gene families, many of which are known to be involved in stress response. Overall, our results indicate that molecular stress responses can be identified within natural populations and that linking molecular mechanisms with genetic variation at the population level could contribute significantly to adverse outcome frameworks.

Early transcriptional response pathways in Daphnia magna are coordinated in networks of crustacean-specific genes.

Natural habitats are exposed to an increasing number of environmental stressors that cause important ecological consequences. However, the multifarious nature of environmental change, the strength and the relative timing of each stressor largely limit our understanding of biological responses to environmental change. In particular, early response to unpredictable environmental change, critical to survival and fitness in later life stages, is largely uncharacterized. Here, we characterize the early transcriptional response of the keystone species Daphnia magna to twelve environmental perturbations, including biotic and abiotic stressors. We first perform a differential expression analysis aimed at identifying differential regulation of individual genes in response to stress. This preliminary analysis revealed that a few individual genes were responsive to environmental perturbations and they were modulated in a stressor and genotype-specific manner. Given the limited number of differentially regulated genes, we were unable to identify pathways involved in stress response. Hence, to gain a better understanding of the genetic and functional foundation of tolerance to multiple environmental stressors, we leveraged the correlative nature of networks and performed a weighted gene co-expression network analysis. We discovered that approximately one-third of the Daphnia genes, enriched for metabolism, cell signalling and general stress response, drives transcriptional early response to environmental stress and it is shared among genetic backgrounds. This initial response is followed by a genotype- and/or condition-specific transcriptional response with a strong genotype-by-environment interaction. Intriguingly, genotype- and condition-specific transcriptional response is found in genes not conserved beyond crustaceans, suggesting niche-specific adaptation.

Deciphering the Combined Effects of Environmental Stressors on Gene Transcription: A Conceptual Approach.

The use of classical mixture toxicity models to predict the combined effects of environmental stressors based on toxicogenomics (OMICS) data is still in its infancy. Although several studies have made attempts to implement mixture modeling in OMICS analysis to understand the low-dose interactions of stressors, it is not clear how interactions occur at the molecular level and how results generated from such approaches can be better used to inform future studies and cumulative hazard assessment of multiple stressors. The present work was therefore conducted to propose a conceptual approach for combined effect assessment using global gene expression data, as illustrated by a case study on assessment of combined effects of gamma radiation and depleted uranium (DU) on Atlantic salmon ( Salmo salar). Implementation of the independent action (IA) model in reanalysis of a previously published microarray gene expression dataset was performed to describe gene expression patterns of combined effects and identify key gene sets and pathways that were relevant for understanding the interactive effects of these stressors. By using this approach, 3120 differentially expressed genes (DEGs) were found to display additive effects, whereas 279 (273 synergistic, 6 antagonistic) were found to deviate from additivity. Functional analysis further revealed that multiple toxicity pathways, such as oxidative stress responses, cell cycle regulation, lipid metabolism, and immune responses were enriched by DEGs showing synergistic gene expression. A key toxicity pathway of DNA damage leading to enhanced tumorigenesis signaling is highlighted and discussed in detail as an example of how to take advantage of the approach. Furthermore, a conceptual workflow describing the integration of combined effect modeling, OMICS analysis, and bioinformatics is proposed. The present study presents a conceptual framework for utilizing OMICS data in combined effect assessment and may provide novel strategies for dealing with data analysis and interpretation of molecular responses of multiple stressors.

Transgenerational DNA Methylation Changes in Daphnia magna Exposed to Chronic γ Irradiation.

Our aim was to investigate epigenetic changes in Daphnia magna after a 25-day chronic external γ irradiation (generation F0 exposed to 6.5 µGy·h-1 or 41.3 mGy·h-1) and their potential inheritance by subsequent recovering generations, namely, F2 (exposed as germline cells in F1 embryos) and F3 (the first truly unexposed generation). Effects on survival, growth, and reproduction were observed and DNA was extracted for whole-genome bisulfite sequencing in all generations. Results showed effects on reproduction in F0 but no effect in the subsequent generations F1, F2, and F3. In contrast, we observed significant methylation changes at specific CpG positions in every generation independent of dose rate, with a majority of hypomethylation. Some of these changes were shared between dose rates and between generations. Associated gene functions included gene families and genes that were previously shown to play roles during exposure to ionizing radiation. Common methylation changes detected between generations F2 and F3 clearly showed that epigenetic modifications can be transmitted to unexposed generations, most likely through the germline, with potential implications for environmental risk.

Transgenerational Inheritance of DNA Hypomethylation in Daphnia magna in Response to Salinity Stress.

Epigenetic mechanisms have been found to play important roles in environmental stress response and regulation. These can, theoretically, be transmitted to future unexposed generations, yet few studies have shown persisting stress-induced transgenerational effects, particularly in invertebrates. Here, we focus on the aquatic microcrustacean Daphnia, a parthenogenetic model species, and its response to salinity stress. Salinity is a serious threat to freshwater ecosystems and a relevant form of environmental perturbation affecting freshwater ecosystems. We exposed one generation of D. magna to high levels of salinity (F0) and found that the exposure provoked specific methylation patterns that were transferred to the three consequent nonexposed generations (F1, F2, and F3). This was the case for the hypomethylation of six protein-coding genes with important roles in the organisms' response to environmental change: DNA damage repair, cytoskeleton organization, and protein synthesis. This suggests that epigenetic changes in Daphnia are particularly targeted to genes involved in coping with general cellular stress responses. Our results highlight that epigenetic marks are affected by environmental stressors and can be transferred to subsequent unexposed generations. Epigenetic marks could therefore prove to be useful indicators of past or historic pollution in this parthenogenetic model system. Furthermore, no life history costs seem to be associated with the maintenance of hypomethylation across unexposed generations in Daphnia following a single stress exposure.

Systematic Evaluation of Chronic Metal-Mixture Toxicity to Three Species and Implications for Risk Assessment.

Metal contamination generally occurs as mixtures. However, it is yet unresolved how to address metal mixtures in risk assessment. Therefore, using consistent methodologies, we have set up experiments to identify which mixture model applies best at low-level effects, i.e., the independent action (IA) or concentration addition (CA) reference model. The toxicity of metal mixtures (Ni, Zn, Cu, Cd, and Pb) to Daphnia magna, Ceriodaphnia dubia, and Hordeum vulgare was investigated in different waters or soils, totaling 30 different experiments. Some mixtures of different metals, each individually causing <10% inhibition, yielded much larger inhibition (up to 66%) when dosed in combination. In general, IA was most accurate in predicting mixture toxicity, while CA was the most conservative. At low-effect levels important in risk assessments, CA overestimated mixture toxicity to daphnids and H. vulgare, on average, with a factor 1.4 to 3.6. Observed mixture interactions could be related to bioavailability or by competition interactions, either for binding sites of dissolved organic carbon or for biotic ligand sites. Our study suggests that the current metal-by-metal approach in risk evaluations may not be conservative enough for metal mixtures.

Multigenerational Effects of the Antibiotic Tetracycline on Transcriptional Responses of Daphnia magna and Its Relationship to Higher Levels of Biological Organizations.

Given the risk of environmental pollution by pharmaceutical compounds and the effects of these compounds on exposed ecosystems, ecologically relevant and realistic assessments are required. However, many studies have been mostly focused on individual responses in a single generation exposed to one-effect concentrations. Here, transcriptional responses of the crustacean Daphnia magna to the antibiotic tetracycline across multiple generations and effect concentrations were investigated. The results demonstrated that tetracycline induced different transcriptional responses of daphnids that were dependent on dose and generation. For example, reproduction-related expressed sequence tags (ESTs), including vitellogenin, were distinctly related to the dose-dependent tetracycline exposure, whereas multigenerational exposure induced significant change of molting-related ESTs such as cuticle protein. A total of 65 ESTs were shared in all contrasts, suggesting a conserved mechanism of tetracycline toxicity regardless of exposure concentration or time. Most of them were associated with general stress responses including translation, protein and carbohydrate metabolism, and oxidative phosphorylation. In addition, effects across the dose-response curve showed higher correlative connections among transcriptional, physiological, and individual responses than multigenerational effects. In the multigenerational exposure, the connectivity between adjacent generations decreased with increasing generation number. The results clearly highlight that exposure concentration and time trigger different mechanisms and functions, providing further evidence that multigenerational and dose-response effects cannot be neglected in environmental risk assessment.

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.

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.

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.

Genome-wide transcription profiles reveal genotype-dependent responses of biological pathways and gene-families in Daphnia exposed to single and mixed stressors.

The present study investigated the possibilities and limitations of implementing a genome-wide transcription-based approach that takes into account genetic and environmental variation to better understand the response of natural populations to stressors. When exposing two different Daphnia pulex genotypes (a cadmium-sensitive and a cadmium-tolerant one) to cadmium, the toxic cyanobacteria Microcystis aeruginosa, and their mixture, we found that observations at the transcriptomic level do not always explain observations at a higher level (growth, reproduction). For example, although cadmium elicited an adverse effect at the organismal level, almost no genes were differentially expressed after cadmium exposure. In addition, we identified oxidative stress and polyunsaturated fatty acid metabolism-related pathways, as well as trypsin and neurexin IV gene-families as candidates for the underlying causes of genotypic differences in tolerance to Microcystis. Furthermore, the whole-genome transcriptomic data of a stressor mixture allowed a better understanding of mixture responses by evaluating interactions between two stressors at the gene-expression level against the independent action baseline model. This approach has indicated that ubiquinone pathway and the MAPK serine-threonine protein kinase and collagens gene-families were enriched with genes showing an interactive effect in expression response to exposure to the mixture of the stressors, while transcription and translation-related pathways and gene-families were mostly related with genotypic differences in interactive responses to this mixture. Collectively, our results indicate that the methods we employed may improve further characterization of the possibilities and limitations of transcriptomics approaches in the adverse outcome pathway framework and in predictions of multistressor effects on natural populations.

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.

Extrapolation of Metal Toxicity Data for the Rotifer Brachionus calyciflorus Using an Individual-Based Population Model.

Ecological risk assessment (ERA) of metals typically starts from standardized toxicity tests, the data from which are then extrapolated to derive safe concentrations for the envisioned protection goals. Because such extrapolation in conventional ERA lacks ecological realism, ecological modeling is considered as a promising new approach for extrapolation. Many published population models are complex, that is, they include many processes and parameters, and thus require an extensive dataset to calibrate. In the present study, we investigated how individual-based models based on a reduced version of the Dynamic Energy Budget theory (DEBkiss IBM) could be applied for metal effects on the rotifer Brachionus calyciflorus. Data on survival over time and reproduction at different temperatures and food conditions were used to calibrate and evaluate the model for copper effects. While population growth and decline were well predicted, the underprediction of population density and the mismatch in the onset of copper effects were attributed to the simplicity of the approach. The DEBkiss IBM was applied to toxicity datasets for copper, nickel, and zinc. Predicted effect concentrations for these metals based on the maximum population growth rate were between 0.7 and 3 times higher in all but one case (10 times higher) than effect concentrations based on the toxicity data. The size of the difference depended on certain characteristics of the toxicity data: both the steepness of the concentration-effect curve and the relative sensitivity of lethal and sublethal effects played a role. Overall, the present study is an example of how a population model with reduced complexity can be useful for metal ERA. Environ Toxicol Chem 2024;43:324-337. © 2023 SETAC.

Modeling Full Life-Cycle Effects of Copper on Brook Trout (Salvelinus fontinalis) Populations.

Population models are increasingly used to predict population-level effects of chemicals. For trout, most toxicity data are available on early-life stages, but this may cause population models to miss true population-level effects. We predicted population-level effects of copper (Cu) on a brook trout (Salvelinus fontinalis) population based on individual-level effects observed in either a life-cycle study or an early-life stage study. We assessed the effect of Cu on predicted trout densities (both total and different age classes) and the importance of accounting for effects on the full life cycle compared with only early-life stage effects. Additionally, uncertainty about the death mechanism and growth effects was evaluated by comparing the effect of different implementation methods: individual tolerance (IT) versus stochastic death (SD) and continuous versus temporary growth effects. For the life-cycle study, the same population-level no-observed-effect concentration (NOECpop) was predicted as the lowest reported individual-level NOEC (NOECind; 9.5 µg/L) using IT. For SD, the NOECpop was predicted to be lower than the NOECind for young-of-the-year and 1-year-old trout (3.4 µg/L), but similar for older trout (9.5 µg/L). The implementation method for growth effects did not affect the NOECpop of the life-cycle study. Simulations based solely on the early-life stage effects within the life-cycle study predicted unbounded NOECpop values (≥32.5 µg/L), that is, >3.4 times higher than the NOECpop based on all life-cycle effects. For the early-life stage study, the NOECpop for both IT and SD were predicted to be >2.6 times higher than the lowest reported NOECind. Overall, we demonstrate that effects on trout populations can be underestimated if predictions are solely based on toxicity data with early-life stages. Environ Toxicol Chem 2024;43:1662-1676. © 2024 SETAC.

Population Modeling in Metal Risk Assessment: Extrapolation of Toxicity Tests to the Population Level.

Population models can be a useful tool for ecological risk assessment to increase ecological realism. In the present study, population models were used to extrapolate toxicity test results of four metals (Ag, Cu, Ni, Zn) to the population level. In total, three primary producers, five invertebrate species, and five fish species were covered. The ecological modeling-based laboratory to population effect extrapolation factor (ECOPEX factor), defined as the ratio of the predicted 10% effect concentration (EC10) at the population level and the observed EC10 for the laboratory toxicity test, ranged from 0.7 to 78.6, with a median of 2.8 (n = 27). Population modeling indicated clearly higher effect concentrations in most of the cases (ECOPEX factor >2 in 14 out of 27 cases), but in some cases the opposite was observed (in three out of 27 cases). We identified five main contributors to the variability in ECOPEX factors: (1) uncertainty about the toxicity model, (2) uncertainty about the toxicity mechanism of the metal, (3) uncertainty caused by test design, (4) impact of environmental factors, and (5) impact of population endpoint chosen. Part of the uncertainty results from a lack of proper calibration data. Nonetheless, extrapolation with population models typically reduced the variability in EC10 values between tests. To explore the applicability of population models in a regulatory context, we included population extrapolations in a species sensitivity distribution for Cu, which increased the hazardous concentration for 5% of species by a factor 1.5 to 2. Furthermore, we applied a fish population model in a hypothetical Water Framework Directive case using monitored Zn concentrations. This article includes recommendations for further use of population models in (metal) risk assessment. Environ Toxicol Chem 2024;00:1-21. © 2024 SETAC.

The influence of pH and dissolved organic carbon on the ecotoxicity of ampicillin and clarithromycin.

The impacts of water chemistry properties including pH and dissolved organic carbon (DOC) on the ecotoxicity of active pharmaceutical ingredients (APIs) are increasingly evident. These impacts are a result of alterations in API bioavailability: pH regulates the bioavailability of many ionizable APIs via chemical speciation, whereas DOC interacts with several APIs to inhibit the APIs from traversing the membrane system of organisms. In this study, we examined the influences of pH and DOC on the bioavailability of ampicillin (AMP) and clarithromycin (CLA) with the help of a bioavailability model. The effects on bioavailability were quantified by ecotoxicity observed in cyanobacteria growth inhibition tests with Microcystis aeruginosa PCC7806. The median effect concentration (96 h-EC50total) of AMP increased by 5-fold when pH raised from 7.4 to 9.0, suggesting the zwitterionic AMP+/- species being higher in bioavailability than the negatively charged AMP- species. CLA ecotoxicity showed no significant pH-dependency, suggesting CLA+ and CLA0 species to be equally bioavailable, albeit it correlated significantly with M. aeruginosa growth rate in negative controls. In addition, DOC demonstrated no significant effects on the ecotoxicity of AMP or CLA. Overall, together with earlier results on ciprofloxacin, our data show that bioavailability relations with pH and DOC are variable among different antibiotics. Factors other than chemical speciation alone could play a role in their bioavailability, such as their molecular size and polarity.