Environmental stressors, such as contaminants and physical factors, rarely act in isolation, and studying their joint effects provides a more accurate reflection of real-world scenarios. To capture these interactions and disentangle the direct and indirect influences on algal responses, we applied partial least squares structural equation modeling (PLS-SEM), allowing us to reveal the hierarchical relationships among stressors and their cumulative impact on algal physiology. We examined combined effects of microplastics (MP; presence/absence), polycyclic aromatic hydrocarbons (PAHs; a mixture of acenaphthene, fluorene, phenanthrene, and fluoranthene at a total chemical activity in the sediment of 0 or 0.14), and sediment resuspension (turbidity: 0.8–3.9 NTU) on Ceramium tenuicorne, a coastal macroalga that is likely to encounter all these stressors in its natural habitats. Mechanical mixing at two intensities (low and high) was applied as an experimental treatment to induce resuspension. The analysis separated the effects of mechanical mixing and turbidity, given their nonlinear relationship, as stronger mechanical mixing did not consistently result in proportional turbidity increases. The algal physiological responses were evaluated using changes in pigment composition (Chl a, Chl c, and carotenoids), photosystem II (PSII) performance, total antioxidant capacity, and algal stoichiometry measured as elemental (%C, %N, %H, and C/N) ratios. We found that PAH exposure was the main suppressor of pigment concentrations and PSII performance, underscoring the mechanisms of its adverse effects on the photosynthetic machinery and nutrient assimilation. Moreover, stronger turbulence further decreased pigment concentrations, while sediment resuspension increased antioxidant capacity in algae, possibly due to physical damage from abrasion and scouring. We also found that MP addition significantly increased turbidity, thus aggravating the effects of the sediment resuspension. In conclusion, we provide a mechanistic explanation of how the combined exposure to MPs, PAHs, and sediment resuspension can impact pigment composition, photosynthesis, and stoichiometry of the algae, leading to decreased productivity.

Guidelines for ecotoxicity testing with Daphnia magna specify particular feeding protocols during the exposure, yet standardization for preexposure feeding remains ambiguous despite its recognized significance in affecting organismal metabolic capacity and tolerance. This ambiguity may contribute to disparate responses and heightened uncertainty in determining the effect concentrations of test chemicals, particularly those inducing metabolic effects through narcosis. Here, we address this gap through a three-step doseresponse experiment with neonates of D. magna subjected to two alternative feeding regimes in the preexposure phase: starved and moderately fed during the first 24 hr after birth. Following this treatment, the daphnids were exposed to narcosis-inducing substances (polycyclic aromatic hydrocarbons; PAHs) for 72 hr before being transferred to clean media with algal food ad libitum for a 48 hr recovery phase. Daphnid survivorship, individual protein content, and body size at the end of each experiment phase-pre-exposure, postexposure and postrecovery-were compared between the treatments. Significant treatment effects were observed, including lower and less variable protein content in the starved daphnids entering the PAH exposure phase, yet higher survivorship and greater recovery potential in these daphnids compared with the fed individuals. Our findings underscore the importance of early-life food access and advocate for mandatory reporting of pre-exposure feeding regimes, particularly when testing substances acting via nonpolar narcosis.

Guidelines for ecotoxicity testing with Daphnia magna specify particular feeding protocols during the exposure, yet standardization for preexposure feeding remains ambiguous despite its recognized significance in affecting organismal metabolic capacity and tolerance. This ambiguity may contribute to disparate responses and heightened uncertainty in determining the effect concentrations of test chemicals, particularly those inducing metabolic effects through narcosis. Here, we address this gap through a three-step dose-response experiment with neonates of D. magna subjected to two alternative feeding regimes in the preexposure phase: starved and moderately fed during the first 24 hr after birth. Following this treatment, the daphnids were exposed to narcosis-inducing substances (polycyclic aromatic hydrocarbons; PAHs) for 72 hr before being transferred to clean media with algal food ad libitum for a 48 hr recovery phase. Daphnid survivorship, individual protein content, and body size at the end of each experiment phase—pre-exposure, postexposure and postrecovery—were compared between the treatments. Significant treatment effects were observed, including lower and less variable protein content in the starved daphnids entering the PAH exposure phase, yet higher survivorship and greater recovery potential in these daphnids compared with the fed individuals. Our findings underscore the importance of early-life food access and advocate for mandatory reporting of pre-exposure feeding regimes, particularly when testing substances acting via nonpolar narcosis.

Hydrophobic organic contaminants (HOCs) are conventionally screened by matching electron ionization (EI) mass spectra acquired using gas chromatography-mass spectrometry (GC-MS) to reference spectra. However, extensive in-source fragmentation hampers de novo structure elucidation of novel substances that are absent from EI databases. To address this problem, a new method based on GC-atmospheric pressure chemical ionization (APCI) coupled to ion mobility-high resolution mass spectrometry (IM-HRMS) was developed for simultaneous target, suspect, and nontarget screening of HOCs. Of 102 target chemicals, 85.3% produced (quasi-)molecular ions as base peaks, while 71.6% displayed method detection limits lower than those of GC-EI-low resolution MS. The optimized method was applied to standard reference sediment and sediments from the Baltic Sea, an Arctic shelf, and a Norwegian lake. In total, we quantified 56 target chemicals with concentrations ranging from 4.86 pg g-1 to 124 ng g-1 dry weight. Further, using a combination of full scan mass spectrum, retention time, collision cross section (CCS), and fragmentation spectrum, a total of 54 suspects were identified at Confidence Level (CL) 2. Among the remaining features, 169 were prioritized using a halogen-selective CCS cutoff (100 Å2 + 20% mass), leading to annotation of 54 substances (CL ≤ 3). Notably, a suite of fluorotelomer thiols, disulfides, and alkyl sulfones were identified in sediment (CL 1-2) for the first time. Overall, this work demonstrates the potential of GC-APCI-IM-HRMS as a next-generation technique for resolving complex HOC mixtures in environmental samples through exploitation of molecular ions.

Sediments can act as reservoirs for hydrophobic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), but can also release these pollutants into the water, posing risks to aquatic life. Conventional risk assessments typically focus on total sediment concentrations, even though the bioavailable fraction provides a more accurate measure of ecological risk. This study aimed to explore how sediment resuspension and contaminant hydrophobicity influence contaminant release into the water. We conducted an experiment where the release of four PAHs (acenaphthene, fluorene, phenanthrene, and fluoranthene) from an artificial sediment was studied at different resuspension treatments. Polyethylene passive samplers were used to sample PAHs released from sediment and water turbidity was used as a proxy for sediment resuspension. We identified a turbidity threshold at 2.7 NTU, below which PAH release was primarily driven by diffusion from sediment pore water, whereas at higher turbidity, resuspension and desorption processes played a more dominant role. Hydrophobicity was a critical factor for contaminant release: contaminants with a log K_OW below 4.3 were more likely to be released at low turbidity, whereas those with a log K_OW above 4.5 were released more at higher turbidity. Taken together, the results show that the release of contaminants from sediment into the water column is influenced by contaminant hydrophobicity and turbidity.

Suspect screening strives to rapidly monitor a large number of substances in a sample using mass spectral libraries. For hydrophobic organic contaminants (HOCs), these libraries are traditionally based on electron ionization mass spectra. However, with the growing use of state-of-the-art mass spectrometers, which often use alternative ionization approaches and separation techniques, new suspect screening workflows and libraries are urgently needed. This study established a new suspect screening library for 1,590 HOCs, including exact mass and a combination of measured and model-predicted values for retention time (RT) and collision cross section (CCS). The accuracy of in silico predictions was assessed using standards for 102 HOCs. Thereafter, using gas chromatography-atmospheric pressure chemical ionization-ion mobility-mass spectrometry, a suspect screening workflow constrained by the full scan mass spectrum of (quasi-)molecular ions (including isotope patterns), RT, CCS, and fragmentation mass spectra, together with a continuous scoring system, was established to reduce false positives and improve identification confidence. Application of the method to fortified and standard reference sediment samples demonstrated true positive rates of 79% and 64%, respectively, with all false positives attributed to suspect isomers. This study offers a new workflow for improved suspect screening of HOCs using multidimensional information and highlights the need to enrich mass spectral databases and extend the applicable chemical space of current in silico tools to hydrophobic substances.

Ecotoxicity assessments often struggle with contaminant mixtures. This study explored combining chemical activity of hydrophobic organic contaminants (HOCs) and metals, using zinc as a model. An acute Daphnia magna immobilization test, with protein content as an additional endpoint, revealed an additive sublethal effect. The findings suggest chemical activity could serve as a unified approach for assessing HOCs and metals together, offering a promising method for more accurate environmental toxicity evaluations.

Ecotoxicity assessments often struggle with contaminant mixtures. This study explored combining chemical activity of hydrophobic organic contaminants (HOCs) and metals, using zinc as a model. An acute Daphnia magna immobilization test, with protein content as an additional endpoint, revealed an additive sublethal effect. The findings suggest chemical activity could serve as a unified approach for assessing HOCs and metals together, offering a promising method for more accurate environmental toxicity evaluations.

Persistent organic pollutants (POPs) pose a risk in aquatic environments. In sediment, this risk is frequently evaluated using total or organic carbon-normalized concentrations. However, complex physicochemical sediment characteristics affect POP bioavailability in sediment, making its prediction a challenging task. This task can be addressed using chemical activity, which describes a compound's environmentally effective concentration and can generally be approximated by the degree of saturation for each POP in its matrix. We present a proof of concept to load artificial sediments with POPs to reach a target chemical activity. This approach is envisioned to make laboratory ecotoxicological bioassays more reproducible and reduce the impact of sediment characteristics on the risk assessment. The approach uses a constantly replenished, saturated, aqueous POP solution to equilibrate the organic carbon fraction (e.g., peat) of an artificial sediment, which can be further adjusted to target chemical activities by mixing with clean peat. We demonstrate the applicability of this approach using four polycyclic aromatic hydrocarbons (acenaphthene, fluorene, phenanthrene, and fluoranthene). Within 5 to 17 weeks, the peat slurry reached a chemical equilibrium with the saturated loading solution. We used two different peat batches (subsamples from the same source) to evaluate the approach. Variations in loading kinetics and eventual equilibrium concentrations were evident between the batches, which highlights the impact of even minor disparities in organic carbon properties within two samples of peat originating from the same source. This finding underlines the importance of moving away from sediment risk assessments based on total concentrations. The value of the chemical activity-based loading approach lies in its ability to anticipate similar environmental impacts, even with varying contaminant concentrations. 

Hydrophobic organic contaminants (HOCs) affect phytoplankton at cellular to population levels, ultimately impacting communities and ecosystems. Baseline toxicants, such as some HOCs, predominantly partition to biological membranes and storage lipids. Predicting their toxic effects on phytoplankton populations therefore requires consideration beyond cell uptake and diffusion. Functional traits like lipid content and profile can offer insights into the diverse responses of phytoplankton populations exposed to HOCs. Our study investigated the vulnerability of five phytoplankton species populations to varying chemical activities of a mixture of polycyclic aromatic hydrocarbons (PAHs). Population vulnerability was assessed based on intrinsic sensitivities (toxicokinetic and toxicodynamic), and demography. Despite similar chemical activities in biota within the exposed algae, effects varied significantly. According to the chemical activity causing 50% of the growth inhibition (Ea₅₀), we found that the diatom Phaeodactylum tricornutum (Ea₅₀ = 0.203) was the least affected by the chemical exposure and was also a species with low lipid content. In contrast, Prymnesium parvum (Ea₅₀ = 0.072) and Rhodomonas salina (Ea₅₀ = 0.08), both with high lipid content and high diversity of fatty acids in non-exposed samples, were more vulnerable to the chemical mixture. Moreover, the species P. parvum, P. tricornutum, and Nannochloris sp., displayed increased lipid production, evidenced as 5–10% increase in lipid fluorescence, after exposure to the chemical mixture. This lipid increase has the potential to alter the intrinsic sensitivity of the populations because storage lipids facilitate membrane repair, reconstitution and may, in the short-term, dilute contaminants within cells. Our study integrated principles of thermodynamics through the assessment of membrane saturation (i.e. chemical activity), and a lipid trait-based assessment to elucidate the differences in population vulnerability among phytoplankton species exposed to HOC mixtures.