Cryptic species are rarely considered in ecotoxicology, resulting in misleading outcomes when using a single morphospecies that encompasses multiple cryptic species. This oversight contributes to the lack of reproducibility in ecotoxicological experiments and promotes unreliable extrapolations. The important question of ecological differentiation and the sensitivity of cryptic species is rarely tackled, leaving a substantial knowledge gap regarding the vulnerability of individual cryptic species within species complexes. In times of agricultural intensification and the frequent use of pesticides, there is an urgent need for a better understanding of the vulnerability of species complexes and possible differences in adaptive processes. We used the cryptic species complex of the aquatic amphipod Gammarus roeselii, which comprises at least 13 genetic mtDNA lineages and spans from small-scale endemic lineages in Greece to a large-scale widely distributed lineage in central Europe. We exposed eleven populations belonging to four lineages to the neonicotinoid thiacloprid in an acute toxicity assay. We recorded various environmental variables in each habitat to assess the potential pre-exposure of the populations to contaminants. Our results showed that the populations differed up to 4-fold in their tolerances. The lineage identity had a rather minor influence, suggesting that the cryptic species complex G. roeselii does not differ significantly in tolerance to the neonicotinoid thiacloprid. However, the observed population differentiation implies that recent pre-exposure to thiacloprid (or similar substances) or general habitat contamination has triggered adaptive processes. Though, the extent to which these mechanisms are equally triggered in all lineages needs to be addressed in the future. Our study provides two key findings: Firstly, it shows that observed phylogenetic differences within the G. roeselii species complex did not reveal differences in thiacloprid tolerance. Second, it confirms that differentiation occurs at the population level, highlighting that susceptibility to toxicants is population-dependent. The population-specific differences were within the range of accepted intraspecific variability from a regulatory standpoint. From an evolutionary-ecological perspective, it remains intriguing to observe how persistent stresses will continue to influence tolerance and whether different populations are on distinct pathways of adaptation. Given that the potential selection process has only lasted a relatively short number of generations, it is crucial to monitor these populations in the future, as even brief exposure periods significantly impact evolutionary responses.

Oil spills pose significant environmental risks, particularly in cold seas. In the Baltic Sea, the low salinity (from 0 to 2 up to 18) affects the behaviour of the spilled oil as well as the efficiency and ecological impacts of oil spill response methods such as mechanical collection and the use of dispersants. In the present study, mussels (Mytilus trossulus) were exposed under winter conditions (5 °C) to the water-accommodated fraction (WAF) of Naphthenic North Atlantic crude oil prepared by mechanical dispersion or to the chemically enhanced fraction (CEWAF) obtained using the dispersant Finasol OSR 51 at salinities of 5.6 and 15.0. Especially at the lower salinity, high bioaccumulation of polycyclic aromatic hydrocarbons was recorded in mussels in the CEWAF treatments, accompanied by increased biomarker responses. In the WAF treatments these impacts were less evident. Thus, the use of dispersants in the Baltic Sea still needs to be carefully considered.

Sticklebacks (Gasterosteiformes) are increasingly used in ecological and evolutionary research and have become well established as role model species for biologists. However, ecotoxicology studies concerning behavioural effects in sticklebacks regarding stress responses, mainly induced by chemical mixtures, have hardly been addressed. For this purpose, we investigated the swimming behaviour (including mortality rate based on 96-h LC50 values) of two ecologically similar three-spined (Gasterosteus aculeatus) and nine-spined sticklebacks (Pungitius pungitius) to short-term (up to 24 h) metal mixture (MIX) exposure. We evaluated the relevance and efficacy of behavioural responses of test species in the early toxicity assessment of chemical mixtures. Fish exposed to six (Zn, Pb, Cd, Cu, Ni, and Cr) metals in the mixture were either singled out by the Water Framework Directive as priority or as relevant substances in surface water, which was prepared according to the environmental quality standards (EQSs) of these metals set for inland waters in the European Union (EU) (Directive 2013/39/EU). The performed behavioural analysis showed the main effect on the interaction between time, species, and treatment variables. Although both species exposed to MIX revealed a decreasing tendency in swimming activity, these species' responsiveness to MIX was somewhat different. Substantial changes in the activity of G. aculeatus were established after a 3-h exposure to MIX solutions, which was 1.43-fold lower, while in the case of P. pungitius, 1.96-fold higher than established 96-h LC50 values for each species. This study demonstrated species-specific differences in response sensitivity to metal-based water pollution, indicating behavioural insensitivity of P. pungitius as model species for aquatic biomonitoring and environmental risk assessments.v.

This study represents the first attempt to assess genotoxicity and cytotoxicity effects in herring (Clupea harengus membras), flounder (Platichthys flesus), and cod (Gadus morhua callarias) caught at 47 study stations, located close to chemical munition dumpsites in the Gotland Basin, the Baltic Sea. Herring sampled from stations located in the center of chemical munition dumpsites exhibited the highest levels of micronuclei (MN) and total genotoxicity (ΣGentox), which is defined as the sum of frequencies of such nuclear abnormalities as micronuclei, nuclear buds, nuclear buds on the filament, and bi-nucleated erythrocytes with nucleoplasmic bridges. Exceptionally high and high ΣGentox risks were determined for flounder (89.47%), herring (79.31%), and cod (50%) caught at the stations located close to the chemical munition dumpsites.

Bivalve molluscs represent the most recognized bioindicators of freshwater pollution. However, their ability to indicate specific xenobiotics in complex exposures is unclear. In this study, we aimed to track the particular effects of the pesticide Roundup (Rnd) and the antipsychotic drug chlorpromazine (Cpz) on the mussel Unio tumidus at the simpler environmentally relevant models. We treated the mussels by Rnd (17 μg L−1), Cpz (18 μg L−1), the mixture of Rnd and Cpz at 18 °C (RndCpz), and Rnd at 25 °C (RndT) and examined their digestive glands after 14 days of exposure. We analyzed total antioxidant capacity, glutathione (GSH&GSSG) and protein carbonyls levels, total and Zn-related concentrations of metallothioneins (MT and Zn-MT, respectively), the activities of CYP450-related EROD, glutathione S-transferase, cholinesterase, caspase-3, citrate synthase (CS), lysosomal membrane integrity (NRR), and Zn level in the tissue. Shared responses were indicated as the increase of the antioxidant, Zn-MT, and EROD levels, whereas the changes of Zn concentration, NRR, and caspase-3 activity were most diverse compared to control. According to discriminant analysis, complex exposures abolished the individual response traits and intensified the harmful effects that caused a decrease in the Zn level in the RndCpz- and RndT-groups and the loss of lysosomal integrity in the RndT-group. We concluded that multi-marker expertise with the application of integrated indices had benefits when evaluating the effects of complex exposures. Graphical abstract: [Figure not available: see fulltext.].

In the brackish water Baltic Sea, oil pollution is an ever-present and significant environmental threat mainly due to the continuously increasing volume of oil transport in the area. In this study, effects of exposure to crude oil on two common Baltic Sea species, the mussel Mytilus trossulus and the amphipod Gammarus oceanicus, were investigated. The species were exposed for various time periods (M. trossulus 4, 7, and 14 days, G. oceanicus 4 and 11 days) to three oil concentrations (0.003, 0.04, and 0.30 mg L−1 based on water measurements, nominally aimed at 0.015, 0.120, and 0.750 mg L−1) obtained by mechanical dispersion (oil droplets). Biological effects of oil exposure were examined using a battery of biomarkers consisting of enzymes of the antioxidant defense system (ADS), lipid peroxidation, phase II detoxification (glutathione S-transferase), neurotoxicity (acetylcholinesterase inhibition), and geno- and cytotoxicity (micronuclei and other nuclear deformities). In mussels, the results on biomarker responses were examined in connection with data on the tissue accumulation of polycyclic aromatic hydrocarbons (PAH). In M. trossulus, during the first 4 days of exposure the accumulation of all PAHs in the two highest exposure concentrations was high and was thereafter reduced significantly. Significant increase in ADS responses was observed in M. trossulus at 4 and 7 days of exposure. At day 14, significantly elevated levels of geno- and cytotoxicity were detected in mussels. In G. oceanicus, the ADS responses followed a similar pattern to those recorded in M. trossulus at day 4; however, in G. oceanicus, the elevated ADS response was still maintained at day 11. Conclusively, the results obtained show marked biomarker responses in both study species under conceivable, environmentally realistic oil-in-seawater concentrations during an oil spill, and in mussels, they are related to the observed tissue accumulation of oil-derived compounds.