In the life of the endangered but still fished Anguilla anguilla, glass eels are recruited through estuaries. These fragile ecosystems are among the most disturbed on the planet. Here, heavy metals and microplastics were measured in estuary water and European glass eels entering bay of Biscay rivers of different size and anthropogenic stress. Eels from all the estuaries exhibited cadmium exceeding legal European limits, as happened with lead in samples from the highly disturbed Avilés estuary. Several water samples from small rivers surpassed the estimated limit of microplastic for ecotoxicological safety. In multiple regression analysis, both eel lead and microplastic content were significantly explained from shipping activity in the estuaries. Eel cadmium content was not associated with estuary stressors, being probably acquired during the oceanic migration of eel larvae. The presence in eels of new white polyethylene particles that had not been found previously in the region could be explained from the marine plastic spill of "Toconao" cargo in December. The same spill could explain a significant increase of microplastic bioconcentration in the glass eels in comparison with previous surveys. These risks for critically endangered eels, and for the consumers, highlight the urgent need for reducing the impact of contaminants on both local and global scales.

The European eel Anguilla anguilla is a critically endangered catadromous species. There is an urgent need for close surveillance of the populations that are still viable in European rivers. The species is difficult to observe in freshwater because of its bottom-dwelling behavior; the currently employed methods of eel monitoring in Europe based on the physical capture of individuals are stressful and may cause mortality. Here, we present a new highly sensitive method based on an A. anguilla-specific qPCR marker designed within the cytochrome oxidase I mitochondrial gene for application on environmental DNA (eDNA). Since the detectability of eDNA depends on the hydrographic conditions, we applied correction for altitude and a linear model and were able to predict the eel biomass from the eDNA in the different rivers of northern Spain still holding wild populations. The method was validated by electrofishing surveys. This novel eDNA-based marker allows for estimating the European eel biomass in running waters from small 1.5 L water samples and could complement, or replace in some cases, current eel surveys without disturbing wild populations.

In the present study, the effects of emerging and legacy pollutants such as hazardous microplastics (hMP) and toxic elements (As, Cd, Hg and Pb) were investigated in wild Mediterranean mussel Mytilus galloprovincialis (n = 40) from three estuaries with different anthropogenic uses in the Asturias region (SW Bay of Biscay). The expression levels of six candidate genes related with oxidative stress and/or heavy metal detoxification (sod1, sod2, cat, hsp70, mt10 & mt20) were measured using qPCR. The relationship between their expression levels, the Condition Index (CI), and the concentration of these concurrent pollutants was assessed through linear mixed models (LMM). PERMANOVA revealed significant differences between polluted and clean locations for both pollutants and gene expression levels in mussels. However, no differences were found between Nalón and Sella estuaries despite their distinct historical uses and pollution levels, suggesting recovery in post-mining times. Overall, the expression of the antioxidant gene sod2 and the detoxification genes mt10 and mt20 were upregulated in mussels from the most industrialized and heavy metal polluted estuary of Avilés, with Cd and Pb significantly predicting mt10 and mt20 increase. Hg and the hMP content significantly explained the expression patterns of sod1 and sod2 genes. To the best of our knowledge, this is the first study examining the combined molecular effects of legacy and emerging pollutants on wild populations of the bioindicator Mediterranean mussel. Additionally, it represents the first application of this molecular approach to monitor the ecological status of estuaries in the region that could be applied elsewhere.

Understanding the effects of global change is crucial to aid in the conservation of critically endangered species such as the European eel (Anguilla anguilla). Emerging pollutants (microplastics) and legacy ones such as heavy metals, as well as thermal pollution, are deleterious for this species; however, their molecular impacts on glass eels entering estuaries remain understudied. Investigating the molecular signatures of environmental stress can provide insights into some of the factors contributing to A. anguilla population decline. In this study, expression changes in candidate genes associated with general and thermal stress (hsp70 & hsp90), oxidative stress (sod1, sod2, cat, gpx1), heavy metal detoxification (mt2), and neurotoxicity (ache) were measured in gill tissue of glass eels exposed to polystyrene (PS) microbeads in an aquarium, and also in individuals sampled from six Bay of Biscay estuaries with different levels of microplastics, heavy metals, nitrites, nitrates and thermal pollution. In the aquarium experiment, a significant positive correlation was found between hsp70 expression and PS concentration. On the other hand, in the glass eels sampled from the wild results showed that gene expression patterns tend to cluster based on estuary's pollution levels. Specifically, hsp70 gene expression was upregulated in eels from the most polluted estuaries, suggesting signs of damage. This study highlights the molecular effects and signatures of emerging and legacy pollutants on the critically endangered European eel, under natural and controlled conditions.