Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 μg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 μg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 μg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

Despite the key role of zooplankton communities in regulating the water quality of lentic ecosystems, they are absent from the list of biological elements of the Water Framework Directive (WFD) for the assessment of ecological status. Based on this, the present work was a case study that aimed to understand the relevance of zooplankton as a bioindicator for classifying the water quality of reservoirs. For one year and in each season, the water quality of the Torrão reservoir was assessed using the mandatory elements proposed by the WFD in the sampling year (second cycle) and the legislation currently applicable (third cycle). Additionally, zooplankton samples were collected to characterize the community dynamic. The water quality of the Torrão reservoir does not reach the WFD good ecological potential. Furthermore, with the updating of the criteria, the classification tends to get worse. Concerning the zooplankton, the occurrence of Cyclopoida and Bosmina are associated with lower water quality, as well as the collapse or low abundance of the Daphnia population, in the same periods. Low abundances of zooplankton are associated with better water quality, and the Shannon-Wiener diversity values decrease with the increase of the trophic state. High-efficiency feed filters and macrofiltrator organisms dominate the Torrão reservoir in all seasons, which is associated with low water quality. The Calanoida/Cyclopoida ratio shows a strong and negative association with the trophic state. The occurrence and abundance of species, Shannon-Wiener diversity, functional groups (high and low efficiency feed filters and macrofiltrators), and different ratios (large Cladocera/total Cladocera and Calanoida/Cyclopoida) are promising and valuable tools to determine the water quality status, and should be considered within the WFD metrics. Notwithstanding this, further research including reservoirs from different geographical areas with various trophic status and pressures must be analyzed.

Reservoirs are dynamic ecosystems subject to different pressures that influence and compromise their ecological structure. The main objective of this study was to evaluate the potential of using the macroinvertebrate to assess the water quality of four reservoirs (one site in Miranda—M and Pocinho—P; four sites in Aguieira—Ag1 to Ag4; and five sites in Alqueva—Al1 to Al5). The sites were sampled in autumn 2018 (A18), spring and autumn 2019 (S19 and A19) and spring 2020 (S20). In situ physical and chemical parameters were measured and a sample of water and macroinvertebrate were collected for further analyses. Total phosphorus exceeded the allowed concentrations (maximum values recorded: M—0.13 mg/L, P—0.09 mg/L, Ag3—0.22 mg/L and Al5—0.18 mg/L). Total abundance varied between 4 and 3088. Taxonomic richness was always low, between 1 and 12 taxa. The highest Shannon–Wiener value (1.91) was recorded in Ag1_A18 and Al2_A18. Pielou’s evenness varied widely across all reservoirs, from 0.06 to 0.92. Almost all the organisms found were associated with polluted water, according to the index ratings. Organisms tolerant of disturbances (e.g., Chironomidae and Oligochaeta) were associated with sites with the worst water quality, according to the Water Framework Directive (WFD), (M, Ag3, Ag4 and Al5) while organisms with moderate tolerance to disturbances (e.g., Cordullidae and Polycentropodidae) were associated with sites with better water quality (P, Ag1, Ag2 and Al1 to Al4). The macrozoobenthos index (MZB) used proved to be a sensitive tool to Portuguese reservoirs, corroborating most of the results obtained in the remaining analyses, as well as providing a clear ecological potential complementing the analysis carried out by the WFD. Based on this, the macroinvertebrate community appeared to be sensitive and able to characterize the reservoirs’ water quality.

The Water Framework Directive (WFD) is used to evaluate the water quality of aquatic ecosystems. Phytoplankton is the only biological element considered in the reservoirs water quality assessment. In this study, we aimed to assess the use of the bacterioplankton community as an indicator of water quality, using a culture-independent assay (denaturing gradient gel electrophoresis, DGGE). Four Portuguese reservoirs (Miranda, Pocinho, Aguieira and Alqueva) were analysed in four periods (autumn 2018, spring and autumn 2019, and spring 2020). Bacterial total abundance had similar values for Miranda, Pocinho and Aguieira, and generally lower values for Alqueva. Diversity and richness values did not show a clear trend. Negative correlations were observed between some nutrients and the bacterial community. Overall, members of Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospirae and Proteobacteria were identified in all sampling sites. In Alqueva, no spatial, temporal or water body quality relationships with bacterial community were observed, which may be due to its higher size, low water velocity rate and higher residence times. However, in Miranda, Pocinho and Aguieira, a strong spatial and temporal bacterial community dynamic was observed. Furthermore, the presence of some species (e.g., Acinetobacter sp.) may reflect the poor water quality that was not detected by the WFD approach.

At the global level, numerous reservoirs exhibit a pronounced water degradation. Inadequate land use and climate change effects contribute to freshwater degradation and disrupt the ecosystem balances. This study aimed to evaluate the temporal and spatial effects of the surrounding area on two Portuguese reservoirs: Rabagão and Aguieira. For each reservoir sub-watershed scale, the evolution of land use and soil occupation and the pressures reported over the past decade were analyzed. Additionally, official records of water quality parameters were collected, and water quality was assessed according to the Water Framework Directive (WFD). Both reservoirs show anthropogenic pressure, reflected in the water quality. Rabagão has good water quality, associated with undeveloped lands (47%), agriculture (26%), and one pressure on the aquaculture sector. Aguieira is characterized by high nutrient concentrations, low transparency, and phytoplankton. This is linked to various land uses, including forestry (75%), and agriculture (19%), as well as multiple environmental pressures. Key contributors include urban discharge (27 sites) and water catchments allocated for agricultural purposes (89 sites) and others. The long-term data showed an increase in chlorophyll a concentration, water temperature, and pH values, and a decrease in the concentration of total phosphorus, but higher than the reference value. Additionally, the usage of the surrounding area of the hydrographic basin shows that it is extremely important for water quality and should be included in the WFD. Addressing the problems in the surrounding areas reservoirs is essential to adopting measures that improve water quality, therefore guaranteeing the health of the environment as expected under the One Health concept.

This work intended to assess the adaptability of bioassay with Raphidocelis subcapitata to be used as a complement to the water quality assessment parameters of reservoirs imposed by the European Water Framework Directive (WFD). Thus, water samples of Portuguese reservoirs (Miranda, Pocinho, Aguieira, and Alqueva) were analyzed in three sampling periods (spring and autumn 2019, and spring 2020). A physical and chemical report of waters was also performed. R. subcapitata assay proved to be sensitive, indicating the presence of a potential perturbation that was not always associated with chemical analysis performed. In general, in the spring samplings, the water samples showed more disturbances to R. subcapitata, which in some situations may be associated with the higher content of nutrients and metals. Microalgae assay can be an effective complementary tool to indicate the ecotoxicological potential since they responded quickly to all sample components of water samples, in a wide-ranging variety of water conditions (different sites in several reservoirs). High similarities between the final ecotoxicological and the ecological potentials, according to the WFD parameters, were detected. The ecotoxicological approach based on our results allowed to confirm that bioassays with R. subcapitata are suitable and sensible to detect perturbations.