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.

Tryptophan (Trp) is an essential amino acid, commercially available as a feed-grade product, and is a precursor to serotonin and melatonin, which are both important molecules in stress mitigation. Meagre have a high potential for marine aquaculture diversification but are highly susceptible to stressful conditions. This study aimed to assess the potential role of dietary tryptophan supplementation in meagre juveniles in order to minimize the deleterious effect of potential stress conditions. For this, a growth trial was performed wherein meagre juveniles were fed four isoproteic (45%DM) and isolipidic (16%DM) diets; namely, a control diet, and three diets similar to the control diet but supplemented with varying levels of tryptophan, graded according to the resulting percentage in each diet’s dry matter (Control, 0.25%Trp, 0.5%Trp, and 1%Trp), corresponding to a total dietary tryptophan of 1.06, 1.70, 2.08, and 3.24 g 16 g⁻¹ N, respectively. Diets were tested in triplicate, and fish were fed twice a day, six days a week, for eight weeks. Five days after the end of the growth trial, a time-course blood sampling was performed at 0 h, 1 h, 3 h, 6 h, and 12 h after feeding. At the 6 h sampling point, the liver was also collected. Overall, our results indicate that 1Trp supplementation (total dietary Trp 3.24 g 16 g⁻¹ N) may be harmful to fish, decreasing growth performance and feed utilization, although doses up to 0.5Trp do not influence these parameters. Voluntary feed intake lineally decreased with the increase in the level of dietary Trp. Whole-body lipid content decreased at the highest tryptophan inclusion, whereas no changes were observed in protein levels. After 12 h from feeding, plasma glucose levels were lower in all dietary treatments supplemented with tryptophan compared to those observed in the control. Hepatic enzyme activity of protein catabolism decreased with dietary Trp inclusion. Overall, our results suggested that while a dietary Trp level increase of up to 2.08 g 16 g⁻¹ N did not affect growth performance and feed efficiency, both these parameters were severely compromised with a Trp level of 3.24 g 16 g⁻¹ N.

Environmental exposure to caffeine (CAF) poses potential risks to aquatic ecosystems, affecting non-target species. This study investigated the chronic effects of environmentally relevant CAF concentrations, ranging from 0.16–50 µg/L, on zebrafish behaviour. A Kohonen-type artificial neural network classified zebrafish behaviour into nine behavioural classes based on a set of movement descriptors (mean meander, mean velocity, instantaneous velocity, distance to centre point, mean angular velocity and instantaneous acceleration), while a comprehensive analysis integrated behavioural classes previously defined and biochemical markers of oxidative stress, lipid peroxidation, reserve energy content, energetic pathways, and neurotoxicity. The discriminant analysis demonstrated that behaviour descriptors and biomarkers individually explained 38% and 67% of data variation, respectively, while the combination resulted in 19 models with 100% correct diagnosis. One of the models (Model A) seemed to suit the best dose-response relationship, incorporating key biomarkers including superoxide dismutase, catalase, glutathione peroxidase activities, and behavioural characteristics such as movement distance and velocity. This suggested methodology offers a different approach to evaluating CAF’s ecological impact, highlighting behavioural analysis as a valuable complement to traditional ecotoxicological assessments. This study provides a novel framework for understanding organism-level responses to environmental stressors (e.g., several anthropogenic compounds), utilising Mahalanobis distance as an integrative response index. This approach shows promise for broader application in assessing the impact of various aquatic contaminants on aquatic organisms (from bacteria to fish), potentially extending to pharmaceuticals, pesticides, and industrial pollutants.

Triops longicaudatus is a crustacean typically inhabiting temporary freshwater bodies in regions with a Mediterranean climate. These crustaceans are easily maintained in the laboratory and show a set of biological features that make them good candidates for diagnosing environmental quality and health. However, information about their responses to environmental contamination is scarce. This study characterised the locomotor responses of juvenile and adult/mature T. longicaudatus to low concentrations of five model toxicants upon a very short 1.5 h exposure: tributyltin, mercury, lindane, sodium hypochlorite and formaldehyde. A video-tracking system was used to record the locomotor behaviour. The data were analysed with an artificial neural network to identify distinct behaviours, followed by Chi-square and Correspondence analysis to characterise the response to each toxicant. The results showed that T. longicaudatus is sensitive to aquatic contamination, particularly sodium hypochlorite. Six behaviour types were defined, which allowed for the characterisation and discrimination of the test toxicants. The results support the need for more investigation into this species and its behaviour types as an alternative to animal testing and the more apical and often invasive endpoints commonly recommended in standard guidelines.