João Soares Carrola

Background: The consumption of psychoactive substances (PASs), including ketamine (KET), metaphedrone (3-MMC) and methylenedioxypyrovalerone (MDPV), has increased globally. Their presence in sewage sludge, biosolids and other soil amendments raises concerns about potential impacts on soil organisms and ecosystem functioning. However, their effects on terrestrial ecosystems remain poorly understood. Methods: This study assessed the acute and chronic toxicity of KET, 3-MMC and MDPV in the earthworm Eisenia fetida by evaluating avoidance behaviour, biomass, mortality, genotoxicity and reproduction. Results: Significant avoidance responses were observed only for 3-MMC (25 µg/kg) and MDPV (2500 µg/kg). No significant effects on adult mortality were detected, although MDPV (250 µg/kg) reduced biomass. All three compounds induced genotoxicity in coelomocytes at 250 and 2500 µg/kg. While DNA damage occurred at concentrations above those reported in environmental studies, MDPV significantly reduced reproduction at 25 µg/kg, a concentration detected under real environmental conditions. Conclusions: These findings demonstrate that PASs, particularly MDPV, may pose risks to soil invertebrates, highlighting the need for further ecotoxicological studies across different terrestrial species, trophic levels and environmental compartments to better understand their ecological impacts.

The simultaneous occurrence of amphetamine (AMP) and other illicit drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), butylone (BTL), and 3,4-dimethylmethcathinone (3,4-DMMC), in wastewater and aquatic ecosystems has been frequently reported. These psychoactive substances share similar modes of action and pose a significant threat to freshwater organisms and human health due to their neurotoxicity and physiological harmful effects. The individual adverse effects of AMP and illicit drugs on non-target aquatic organisms have been demonstrated; however, the combined effects of these drugs have been considerably less explored. This study investigated the ecotoxicity of a mixture of these substances at an environmentally relevant concentration (0.1 μg L⁻¹) and at a 10-fold higher concentration (1.0 μg L⁻¹), using the freshwater microcrustacean water flea (Daphnia magna) exposed for 14 days. Several endpoints were evaluated. No significant changes were observed in swimming behavior or reproduction parameters, but some changes were noted for morphophysiological and biochemical endpoints. A significant increase was observed in the organisms’ heart rate and in catalase activity. Overall, the mixture did not induce significant ecotoxicological effects under the tested conditions. Nevertheless, the presence of other contaminants and longer exposure periods in natural environments may increase the ecological risks associated with these psychoactive substances.

Introduction: Biological invasions constitute one of the main threats to freshwater ecosystems, causing significant ecological changes through the introduction of exotic species that compete with or prey upon native species. In Portugal, the introduction and spread of exotic species in lotic and lentic ecosystems, such as pike (Esox lucius), European catfish (Silurus glanis), and largemouth bass (Micropterus salmoides), all top predators, may have a big impact on autochthonous species. In contrast, bleak (Alburnus alburnus), European perch (Perca fluviatilis), and common carp (Cyprinus carpio) compete aggressively for food resources. In the Sousa River basin, gudgeon (Gobio lozanoi) is considered an exotic species with potential ecological impact, with the minnow (Phoxinus phoxinus) stand having been recently identified in Portugal and, so far, recorded only in this river basin, and not yet being classified as an invasive species in Portugal. Public knowledge regarding invasive aquatic biodiversity remains a significant bottleneck for conservation. Because recreational angling is a prominent dispersal vector, initiatives that directly target this community are relevant. Objective: The aim is to carry out a bibliographic review on the exotic ichthyofauna species present in the Sousa River, with special focus on the invasion potential of the minnow (P. phoxinus). Methodology: The literature review was conducted based on the ScienceDirect, Springer Nature Link, and Fauna Norvegica databases, selecting publications between 2006 and 2025 concerning relevant studies on the potentially invasive characteristics of the minnow (P. phoxinus). The methodology is based on the analysis of studies regarding the impacts caused on riparian ecosystems. Results: The species P. phoxinus presents a generalist diet and high adaptive capacity, allowing it to colonise new habitats and compete aggressively with native species for trophic resources. Its presence is associated with negative impacts on brown trout populations (Salmo trutta), reducing growth and productivity, especially in mountain ecosystems. Increased species density also causes a significant decrease in benthic macroinvertebrate biodiversity. Studies conducted in the Douro basin indicate that the arrival of minnow in Portugal resulted from human action, probably associated with its use as live bait in recreational fishing. Conservation programmes use diverse tactics to bridge the awareness gap. Recent initiatives feature electrofishing demonstrations to visually differentiate species, theatrical performances, and even culinary show-cooking events using invasive predators like the European catfish to promote harvesting. Conclusions: The potential transition of P. phoxinus into an exotic and invasive species may be associated with the ecological pressure exerted on native communities, particularly through competition for trophic resources, highlighting the need to assess its dispersion in the Sousa basin and its impacts on fish fauna and benthic macroinvertebrates. It is important to do more sampling to understand its real distribution in the Sousa Basin. Additionally is important to explain to recreational anglers and the general population the impacts of fish transfer and the adverse effects of invasive species on freshwater Portuguese ecosystems.

Introduction: Synthetic cathinones (SC) are an emerging class of neuroactive contaminants increasingly detected in aquatic systems due to their widespread recreational use. Their continuous release at ng–µg L⁻¹ levels is particularly concerning, as these compounds are specifically designed to alter neural function, raising the likelihood of subtle yet ecologically relevant effects in non-target organisms. Among them, 3,4-methylenedioxypyrovalerone (MDPV) is one of the most-reported SC in wastewater and surface waters. Nevertheless, its chiral nature has been largely overlooked in ecotoxicological studies, despite growing evidence that enantiomers can differ markedly in biological activity, potentially leading to underestimated environmental risks. Objective: The ecotoxicological impact of racemic MDPV ((R,S)-MDPV) and its separate enantiomers ((R)-MDPV and (S)-MDPV) were examined using zebrafish (Danio rerio) as a model, focusing on survival and embryonic development. Methodology: Zebrafish embryos, at approximately 3-hours post-fertilization (hpf), were exposed over 96 h to environmentally relevant concentrations of MDPV forms (0.18−2.8 μg L⁻¹). Each treatment and control group included 50 animals distributed across 5 replicates. Mortality was assessed at multiple developmental stages (7, 24, 48, 72, and 96 h), along with cumulative mortality. Developmental endpoints included spontaneous movements (24 h), heartbeat (48 h), and hatching rate (48 and 72 h), quantified using stereomicroscopy and video analysis. Results: MDPV showed concentration and enantioselective effects, with (S)-MDPV being the most toxic. Behavioral and cardiac responses varied across forms, while hatching depended on concentration and time without a clear enantioselective pattern. Conclusions: MDPV disrupts early zebrafish development, impairing survival and embryonic development in a concentration-dependent and enantioselective manner, with (S)-MDPV demonstrating greater toxicity. These findings emphasize the importance of considering chirality in the environmental risk assessment of psychoactive contaminants such as SC, as enantiomer-specific effects may influence organism fitness, survival, and broader ecological outcomes.

Introduction: Chemical pollution of water bodies constitutes a global problem with huge impacts on fish populations. Consequently, the assessment of the effects of contaminants, especially on the nervous system, has become essential. The zebrafish (Danio rerio) has emerged as a prominent vertebrate model in ecotoxicology and neuroscience, in large part owing to the availability of genetic resources, including a high level of genome sequencing and annotation, plus the similarity of its neuron types and neurotransmitters to other vertebrates, including humans, and its stereotyped behaviour. Objective: The main objective of this mini-review is to present a synthesis of the key behavioural assays used in zebrafish larvae to assess neurotoxicity, focusing on developmental neurotoxicity. Methodology: A literature review was conducted based on the ScienceDirect and PubMed databases, covering publications between 2000 and 2025, selecting relevant studies on larval (up to 120 hpf) behaviour and contaminant exposure. The methodology was based on the analysis of behavioural tests applied to larvae, which evaluate responses to various stimuli, including visual, acoustic, tactile, and social stimuli. Results: Established, commonly used key assays include the light/dark test and locomotor, touch, photomotor, acoustic, and social response tests. The literature results confirm that zebrafish larvae exhibit complex behavioural patterns comparable to those of higher vertebrates, making them suitable for neurobehavioural studies. Changes in locomotor behaviour, responses to stimuli, or social patterns are extremely sensitive indicators of early neurotoxic effects, often before morphological changes are observed. Furthermore, the developing nervous system is particularly sensitive to chemicals, with high potential for irreversible effects, even with short-term exposures. Conclusions: Overall, our findings demonstrate that behavioural assays in zebrafish larvae constitute an effective, sensitive, and economically viable tool for assessing the neurotoxicity of compounds, contributing to a better understanding of the mechanisms of action and advancing environmental protection and public health strategies, considering also the “one health” approach.

Introduction: Portugal is, historically, one of the countries with the highest fish consumption worldwide, reflecting strong cultural and dietary traditions. However, shifts in food systems, economic conditions, and globalization may have altered its availability and nutritional contribution to population’s dietary intake over the time. Understanding these long-term dynamics is essential to inform public health and food policy strategies. This work aims to analyze temporal trends and identify structural changes in fish availability in Portugal between 1961 and 2023. Methodology: Data were obtained from FAOSTAT Food Balance Sheets for the period 1961–2023. Fish availability was expressed in grams per capita per day, alongside energy and macronutrient contributions. Descriptive statistics were calculated. Temporal trends were assessed using segmented linear regression models, considering structural breakpoints in 1975, 1986, 2001, and 2014. Model significance was evaluated using ANOVA. Results: Mean fish availability was 147.36 g/capita/day (SD = 26.58), ranging from 67.53 to 195.84 g/capita/day. Fish contributed on average 77.28 kcal/day, 13.25 g/day of protein, and 2.48 g/day of fat. The segmented regression model was statistically significant (p = 0.029), explaining 16.7% of the variability. Significant changes in trends were observed after 1975 (β = −2.185; p = 0.043), indicating a decrease, followed by a significant increase after 1986 (β = 5.164; p = 0.007), and a subsequent decline after 2001 (β = −4.163; p = 0.033). No significant changes were identified after 2014 (p = 0.684). Conclusions: Fish availability in Portugal shows marked structural changes over time, associated with probability, socio-economic and political transitions. Despite fluctuations, fish remains a nutritionally relevant component of the Portuguese diet. These findings highlight the importance of considering long-term trends in food availability when designing nutrition and sustainability policies.

The European seabass (Dicentrarchus labrax) is one of the most emblematic coastal fish species in the Northeast Atlantic, with high commercial value for fisheries and aquaculture, and importance for sport and recreational fishing. Despite its socio-economic importance, the Iberian divisions, Cantabrian Sea (8c) and the Atlantic Iberian waters (9a), defined by the International Council for the Exploration of the Sea (ICES), lack stock delimitation data. Moreover, this species is missing basic biological information, a seasonal reproductive fishing ban, and the annual landings in this region are more than double the levels recommended by ICES. To investigate the population structure of D. labrax in these areas, 140 adult individuals (36–51 cm of total length) were collected between January and March 2025 in three locations along the Atlantic coast of the Iberian Peninsula: Avilés (n = 47), Peniche (n = 48), and Lagos (n = 45). Fish from each location were analyzed for body geometric morphometrics (truss network) and otolith shape contour (Elliptical Fourier Descriptors). Data were evaluated using univariate and multivariate tests to assess spatial differences and reclassification success among locations. Results revealed regional differences using body morphometry and otolith shape analyses. The overall reclassification success was 68% for truss networking, 51% for otolith shape, and 65% when both methods were combined. Despite the observed differences, the absence of clear, isolated populations supports the ICES definition of a single, though not homogeneous, European seabass stock in the Atlantic Iberian coastal waters. Nevertheless, individuals from Avilés exhibited distinctive morphometric patterns and otolith shapes, suggesting possible adaptations to local selective pressures in slightly different environments. Further studies integrating genetic tools, otolith chemistry, parasitic fauna and telemetry analyses, as well as other fish samples from adjacent areas such as the Bay of Biscay, are recommended to achieve a more comprehensive understanding of the population structure and migration patterns of this key species in the Atlantic Iberian coastal waters.

Chiral pharmaceuticals, racemic or enantiomerically pure forms and their metabolites, can reach aquatic ecosystems via wastewater effluents (inefficient treatment operations) or by direct human disposal. They may negatively affect nontarget organisms even at low environmental concentrations. To make an accurate risk evaluation, the (eco)toxicity of both enantiomers needs to be assessed. Promethazine (PMZ) is a chiral antihistamine that has been detected in aquatic ecosystems owing to its high consumption. Promethazine undergoes metabolism in the liver, producing chiral metabolites such as promethazine sulfoxide (PMZSO) and N-desmethylpromethazine (DMPMZ) that reach water bodies. However, knowledge regarding the enantioselective toxicity of PMZ and its metabolites on aquatic organisms is missing. This study aimed to explore the potential enantioselective toxicity of PMZ and its metabolites on two relevant freshwater organisms, daphniid and fish, representing different trophic levels. The half maximal effect concentrations (EC50s) in Daphnia magna of PMZ, DMPMZ, and PMZSO were 2.33, 2.31, > 4 mg L⁻¹, respectively, > 4 and 2.50 mg L⁻¹ for (R) and (S)-PMZ, respectively, and > 4 mg L⁻¹ for the enantiomers of DMPMZ and PMZSO. In studies involving zebrafish, Danio rerio, (R, S)-PMZ showed a median lethal concentration (LC50) of .72 mg L⁻¹, and specific assays revealed that (R)-PMZ exhibited more pronounced adverse effects on larvae at the embryonic, morphological, and biochemical level than the racemate and (S)-PMZ. Toxicity and potential bioaccumulation of these compounds in daphniids and fish were also conducted using in silico tests through proprietary software. The results revealed a concordance between the experimental and predicted EC50 and LC50 values in both species.

Climate changes linked to extreme events pose a threat to freshwater biodiversity, impacting organs, behaviour, and reproduction. Moreover, these changes can be amplified by pollution. Copper sulphate (CuSO₄) is used in agriculture and aquaculture, so the copper can reach streams, rivers, and lakes impacting fish. This study evaluated the combined effects of temperature and copper on gills and liver histology biomarkers and in the behaviour of Mozambique tilapia over 28 days. Tilapias were exposed to different water temperatures (25 °C and 32 °C) and CuSO₄ concentrations (1.1 and 3.6 mg/L). Fish from the control group were exposed to water without copper sulphate and at 25 °C (within their optimum range). Histopathological analysis revealed significant tissue lesions, namely aneurysms and bending of gill lamellae, and hyalinization and vacuolization in the liver at the higher temperature and CuSO₄ level. Moreover, behavioural observations revealed increased stress changes under the same conditions. These findings highlight the effects of fast climate change, and rising temperatures on copper toxicity, underlining the necessity for strict monitoring and regulation of copper use due to future climate change scenarios to protect aquatic ecosystems, ichthyofauna population and trophic web dynamics. This data also alerts for similar problems with other toxic metals or chemicals, at short term, in streams and rivers, under rapid climate changes and more frequent extreme events.