Alexandre Campos

Aquatic organisms in eutrophic brackish and freshwater systems are chronically exposed to natural and anthropogenic contaminants, including cyanotoxins, metals and pesticides, mainly driven by cyanobacterial blooms and human activities. However, the environmental risks posed by their combinations to the aquatic environment remain poorly understood. This study investigated the growth response of Chlorella vulgaris exposed to four single-chemical compounds (microcystin-LR, cylindrospermopsin, terbuthylazine, and cadmium) and five binary mixtures for 7 days. In single-chemical exposures, terbuthylazine was the most toxic to C. vulgaris , followed by cadmium, cylindrospermopsin and microcystin-LR. The obtained growth data were compared with concentration addition predictions based on individual toxicities using the MIXTOX tool, testing deviations for synergistic/antagonistic interactions, dose ratio and dose level dependencies. Mixture effects varied with composition and exposure time. Antagonism was the prevailing type of interaction between the chemical compounds after 4 days of exposure, while dose level dependency was the only deviation observed after 7 days. Regarding dose level dependency, synergism was observed at low doses of each chemical in three of the five tested binary mixtures. Time was an important factor in the change in the toxicity pattern. This synergistic effect poses a significant risk to aquatic organisms exposed simultaneously to cyanotoxins and anthropogenic contaminants in the environment.

Cyanobacterial blooms and the associated production of cyanotoxins represent a growing concern for freshwater ecosystems, particularly in regions increasingly affected by eutrophication and climate change. In Portugal, data on the occurrence and distribution of cyanotoxins remain limited, especially in southern multipurpose reservoirs that support the water supply and irrigation in a region severely affected by water scarcity. This study investigates the presence of cyanobacteria and their major cyanotoxin groups—microcystins (MCs), cylindrospermopsin (CYN), and anatoxin-a (ANTX)—in three reservoirs of the Alqueva water storage system (EFEMA), combining chemical analyses with molecular tools to assess toxin-producing cyanobacteria. Liquid chromatography–mass spectrometry (LC–MS) detected MC-LR, MC-RR, and MC-YR exclusively in the particulate fraction of water samples, while no toxins were detected in the dissolved fraction. MC concentrations were generally low (Microcystis in all reservoirs. Several genes from the microcystin biosynthesis cluster (mcyA, mcyB, mcyD, mcyE, and mcyG) were amplified, and sequencing of the mcyA gene revealed high homology with Microcystis aeruginosa. Although the detected MC concentrations remained below World Health Organization (WHO) guideline values for drinking water, their recurrent detection and the confirmed presence of toxin-producing genotypes highlight the need for continued monitoring. This study underscores the value of integrating chemical and molecular approaches for assessing cyanotoxin risks in large multipurpose reservoir systems under increasing environmental pressure.

Nuclear receptors (NRs) of the NR1J1 group have been described in aquatic invertebrates and proposed as the evolutionary counterparts of vertebrate NR1I receptors, known for their role in xenobiotic-sensing and detoxification. In bivalves, previous studies have shown that NR1J1 activity and gene expression are modulated by pharmaceuticals, toxins, natural compounds, and algal extracts. However, their functional properties and diversification remain poorly understood. Here, we investigated the function and evolution of NR1J1 receptors in the bivalves Mytilus galloprovincialis and Ruditapes decussatus. We first identified four nr1j1 paralogs in R. decussatus and then integrated phylogenetic analysis, domain identity comparisons, tissue distribution profiling, and luciferase-based transactivation assays to characterize NR1J1 paralogs from both species. The receptors displayed distinct but partially overlapping transactivation profiles in response to confirmed ligands of NR1I and NR1H receptors, natural compounds, plant extracts, and fish bile, indicating paralog- and species-specific differences in ligand responsiveness. Allocholic acid emerged as the most consistent agonist across paralogs, whereas curcumin, carnosic acid, and Ptychopetalum olacoides extract showed more selective response patterns. Tissue profiling revealed broad but non-uniform expression of nr1j1 paralogs across bivalve tissues. Together, these findings underscore that bivalve NR1J1 receptors constitute a functionally diversified chemical-sensing system and provide a framework for future studies addressing ligand-dependent regulation and detoxification pathways.

This study evaluates volatile extracts (HE1 and HE2) from the lichen Pseudevernia furfuracea as eco-friendly agents to control algal proliferation, specifically targeting the cyanobacterium Microcystis aeruginosa and the green microalga Chlorella sorokiniana. Both extracts exhibited potent anti-microalgal activity against the two species with a minimum inhibitory concentration (MIC) ranging from 375 to 750 µg/mL. Furthermore, both extracts reduced cell density by more than 98% after eight days of treatment. Chlorophyll a and protein levels decreased significantly (>80%) in both species, indicating suppression of pigment synthesis. However, their physiological responses were distinct: M. aeruginosa underwent early acute oxidative stress and severe membrane damage, while C. sorokiniana exhibited delayed oxidative activation and a negative growth rate, suggesting non-lytic metabolic inhibition. An in silico study by molecular docking of the most abundant compounds identified in these volatile extracts, such as terpenoids (abietatriene, δ-cadinene) and a phenolic compound (atraric acid), showed that these compounds interact with vital cellular targets in M. aeruginosa and C. sorokiniana and likely contribute to the effects observed in these two species. Predictive toxicity by applying the ADMET framework confirmed the favorable bioavailability and low acute toxicity of these volatile compounds. Therefore, P. furfuracea volatiles are promising, species-specific, and environmentally safe candidates for mitigating aquatic algal proliferation through targeted oxidative and metabolic interference.

The increase in multidrug resistance in microorganisms and the rise of emergent infectious diseases worldwide is a threat to human and animal health. Therefore, research on new molecules with antibiotic potential is a priority. Lichens have a unique secondary metabolism with relatively untapped potential, yet their essential oils (EOs) and volatile organic compounds (VOCs) remain a relatively untapped resource. This systematic review was conducted following PRISMA 2020 guidelines, with a comprehensive search performed in the Web of Science database for studies published up to 2023. From 254 identified records, six studies involving nine lichen species (Evernia prunastri, Evernia divaricata, Cladonia rangiformis, Cladonia furcata, Parmotrema perlatum, Lichina pygmaea, Parmelia perlata, Hypogymnia physodes, and Parmelia sulcata) met the eligibility criteria. The synthesized data show that these volatile fractions possess significant antimicrobial potential, with minimum inhibitory concentrations (MICs) generally lower than 1 mg/mL. Major bioactive constituents identified include atraric acid, orsellinates, and various sesquiterpenes. While the current evidence highlights a strong potential of lichen volatiles against pathogens, research is limited to a small fraction of known species. This review identifies a critical gap in testing these compounds directly against MDR clinical isolates and suggests that future research should focus on high-biomass species and the heterologous expression of lichen biosynthetic genes to develop sustainable antimicrobial applications.

Marine biofouling remains a major challenge for maritime industries, affecting submerged structures and vessels worldwide. The long-standing reliance on biocidal coatings, together with their documented environmental impacts, has led to increasingly restrictive regulations and an urgent demand for environmentally compatible antifouling (AF) solutions. This study evaluates the AF potential and toxicological profile of two nucleoside analogues, hypoxanthine arabinoside (1′) and 2′-deoxyinosine (2′), selected based on the previously reported non-lethal AF activity of the naturally occurring nucleosides adenosine and 2′-deoxyadenosine from cyanobacteria. Both analogues inhibited the growth of Navicula sp. by approximately 60% without inducing mortality and significantly reduced settlement of Mytilus galloprovincialis plantigrades, with EC₅₀ values of 5.50 µM (1′) and 8.54 µM (2′), and no lethality detected (LC₅₀ > 200 µM). At near-EC₅₀ concentrations, both compounds increased acetylcholinesterase and tyrosinase activities, supported by molecular docking results, suggesting involvement of neurotransmission- and byssal formation-related pathways. Proteomic analysis revealed compound-specific molecular responses. No lethal effects were observed in non-target organisms (LC₅₀ > 32 µM for A. amphitrite and LC₅₀ > 50 µM for A. salina), and environmental fate modelling predicted low bioaccumulation and rapid degradation. Overall, substitution of the amino group by a carbonyl group preserved AF efficacy without increasing toxicity, highlighting nucleosides as promising low-toxicity AF agents.

Microcystin (MC) contamination of surface waters threatens ecosystems and public health. Nature-based solutions such as Multi-Soil-Layering (MSL) systems have been used for MC remediation. However, the biological mechanisms controlling MC degradation remain unclear. The present study investigates microbial community responses in two MSL systems with different clay contents (8% and 54%) exposed to MC-contaminated inputs (well water and eutrophied lake water). Samples were analysed before and after treatment using quantitative PCR (qPCR) to quantify the mlrA gene (encoding microcystinase) and its bacterial hosts. Next-generation sequencing (NGS) was used to assess microbial diversity, while the FAPROTAX database was used to predict functional characteristics. Results showed that MC was mainly adsorbed in pozzolan layers, while mlrA gene abundance and MC-degrading bacteria were higher in soil mixture layers. The presence of mlrA and associated bacteria was most pronounced in lake inflow samples, indicating intrinsic MC Biodegradation potential. Taxonomic analysis revealed dominant phyla including Proteobacteria, Actinobacteriota, Firmicutes, Chloroflexi and Bacteroidota. Functional analysis identified dominant traits such as chemoheterotrophy and aerobic metabolism. These findings provide new insights into microbial interactions in MSL systems and contribute to the optimisation of water treatment strategies for MC-contaminated environments.

Lichens are complex symbiotic systems known for synthesizing diverse secondary metabolites with documented antimicrobial, antioxidant, and antiproliferative activities. The present study focused on Pseudevernia furfuracea, a species widely distributed across Moroccan habitats. Two hydrodistillation-derived extracts (HE1 and HE2) were analyzed through ultra-high-Performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) to characterize their metabolite composition, and their effects were evaluated on Jurkat cells, a representative human cell line of the immune system. As the results of the characterization, the main compounds identified were Caprolactam, N,N-Diethylaniline, Erucamide, and 4-Isopropylaniline. Cytotoxicity assessment revealed that both HE1 and HE2 decreased the viability of Jurkat cells in a concentration-dependent manner. The mean effective concentrations (EC₅₀) after 24 h of treatment were 53.79 ± 2.92 µg/mL for HE1 and 59.76 ± 2.01 µg/mL for HE2. Cell death mechanisms were further examined by flow cytometry, revealing that apoptosis predominated after 24 h of treatment, progressing mainly to late apoptotic stages after 48 h. In parallel, the expression levels of key cytokine genes, including IL-2, TNF-α, and IFN-γ, were quantified at the mRNA level to evaluate potential immunomodulatory effects. Up-regulation was observed in IL-2 after exposure to both extracts for 24 and 48 h, and in the case of IFN-γ after exposure to HE2 for 24 h; in contrast, HE1 and HE2 produced down-regulation in TNF-α at 24 h. These findings suggest that HE1 and HE2 have immunomodulatory activity in Jurkat cells. Further investigations are needed to elucidate the underlying mechanisms and to clarify how HE1 and HE2 influence immune responses in human systems.