Ocean acidification impairs seagrass performance under thermal stress in shallow and deep water.

Despite the effects of ocean acidification (OA) on seagrasses have been widely investigated, predictions of seagrass performance under future climates need to consider multiple environmental factors. Here, we performed a mesocosm study to assess the effects of OA on shallow and deep Posidonia oceanica plants. The experiment was run in 2021 and repeated in 2022, a year characterized by a prolonged warm water event, to test how the effects of OA on plants are modulated by thermal stress. The response of P. oceanica to experimental conditions was investigated at different levels of biological organization. Under average seawater temperature, there were no effects of OA in both shallow and deep plants, indicating that P. oceanica is not limited by current inorganic carbon concentration, regardless of light availability. In contrast, under thermal stress, exposure of plants to OA increased lipid peroxidation and decreased photosynthetic performance, with deep plants displaying higher levels of heat stress, as indicated by the over-expression of stress-related genes and the activation of antioxidant systems. In addition, warming reduced plant growth, regardless of seawater CO2 and light levels, suggesting that thermal stress may play a fundamental role in the future development of seagrass meadows. Our results suggest that OA may exacerbate the negative effects of future warming on seagrasses.

Ecotoxicological effects of the UV-filter 4-MBC on sperms and adults of the mussel Mytilus galloprovincialis.

Present in an increasing number of products, UV-filters are continuously discharged into aquatic environments. Despite potential risks for inhabiting organisms are recognized, the effects of UV-filter 4-methylbenzylidenecamphor (4-MBC) on marine invertebrates are poorly investigated. By combining in vitro/in vivo exposures through a multi-biomarker approach on sperms and adults, the present study evaluated how 4-MBC affect the mussel species Mytilus galloprovincialis, providing ecologically relevant information on organisms' responses. From the obtained results, considering mortality as endpoint, sperms revealed a greater sensitivity (EC50:347 µg/L) than adults (EC50: not calculable). From an ecotoxicological perspective, this resulted in a derived threshold concentration (LOEC) of 100 µg/L and 72 µg/L, respectively. Effects at the cell/molecular level were provided by general redox-status imbalance and oxidative stress. Sperms showed functional and structural impairments, hyperactivation and DNA damage, while adults showed physiological, metabolic/energetic dysfunctions, DNA damage and activation of oxidative and biotransformation enzymes. High 4-MBC bioaccumulation was also observed in exposed mussels (BCFs:14.0-32.0 L/kg). These findings suggest that 4-MBC may impair fitness and survival of the broadcast spawning mussel M. galloprovincialis, affecting reproduction success and population growth.

Polyethylene microplastics reduce filtration and respiration rates in the Mediterranean sponge Petrosia ficiformis.

Microplastic (MP) pollution represents a distinctive mark of the Anthropocene. Despite the increasing efforts to determine the ecological impacts of MP on marine biodiversity, our understanding of their toxicological effects on invertebrate species is still limited. Despite their key functional roles, sponges (Phylum Porifera) are particularly understudied in MP research. These filter-feeders extract and retain particles from the water column, across a broad size range. In this study, we carried out a laboratory experiment to assess the uptake of MPs (polyethylene, PE) by the Mediterranean sponge Petrosia ficiformis, how MPs influence key biological process after different times of exposure (24h and 72h) and whether they can be subsequently eliminated. MP uptake increased with time of exposure, with 30.6% of the inoculated MP particles found in sponge samples after 72h. MPs impaired filtration and respiration rates and these effects were still evident 72h after sponges had been transferred in uncontaminated water. Our study shows that time of exposure represents a key factor in determining MP toxicity in sponges. In addition, our results suggest that sponges are able to incorporate foreign particles and may thus be a potential bioindicator for MP pollutants.

How temperature can alter the combined effects of carbon nanotubes and caffeine in the clam Ruditapes decussatus?

Nowadays, multi-walled carbon nanotubes are considered to be emerging contaminants and their impact in ecosystem has drawn special research attention, while other contaminants, such as caffeine, have more coverage in literature. Despite this, the effects of a combination of the two has yet to be evaluated, especially considering predicted temperature rise. In the present study a typical bioindicator species for marine environment, the clam Ruditapes decussatus, and classical tools, such as biomarkers and histopathological indices, were used to shed light on the species' response to these contaminants, under actual and predicted warming scenarios. The results obtained showed that both contaminants have a harmful effect at tissue level, as shown by higher histopathological index, especially in digestive tubules. Temperatures seemed to induce greater biochemical impacts than caffeine (CAF) and -COOH functionalized multi-walled carbon nanotubes (f-MWCNTs) when acting alone, namely in terms of antioxidant defences and energy reserves content, which were exacerbated when both contaminants were acting in combination (MIX treatment). Overall, the present findings highlight the complex response of clams to both pollutants, evidencing the role of temperature on clams' sensitivity, especially to mixture of pollutants.

Sperm quality assessment in Ficopomatus enigmaticus (Fauvel, 1923): Effects of selected organic and inorganic chemicals across salinity levels.

Contamination by organic and inorganic compounds remains one of the most complex problems in both brackish and marine environments, causing potential implications for the reproductive success and survival of several broadcast spawners. Ficopomatus enigmaticus is a tubeworm polychaete that has previously been used as a model organism for ecotoxicological analysis, due to its sensitivity and ecological relevance. In the present study, the effects of five trace elements (zinc, copper, cadmium, arsenic and lead), one surfactant (sodium dodecyl sulfate, SDS) and one polycyclic aromatic hydrocarbon (benzo(a)pyrene, B(a)P) on the sperm quality of F. enigmaticus were investigated. Sperm suspensions were exposed in vitro to different concentrations of each selected contaminant under four salinity conditions (10, 20, 30, 35). Possible adverse effects on sperm function were assessed by measuring oxidative stress, membrane integrity, viability and DNA damage. Sperm quality impairments induced by organic contaminants were more evident than those induced by inorganic compounds. SDS exerted the largest effect on sperm. In addition, F. enigmaticus sperm showed high tolerance to salinity variation, supporting the wide use of this species as a promising model organism for ecotoxicological assays. Easy and rapid methods on polychaete spermatozoids were shown to be effective as integrated sperm quality parameters or as an alternative analysis for early assessment of marine and brackish water pollution.

Biochemical performance of mussels, cockles and razor shells contaminated by paralytic shellfish toxins.

Marine toxins in bivalves pose an important risk to human health, and regulatory authorities throughout the world impose maximum toxicity values. In general, bivalve toxicities due to paralytic shellfish toxins (PSTs) above the regulatory limit occur during short periods, but in some cases, it may be extended from weeks to months. The present study examines whether cockles (Cerastoderme edule), mussels (Mytilus galloprovincialis) and razor shells (Solen marginatus) naturally exposed to a bloom of Gymnodinium catenatum activated or suppressed biochemical responses as result of the presence of PSTs in their soft tissues. Toxins (C1+2, C3+4, GTX5, GTX6, dcSTX, dcGTX2+3 and dcNEO) and a set of biomarkers (ETS, electron transport system activity; GLY, glycogen; PROT, protein; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferases; LPO, lipid peroxidation; reduced (GSH) and oxidized (GSSG) glutathione contents and AChE, acetylcholinesterase activity) were determined in the three bivalve species. Specimens were harvested weekly in Aveiro lagoon, Portugal, along thirteen weeks. This period included three weeks in which bivalve toxicity exceeded largely the regulatory limit and the subsequence recovery period of ten weeks. Biochemical performance of the surveyed species clearly indicated that PSTs induce oxidative stress and neurotoxicity, with higher impact on mussels and razor shells than in cockles. The antioxidant enzymes CAT and GPx seemed to be the biomarkers better associated with toxin effects.

Relationship between wild-caught organisms for bioassays and sampling areas: Widespread serpulid early-development comparison between two distinct populations after trace element exposure.

Previous studies suggested the suitability of the brackish-water serpulid (Ficopomatus enigmaticus) to be used as model organism for both marine and brackish waters monitoring, by the performance of sperm toxicity and larval development assays. The present study focused on larval development after the exposure of two F. enigmaticus populations (Mediterranean and Atlantic, collected in Italy and Portugal, respectively) to different trace elements (copper, mercury, arsenic, cadmium, and lead) at different concentrations. Results of larval development assays were presented as the percentage of abnormal developed larvae. The effect, measured in terms of EC50 for all toxicants tested, showed that mercury was the most toxic metal for larvae of both populations. Specifically, the tested trace elements may be racked in the following order from the highest to the lowest toxicity: Mediterranean: mercury > copper > lead > arsenic > cadmium; Atlantic: mercury > copper > cadmium > arsenic > lead. Responses of both populations were similar for arsenic. Lead was the least toxic element for the Atlantic population, while cadmium showed the least toxicity for the Mediterranean population. These preliminary results demonstrate the sensitivity and suitability of the organisms to be used in ecotoxicological bioassays and monitoring protocols. Moreover, chemical analyses on soft tissues and calcareous tubes of collected test organisms and their sampling site water were performed, to identify and quantify the concentration of the tested trace elements in these 3 matrices. Populations exhibited less sensitivity to a certain element together with a relevantly higher concentration of the same element in soft tissues. This may indicate a certain resistance to particular contaminant toxic effects by organisms that tend to accumulate the same toxicant. This highlights the potential correlation between wild-caught test organisms' responses and a deep characterization of the sampling site to identify putative abnormalities or differences in model organism response during bioassay execution.

Host genotype and amoxicillin administration affect the incidence of diarrhoea and faecal microbiota of weaned piglets during a natural multiresistant ETEC infection.

Enterotoxigenic Escherichia coli (ETEC) is the aetiological agent of postweaning diarrhoea (PWD) in piglets. The SNPs located on the Mucine 4 (MUC4) and Fucosyltransferase 1 (FUT1) genes have been associated with the susceptibility to ETEC F4 and ETEC F18, respectively. The interplay between the MUC4 and FUT1 genotypes to ETEC infection and the use of amoxicillin in modifying the intestinal microbiota during a natural infection by multiresistant ETEC strains have never been investigated. The aim of this study was to evaluate the effects of the MUC4 and FUT1 genotypes and the administration of amoxicillin through different routes on the presence of diarrhoea and the faecal microbiota composition in piglets naturally infected with ETEC. Seventy-one piglets were divided into three groups: two groups differing by amoxicillin administration routes-parenteral (P) or oral (O) and a control group without antibiotics (C). Faecal scores, body weight, presence of ETEC F4 and F18 were investigated 4 days after the arrival in the facility (T0), at the end of the amoxicillin administration (T1) and after the withdrawal period (T2). The faecal bacteria composition was assessed by sequencing the 16S rRNA gene. We described that MUC4 and FUT1 genotypes were associated with the presence of ETEC F4 and ETEC F18. The faecal microbiota was influenced by the MUC4 genotypes at T0. We found the oral administration to be associated with the presence of diarrhoea at T1 and T2. Furthermore, the exposure to amoxicillin resulted in significant alterations of the faecal microbiota. Overall, MUC4 and FUT1 were confirmed as genetic markers for the susceptibility to ETEC infections in pigs. Moreover, our data highlight that group amoxicillin treatment may produce adverse outcomes on pig health in course of multiresistant ETEC infection. Therefore, alternative control measures able to maintain a healthy faecal microbiota in weaners are recommended.

Engineered nanomaterials: From their properties and applications, to their toxicity towards marine bivalves in a changing environment.

As a consequence of their unique characteristics, the use of Engineered Nanomaterials (ENMs) is rapidly increasing in industrial, agricultural products, as well as in environmental technology. However, this fast expansion and use make likely their release into the environment with particular concerns for the aquatic ecosystems, which tend to be the ultimate sink for this type of contaminants. Considering the settling behaviour of particulates, benthic organisms are more likely to be exposed to these compounds. In this way, the present review aims to summarise the most recent data available from the literature on ENMs behaviour and fate in aquatic ecosystems, focusing on their ecotoxicological impacts towards marine and estuarine bivalves. The selection of ENMs presented here was based on the OECD's Working Party on Manufactured Nanomaterials (WPMN), which involves the safety testing and risk assessment of ENMs. Physical-chemical characteristics and properties, applications, environmental relevant concentrations and behaviour in aquatic environment, as well as their toxic impacts towards marine bivalves are discussed. Moreover, it is also identified the impacts derived from the simultaneous exposure of marine organisms to ENMs and climate changes as an ecologically relevant scenario.

Soluble esterases as biomarkers of neurotoxic compounds in the widespread serpulid Ficopomatus enigmaticus (Fauvel, 1923).

The characterization of soluble cholinesterases (ChEs) together with carboxylesterases (CEs) in Ficopomatus enigmaticus as suitable biomarkers of neurotoxicity was the main aim of this study. ChEs of F. enigmaticus were characterized considering enzymatic activity, substrate affinity (acetyl-, butyryl-, propionylthiocholine), kinetic parameters (Km and Vmax) and in vitro response to model inhibitors (eserine hemisulfate, iso-OMPA, BW284C51), and carbamates (carbofuran, methomyl, aldicarb, and carbaryl). CEs were characterized based on enzymatic activity, kinetic parameters and in vitro response to carbamates (carbofuran, methomyl, aldicarb, and carbaryl). Results showed that cholinesterases from F. enigmaticus showed a substrate preference for acetylthiocholine followed by propionylthiocholine; butyrylthioline was not hydrolyzed differently from other Annelida species. CE activity was in the same range of cholinesterase activity with acetylthiocholine as substrate; the enzyme activity showed high affinity for the substrate p-nytrophenyl butyrate. Carbamates inhibited ChE activity with propionylthiocholine as substrate to a higher extent than with acetylthiocoline. Also CE activity was inhibited by all tested carbamates except carbaryl. In vitro data highlighted the presence of active forms of ChEs and CEs in F. enigmaticus that could potentially be inhibited by pesticides at environmentally relevant concentration.

Physiological and biochemical responses of two keystone polychaete species: Diopatra neapolitana and Hediste diversicolor to Multi-walled carbon nanotubes.

Multi-walled carbon nanotubes (MWCNTs) are one of the most important carbon Nanomaterials (NMs). The production and use of these carbon NMs is increasing rapidly and, therefore, the need to assess their presence in the environment and associated risks has become increasingly important. However, limited literature is available regarding the impacts induced in aquatic organisms by this pollutant, namely in invertebrate species. Diopatra neapolitana and Hediste diversicolor are keystone polychaete species inhabiting estuaries and shallow water bodies intertidal mudflats, frequently used to evaluate the impact of environmental disturbances in these systems. To our knowledge, no information is available on physiological and biochemical alterations on these two species due to MWCNTs exposure. Thus, the present study aimed to assess the toxic effects of different MWCNTs concentrations (0.01; 0.10 and 1.00mg/L) in both species physiological (regenerative capacity and respiration rate) and biochemical (energy reserves, metabolic activities, oxidative stress related biomarkers and neurotoxicity markers) performance, after 28 days of exposure. The results obtained revealed that exposure to MWCNTs induced negative effects on the regenerative capacity of D. neapolitana. Additionally, higher MWCNTs concentrations induced increased respiration rates in D. neapolitana. MWCNTs altered energy-related responses, with higher values of electron transport system activity, glycogen and protein concentrations in both polychaetes exposed to this contaminant. Furthermore, when exposed to MWCNTs both species showed oxidative stress with higher lipid peroxidation, lower ratio between reduced and oxidized glutathione, and higher activity of antioxidant (catalase and superoxide dismutase) and biotransformation (glutathione-S-transferases) enzymes in exposed organisms.

Six-Year Prescription Pattern of Antimicrobial Use in Cats at the Veterinary Teaching Hospital of the University of Pisa.

The use of antimicrobials has greatly contributed to improving animal health. However, their inappropriate use reduces their effectiveness in treating bacterial infections and contributes to the selection of resistance. This study aimed to retrospectively evaluate the six-year pattern (2017-2022) of antimicrobial use in cats visiting the Veterinary Teaching Hospital (VTH) of the University of Pisa (Italy). The total number of prescribed antimicrobials, the number of animals for which an antimicrobial was prescribed, and the total number of antimicrobial prescriptions showed a significant time trend decrease during the study period, except for the fixed-dose combinations. The most frequently prescribed antimicrobials were amoxicillin-clavulanic acid (Synulox) (39.1%) followed by enrofloxacin (29.8%). These antimicrobials were mostly prescribed to treat infections affecting the genitourinary tract (~30%), followed by the gastrointestinal tract, skin, and respiratory system affections. Antimicrobials in empirical associations represented 13.0% of the total antimicrobial prescriptions, and the combination of amoxicillin-clavulanic acid (Synulox) with enrofloxacin accounted for the majority. The oral route represented the main route of administration of prescribed antimicrobials, followed by parenteral and topical ones. Amoxicillin-clavulanic acid (Synulox) (37.2%), ceftriaxone (2.7%), and tobramycin (2.8%) were the most prescribed antimicrobials for the oral, parenteral, and topical routes, respectively. Antimicrobial prescriptions complied with prudent use recommendations in terms of availability of diagnosis, respect to the dose range, duration of treatment, and the use of medicinal products approved for the species. On the contrary, antimicrobial susceptibility tests were used infrequently (5.2%), lacking compliance with the existing guidelines observed in companion animal practice. Overall, additional interventions are required not only to improve the responsible use of antimicrobials in our feline practice but also to implement antimicrobial stewardship programs, enhancing diagnostics such as culture and sensitivity testing in the future.

Pattern of prescriptions and prudent use of antimicrobial in horse practice at a Veterinary Teaching Hospital.

Antimicrobial prescriptions of the University of Pisa and their compliance with prudent use recommendations were investigated over 11 years (2011-2021). At least one antimicrobial was always prescribed in surgical prophylaxis for the suture of wounds and in 33% of horses with signs of disease of a body system. Antimicrobials were administered in monotherapy (48%) in fixed dose combinations (21%) and empirical combinations (31%). Antimicrobials were mostly (63%) administered by parenteral route, while oral and topical antimicrobials accounted for 14% and 23% of prescriptions, respectively. Gentamicin, benzylpenicillin and ceftiofur were the most prescribed antimicrobials; aminoglycosides, penicillins and cephalosporins were the most common class of antimicrobial prescribed. Protected antimicrobials (WHO HPCIA and rifampicin) represented 24% of antimicrobial dispensations. The pattern of classes of antimicrobial used by body system was broad and included up to eight different pharmaceutical classes. The heterogeneity of antimicrobial use was confirmed by the estimate of the prescription diversity index. Antimicrobial prescriptions were in compliance with prudent use recommendations in terms of availability of diagnosis, respect of the dose range and duration of treatment On the contrary, principles of appropriate antimicrobial use have only been partially observed in relation to off-label use, use of antimicrobials in empirical combination, use of antimicrobial susceptibility tests and use of protected antimicrobials, suggesting that additional interventions are required to improve the responsible use of antimicrobials use in our equine practice.

PON1 enzyme activity assays for serum and heparinized plasma in horses and stability evaluation of the enzyme activity over different freeze-thaw cycles and mimic transportation.

Consistent information and standardization procedures regarding the time of storage for frozen samples and the effects of storage time on enzyme activity are still missing in the literature. Thus, we evaluated the effects of different storage temperatures (-20 °C and - 80 °C), three repetitive freeze/thaw cycles, and 24-h mimic transportation on the activities of PON1 (paraoxonase and arylesterase), enzymes involved in the protection and detoxification processes of reactive molecules. PON1 enzymes' activity was validated on serum and heparinized plasma in horses. The results revealed that conditions and time of storage of blood samples for PON1 analyses altered the activities of both enzymes in both sample types, evidencing that these conditions can lead to protein degradation or general alteration. Specifically, paraoxonase and arylesterase activities significantly decreased among storage temperatures, with major effects detected at -20 °C. The repeated freeze/thaw cycles at -20 °C and 24-h mimic transport conditions also generated an expected degradation of the arylesterase in both serum and heparinized plasma while freeze/thaw cycles at -80 °C caused an increase of both arylesterase and paraoxonase activities on both sample types. In general, similar enzyme responses were detected between serum and heparinized plasma.

Interplay of UV-filter pollution and temperature rise scenarios on Mytilus galloprovincialis health: Unveiling sperm quality and adult physiology, biochemistry, and histology insights.

Addressing the impacts of emerging contaminants within the context of climate change is crucial for understanding ecosystem health decline. Among these, the organic UV-filters 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) are widely used in cosmetics and personal care products. Their unique physico-chemical properties, along with their growing commercialization and consumption, have made them ubiquitous in aquatic environments through both direct and indirect releases, raising significant concerns about their potential threats to inhabiting biota. Additionally, increasing surface water temperatures exacerbate ecological risks, making it imperative to understand the implications for non-target species at different biological levels. This study investigated the short- and long-term effects of UV-filters 4-MBC or BP-3, at ecologically relevant concentrations, combined with current and predicted warming scenarios, on the performance and male reproductive health of Mytilus galloprovincialis mussel populations. Using biomarkers across sub-cellular, cellular, tissue, and individual levels, the study revealed significant physiological and biochemical impairments in both sperm cells and adults exposed to UV-filters. Temperature emerged as the primary driver influencing mussel responses and modulating the impacts of 4-MBC/BP-3, emphasizing their sensitivity to temperatures outside the optimal range and interactive effects between stressors. Specifically, sperm motility declined with increasing UV-filter concentrations, while temperature alone influenced ROS production, leading to compromised mitochondrial activity and DNA damage in the presence of combined stressors, indicative of potential reproductive impairments. Adults exhibited high UV-filter bioconcentration potential in whole tissues, compromised physiological status, morphophysiological changes in digestive glands, oxidative stress, and alterations in metabolic capacity, antioxidant defences, and biotransformation mechanisms, correlating with UV-filter exposure and temperature increase. Among the UV-filters tested, 4-MBC was the most detrimental, especially when combined with warming. Overall, this study underscores the vulnerability of M. galloprovincialis to cumulative stressors and highlights the importance of employing a multi-biomarker approach to assess and mitigate the impacts of stressors on coastal ecosystems.

Subcellular effects of imidazolium-based ionic liquids with varying anions on the marine bivalve Mytilus galloprovincialis.

Green Chemistry involves applying a set of principles aimed at minimizing the use of hazardous substances in the design, production, and application of chemical products. In recent decades, Ionic Liquids (ILs) have emerged as more environmentally friendly substitutes for traditional organic solvents. This preference is primarily due to their low vapor pressure, which results in minimal atmospheric pollution and enhanced industrial safety. However, existing literature highlights the toxicity of ILs towards aquatic invertebrates. Consequently, this study points to assess the biochemical effects of a selection of ILs through an in vitro approach. Specifically, digestive gland and gill cellular fractions (S9) of the marine bivalve Mytilus galloprovincialis were exposed to varying concentrations (0.05-2 µM) of three ILs featuring identical cations but different anions. The ILs tested were 1-ethyl-3-methylimidazolium octanoate ([EMIM][Oct]), 1-ethyl-3-methylimidazolium acetate ([EMIM][OAc]), and 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIM][EtSO4]). The results indicate that [EMIM][Oct] induces higher toxicity in both S9 tissues, highlighting a strong effect of the anion. Overall, antioxidant and biotransformation defenses were significantly altered for all three ILs assessed. While acetylcholinesterase activity was significantly inhibited of about half of control activity, indicating neurotoxic damage as part of the toxicity mode of action of these ILs, neither lipid peroxidation nor alterations to DNA integrity were observed (≥100 %). This study supports the use of in vitro techniques as important tools capable of generating reliable ecotoxicological data, which can be further considered as a screening before in vivo testing and used for in silico modeling.

Mytilus galloprovincialis: A valuable bioindicator species for understanding the effects of diclofenac under warming conditions.

Drugs are chemical compounds used to treat and improve organic dysfunctions caused by diseases. These include analgesics, antibiotics, antidepressants, and antineoplastics. They can enter aquatic environments through wastewater streams, where their physico-chemical properties allow metabolites to distribute and accumulate. Current climate change and associated extreme weather events may significantly impact these substances' toxicity and aquatic organisms' sensitivity. Among the chemicals present in aquatic environments is the non-steroidal anti-inflammatory drug diclofenac (DIC), which the EU monitors due to its concentration levels. This study investigated the influence of temperature (control at 17 °C vs. 21 °C) on the effects of DIC (0 µg/L vs. 1 µg/L) in the mussel species Mytilus galloprovincialis. Significant results were observed between 17 and 21 °C. Organisms exposed to the higher temperature showed a decrease in several parameters, including metabolic capacity and detoxification, particularly with prolonged exposure. However, in some parameters, after 21 days, the M. galloprovincialis showed no differences from the control, indicating adaptation to the stress. The results of this study confirm that DIC concentrations in the environment, particularly when combined with increased temperatures, can produce oxidative stress and adversely affect M. galloprovincialis biochemical and physiological performance. This study also validates this species as a bioindicator for assessing environmental contamination with DIC. Beyond its direct impact on aquatic organisms, the presence of pharmaceuticals like DIC in the environment highlights the interconnectedness of human, animal, and ecosystem health, underscoring the One Health approach to understanding and mitigating environmental pollution.

The influence of temperature on the impacts of caffeine in mussels: Evaluating subcellular impacts and model predictions.

In aquatic ecosystems, the presence of pharmaceuticals, particularly caffeine (CAF), has been linked to wastewater discharge, hospital waste, and the disposal of expired pharmaceutical products containing CAF. Additionally, rising temperatures due to climate change are anticipated in aquatic environments. This study aimed to assess the toxicity of various CAF concentrations under current (17 °C) and projected (21 °C) temperature conditions, using the mussel Mytilus galloprovincialis as a bioindicator species. Subcellular impacts were evaluated following 28 days of exposure to four CAF concentrations (0.5; 1.0; 5.0; 10.0 µg/L) at the control temperature (17 °C). Only effects at an environmentally relevant CAF concentration (5.0 µg/L) were assessed at the highest temperature (21 °C). The overall biochemical response of mussels was evaluated using non-metric Multidimensional Scaling (MDS) and the Integrated Biomarker Response (IBR) index, while the Independent Action (IA) model was used to compare observed and predicted responses. Results showed that at 17 °C, increased CAF concentrations were associated with higher metabolism and biotransformation capacity, accompanied by cellular damage at the highest concentration. Conversely, under warming conditions (21 °C), the induction of antioxidant enzymes was observed, although insufficient to prevent cellular damage compared to the control temperature. Regarding neurotoxicity, at 17 °C, the activity of the acetylcholinesterase enzyme was inhibited up to 5.0 µg/L; however, at 10.0 µg/L, activity increased, possibly due to CAF competition for adenosine receptors. The IA model identified a synergistic response for most parameters when CAF and warming acted together, aligning with observed results, albeit with slightly lower magnitudes.

Exploring toxicological interactions in a changing sea: The case of the alkaloids caffeine and caulerpin.

The bisindolic alkaloid caulerpin (CAU) is a bioactive compound isolated from green algae of the genus Caulerpa that are highly invasive in the Mediterranean Sea. On the other side, the purine alkaloid caffeine (CAF) is one of the most globally consumed psychoactive substances and a widespread anthropogenic water pollutant. Both compounds display a large panel of biological properties and are well known to accumulate in the tissues of aquatic organisms and, in certain circumstances, co-occur in the human diet. On this premise, the present study aimed to investigate possible synergistic interactions between CAU and CAF by using the bivalve Mytilus galloprovincialis as a model organism. Mussels were exposed to CAF via medium while they were fed with food enriched with CAU. After treatments, biochemical analysis confirmed the toxic potential of CAF, with increased AChE activity and lipid peroxidation. Also, histopathological alterations were observed in the gills and digestive tubules. The NMR-based metabolomics analysis detected higher levels of free amino acids under CAF treatments. Conversely, the food administration of CAU did not affect the above toxicological biomarkers. In addition, we did not observe any cumulative effect between CAF and CAU toward increased cellular damage and neurotoxicity. On the other hand, a possible action of CAU in decreasing CAF toxicity could be hypothesized based on our results. This hypothesis is supported by the activity of CAU as an agonist of peroxisome proliferator-activated receptors (PPARs). PPARs mediate xenobiotic detoxification via cytochromes P450, which is involved in CAF metabolism. Overall, the results obtained not only rule out any cumulative adverse effects of CAF and CAU but also encourage further research to evaluate the possible use of CAU, a compound easily obtained through the valorization of biomass from invasive species, as a food additive to improve the clearance of xenobiotics.

In vitro screening of imidazolium and pyrrolidinium based ionic liquids toxicity on subcellular fractions of the Mediterranean mussel Mytilus galloprovincialis.

Ionic liquids (ILs) have been considered eco-friendly alternatives to conventional organic solvents. However, several studies have reported that ILs exert toxicity towards aquatic invertebrates. Applying in vitro methodology, the aim of the present study was to evaluate the potential effect of three ILs on the biochemical performance of exposed Mytilus galloprovincialis digestive gland and gills cellular fractions. Carboxylesterase might be involved in the derived toxicity mechanism of ILs as activity levels increased significantly in digestive gland exposed fractions. This group of ILs did not seem to induce genotoxicity, except in gills cellular fractions exposed to 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide. In the literature, in vitro methodology has been suggested as an important complement to animal testing and in silico studies. The present research underlines its efficacy as a quick pre-screening before in vivo testing, particularly with heterogenic groups of substances with high variability in composition, such as ILs and deep eutectic solvents.