Biopesticide spinosad: Unraveling ecotoxicological effects on zebrafish, Danio rerio.

Biopesticides are natural compounds considered more safe and sustainable for the environment. Spinosad (SPI) is a bioinsecticide used in marketed worldwide, to eradicate a variety of pests. This study aimed to assess the impacts of the SPI on the non-target organism zebrafish (Danio rerio). Several concentrations of SPI were tested to evaluate the acute (0.07-1.0 mg/L) and chronic (0.006-0.100 mg/L) ecotoxicological effects. To evaluate sub-individual effects, antioxidant defense, lipid peroxidation, energy sources, and cholinergic biomarkers were quantified. In both exposures, SPI induced significant effects on antioxidant defense indicating oxidative stress, disrupting energy pathways, and exhibiting neurotoxic effects, under environmentally relevant conditions. Integrated Biomarker Response (IBRv2) showed that with increasing SPI concentrations, an increase in impacts on organisms was recorded. This study demonstrates the vulnerability of a non-target organism to SPI, a bioinsecticide considered environmentally safe. Further research is essential to fully understand the implications of spinosad to aquatic biota.

Ecotoxicological Effects of Potassium Dichromate on the Tadpole Shrimp Triops longicaudatus.

The tadpole shrimp Triops longicaudatus is a freshwater crustacean with fast embryonic and larval development, short life cycle, and high fecundity. They are very active swimmers of a reasonable size, easy to spot and record. Such characteristics make it a promising candidate as an experimental model in ecotoxicology to evaluate the effects of aquatic pollutants, particularly using its locomotor behavior as an endpoint. To evaluate the sensitivity of T. longicaudatus and develop endpoints of interest, we conducted exposure experiments with lethal and sub-lethal concentrations of potassium dichromate, a compound known for its ecotoxicological importance and as a hexavalent chromium source. The endpoints evaluated were mortality, growth, sexual maturation, reproductive output, cholinesterase activity and locomotor/swimming behavior. The 96 h median lethal concentration was found to be 65 µg/L. Furthermore, exposure to potassium dichromate at higher concentrations had a significant negative impact on the growth rate of T. longicaudatus in terms of both body mass and length. The time for maturation was also delayed at higher concentrations. In addition, locomotor behavior allowed for the discrimination of all tested chromium concentrations and the control group and from each other, proving to be the most sensitive endpoint. Overall, the data support the potential of T. longicaudatus as a model for ecotoxicity testing, using apical endpoints with impact at the population level; in particular, results suggest that behavior assessments in this species might be useful for detecting hazardous compounds in environmental monitoring of freshwater ecosystems.

Toxic Relationships: Prediction of TBT's Affinity to the Ecdysteroid Receptor of Triops longicaudatus.

Tributyltin (TBT) is a biocide introduced in the 1960s in antifouling paints. Despite legislation banning its use, its persistence in the environment still causes significant harm to organisms. Tributyltin is a ligand of retinoid X receptors (RXR) and ecdysteroid receptors (EcRs), which in arthropods act as homologs of RXR. Focusing on Metazoan species, this study used genomic and proteomic information from different sources to compare their three-dimensional structure, phylogenetic distribution, and amino acid sequence alterations. The objective was to identify possible patterns that relate organisms' sensitivity to TBT using the species Triops longicaudatus as the basis for the comparisons. The results showed great conservation of this protein across several species when comparing the interaction amino acids described to RXR (an EcR analog) in Homo sapiens. The three-dimensional comparison of RXR showed little conformational variation between different sequences by maintaining the interaction pocket. As for the Species Sensitivity Distribution (SSD) curve, an HC05 = 0.2649 [0.0789-0.7082] µg/L was obtained with no specific distribution between the different taxa. Protein-ligand docking analysis was then used to confirm the SSD curve ranking of species. Still, the results showed an opposite trend that may be related, for example, to differences in the LC50 values used in the calculations. This study serves as the first step for applying bioinformatics techniques to produce information that can be used as an alternative to animal or cellular experimentation. These techniques could be adapted to various chemicals and proteins, allowing for observations in a shorter timeframe and providing information on a broader spectrum.

Insights into environmental caffeine contamination in ecotoxicological biomarkers and potential health effects of Danio rerio.

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.

Oral vaccination of fish against vibriosis using spore-display technology.

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.

Novel protein carrier system based on cyanobacterial nano-sized extracellular vesicles for application in fish.

Aquaculture has been one of the fastest-growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP-loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

Environmental diagnosis with Raman Spectroscopy applied to diatoms.

Freshwater quality has been changing due to the ever greater use of water resources and the contamination load resulting from human activities. Management of these systems, thus, requires constant diagnose of water quality with fast and efficient methodologies. The conventional methods adopted are, however, time-consuming, often very expensive, and require specialised expertise. Raman spectroscopy (RS) is a simple, fast and label-free technique that can be applied to environmental diagnosis using diatoms. Here, we developed a diagnostic method based on Raman spectroscopy applied to freshwater diatoms. For this, Raman spectra were recorded from diatoms of three lakes of a natural city park. The data acquired was analysed by chemometrics methods to describe the data (Partial Least Squares Regression), infer relationships in the dataset (Cluster Analysis) and produce classification models (Artificial Neural Network). The classification models developed diagnosed the lakes with excellent accuracy (89%) without requiring taxonomic information about the diatom species recorded. This study provides a proof-of-concept for the application of diatom Raman spectroscopy to diagnosing water quality, laying an important foundation for future environmental studies aiming at assessing freshwater systems, to be replicated at larger scales and to varied geographic settings.

Bacillus spp. Inhibit Edwardsiella tarda Quorum-Sensing and Fish Infection.

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

Dietary tryptophan supplementation does not affect growth but increases brain serotonin level and modulates the expression of some liver genes in zebrafish (Danio rerio).

This study aimed at assessing the effects of the dietary tryptophan (Trp) supplementation on growth and feed utilization, brain serotonin content, and expression of selected liver genes (involved in the liver serotonin pathway, protein synthesis degradation, and antioxidant activity) in zebrafish. A growth trial was conducted with zebrafish juveniles fed five experimental isoproteic (40%DM) and isolipidic (8%DM) fishmeal-based diets containing graded levels of Trp: a Trp-non-supplemented diet (diet Trp0, with 0.22% Trp) and four Trp-supplemented diets containing 2-16 times higher Trp content (diets Trp2, Trp4, Trp8, and Trp16 with 0.40, 0.91, 2.02, and 3.34% Trp, respectively). Diets were tested in quadruplicate, with fish being fed twice a day, 6 days a week for 6 weeks to apparent visual satiation. At the end of the trial, growth performance and feed utilization were assessed, and fish from all experimental groups were sampled for whole-body composition analysis. In addition, fish fed low (Trp0), medium (Trp4), and high (Trp16) Trp diets were also sampled for analysis of brain serotonin content and liver gene expression. Tested tryptophan levels did not influence growth performance nor feed intake. However, values of energy and nitrogen retention as well as body energy content indicate a better feed utilization with diets containing around 0.9% and 2.0% DM Trp. Brain serotonin content increased with increasing dietary tryptophan levels. In addition, regarding liver genes, dietary treatment had a modulatory effect on the expression of Htr1aa and Htr2cl1 genes (encoding for serotonin receptors), TPH1a gene (encoding for tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of serotonin from tryptophan), TOR gene (involved in protein synthesis), and Keap1 gene (involved in antioxidant responses).

Microplastics as a vehicle of exposure to chemical contamination in freshwater systems: Current research status and way forward.

Contamination by microplastics is increasing steadily worldwide, affecting all environments. Additionally, aquatic organisms are often exposed to mixtures of other contaminants, including various chemicals. Numerous studies reported adsorption of chemicals to microplastics, raising concern about their possible role as vehicles of exposure through transfer to biota. Nevertheless, until recently, the studies on the topic were mostly focused on the marine environment. In the past five years, however, plenty of publications contributed empirical data about freshwater ecosystems, raising the need for a critical appraisal of the information. Herein the scientific literature was reviewed and multivariate data analysis was done. The analysed studies employed widely different experimental designs, endpoints, test species, shapes and concentrations of various polymer types and chemicals, often not relevant for the freshwater environment. Our integrated analytical approach revealed unfathomable research gaps, given the theoretical knowledge available and lessons learned from research about the marine environment. Greater harmonization of laboratory studies investigating this topic is needed, as well as testing conditions reflecting real exposure scenarios. Furthermore, standardized testing protocols are urgently required to guide such experiments and improve the comparability of the results obtained.

Raman spectroscopy applied to diatoms (microalgae, Bacillariophyta): Prospective use in the environmental diagnosis of freshwater ecosystems.

Diatom species are good pollution bioindicators due to their large distribution, fast response to changes in environmental parameters and different tolerance ranges. These organisms are used in ecological water assessment all over the world using autoecological indices. Such assessments commonly rely on the taxonomic identification of diatom species-specific shape and frustule ornaments, from which cell counts, species richness and diversity indices can be estimated. Taxonomic identification is, however, time-consuming and requires years of expertise. Additionally, though the diatom autoecological indices are region-specific, they are often applied indiscriminately across regions. Raman spectroscopy is a simpler, fast and label-free technique that can be applied to environmental diagnosis with diatoms. However, this approach has been poorly explored. This work reviews Raman spectroscopy studies involving the structure, location and conformation of diatom cell components and their variation under different conditions. A critical appreciation of the pros and cons of its application to environmental diagnosis is also given. This knowledge provides a strong foundation for the development of environmental protocols using Raman spectroscopy in diatoms. Our work aims at stimulating further research on the application of Raman spectroscopy as a tool to assess physiological changes and water quality under a changing climate.

Linking chemical exposure to lipid homeostasis: A municipal waste water treatment plant influent is obesogenic for zebrafish larvae.

Obesity, a risk factor for the development of type-2 diabetes, hypertension, cardiovascular disease, hepatic steatosis and some cancers, has been ranked in the top 10 health risk in the world by the World Health Organization. Despite the growing body of literature evidencing an association between the obesity epidemic and specific chemical exposure across a wide range of animal taxa, very few studies assessed the effects of chemical mixtures and environmental samples on lipid homeostasis. Additionally, the mode of action of several chemicals reported to alter lipid homeostasis is still poorly understood. Aiming to fill some of these gaps, we combined an in vivo assay with the model species zebrafish (Danio rerio) to screen lipid accumulation and evaluate expression changes of key genes involved in lipid homeostasis, alongside with an in vitro transactivation assay using human and zebrafish nuclear receptors, retinoid X receptor α and peroxisome proliferator-activated receptor γ. Zebrafish larvae were exposed from 4 th day post-fertilization until the end of the experiment (day 18), to six different treatments: experimental control, solvent control, tributyltin at 100 ng/L Sn and 200 ng/L Sn (positive control), and wastewater treatment plant influent at 1.25% and 2.5%. Exposure to tributyltin and to 2.5% influent led to a significant accumulation of lipids, with white adipose tissue deposits concentrating in the perivisceral area. The highest in vitro tested influent concentration (10%) was able to significantly transactivate the human heterodimer PPARγ/RXRα, thus suggesting the presence in the influent of HsPPARγ/RXRα agonists. Our results demonstrate, for the first time, the ability of complex environmental samples from a municipal waste water treatment plant influent to induce lipid accumulation in zebrafish larvae.

Lymnaea stagnalis as a freshwater model invertebrate for ecotoxicological studies.

Lymnaea stagnalis, also referred to as great or common pond snail, is an abundant and widespread invertebrate species colonizing temperate limnic systems. Given the species importance, studies involving L. stagnalis have the potential to produce scientifically relevant information, leading to a better understanding of the damage caused by aquatic contamination, as well as the modes of action of toxicants. Lymnaea stagnalis individuals are easily maintained in laboratory conditions, with a lifespan of about two years. The snails are hermaphrodites and sexual maturity occurs about three months after egg laying. Importantly, they can produce a high number of offspring all year round and are considered well suited for use in investigations targeting the identification of developmental and reproductive impairments. The primary aims of this review were two-fold: i) to provide an updated and insightful compilation of established toxicological measures determined in both chronic and acute toxicity assays, as useful tool to the design and development of future research; and ii) to provide a state of the art related to direct toxicant exposure and its potentially negative effects on this species. Relevant and informative studies were analysed and discussed. Knowledge gaps in need to be addressed in the near future were further identified.

Dietary Protein Requirement During Juvenile Growth of Zebrafish (Danio rerio).

A standard diet for zebrafish, based on their specific nutritional requirements, is of primary importance to improve experimental outcomes with this model organism and optimize its large-scale production. However, the main basic nutritional requirements of zebrafish are yet to be determined. This study aimed at contributing to fill this gap by evaluating the dietary protein requirement of zebrafish juvenile. Ten isoenergetic fishmeal-based diets with increasing protein levels (15%-60%) were formulated, and each diet was assigned to duplicated groups of zebrafish (53.6 mg/17.8 mm initial mean body weight/fork length), fed to apparent satiation during 8 weeks. Weight gain, protein retention, and feed efficiency significantly increased in fish fed diets with increasing protein levels up to 35%-40% and then stabilized. Based on dose-response models, the dietary protein requirement of zebrafish juvenile was estimated at 37.6% and 44.8% for maximum weight gain and maximum protein retention, respectively (with a crude protein-to-energy ratio of about 22.5 g/MJ), corresponding to a protein intake of 14 mg/g average body weight/day. Feed intake increased linearly when fish were fed diets with decreasing protein levels below the estimated requirement, suggesting that zebrafish would regulate feed intake primarily to meet protein needs. On the other hand, the efficiency of protein utilization and retention linearly decreased when fish were fed diets with increasing protein levels above the estimated requirement, indicating that the excess of dietary protein would be deaminated, contributing to increased ammonia excretion. The whole-body composition of fish was affected by the dietary protein level, with fish fed diets with higher protein levels having higher water and protein contents and lower energy content. Considering that zebrafish juveniles are often reared with diets containing excessive amounts of protein, we suggest that the estimated protein requirement should be taken into account to formulate a more suitable, cost-effective, and less pollutant diet for this species.

Effects of Tributyltin and Other Retinoid Receptor Agonists in Reproductive-Related Endpoints in the Zebrafish (Danio rerio).

Both field and experimental data examined the influence of exposure to environmental contaminant tributyltin (TBT) on marine organisms. Although most attention focused on the imposex phenomenon in gastropods, adverse effects were also observed in other taxonomic groups. It has been shown that imposex induction involves modulation of retinoid signaling in gastropods. Whether TBT influences similar pathways in fish is yet to be addressed. In this study, larvae of the model teleost Danio rerio were exposed to natural retinoids, all-trans-retinoic acid, 9-cis-retinoic acid, and all-trans-retinol, as well as to the RXR synthetic pan-agonist methoprene acid (MA) and to TBT. Larvae were exposed to TBT from 5 days post fertilization (dpf) to adulthood, and reproductive capacity was assessed and correlated with mode of action. TBT significantly decreased fecundity at environmentally relevant levels at 1 µg TBT Sn/g in diet. Interestingly, in contrast to previous reports, TBT altered zebrafish sex ratio toward females, whereas MA exposure biased sex toward males. Since fecundity was significantly altered in the TBT-exposed group with up to 62% decrease, the potentially affected pathways were investigated. Significant downregulation was observed in brain mRNA levels of aromatase b (CYP19a1b) in females and peroxisome proliferator activated receptor gamma (PPARg) in both males and females, suggesting an involvement of these pathways in reproductive impairment associated with TBT.

Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress.

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg(-1) of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.

Acute and Chronic Toxicity of Nitrate to Early Life Stages of Zebrafish--Setting Nitrate Safety Levels for Zebrafish Rearing.

Recirculating aquaculture systems (RAS) have been widely used for zebrafish rearing, allowing holding of many thousands of fish at high densities. Water quality in RAS largely depends on biofilters that ultimately convert the extremely toxic ammonia excreted by fish into the much less toxic nitrate. However, when water renewal is minimal in RAS, nitrate can accumulate to high enough levels to negatively impact fish welfare and performance. Therefore, the setting of safety levels of nitrate for zebrafish should be a priority to avoid unwanted effects in both the intensive production of this species and research outputs. The present study aimed to define nitrate safety levels for zebrafish based on acute and chronic toxicity bioassays in early life stages of this species. Acute bioassays revealed ontogenetic changes in response to high nitrate levels. Based on NOEC (no observed effect concentration) values, safety levels should be set at 1450, 1855, and 1075 mg/L NO3(-)-N to prevent acute lethal effects in embryos, newly-hatched larvae, and swim-up larvae, respectively. In the chronic bioassay, larvae were exposed to nitrate concentrations of 50, 100, 200, and 400 mg/L NO3(-)-N during the entire larval period (23 days). No negative effects were observed either on larval performance or condition at concentrations up to 200 mg/L NO3(-)-N. However, at 400 mg/L NO3(-)-N, survival drastically decreased and fish showed reduced growth and evidence of morphological abnormalities. Accordingly, a safety level of 200 mg/L NO3(-)-N is recommended during the larval rearing of zebrafish to prevent negative impacts on juvenile production.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Effects of storage, processing and proteolytic digestion on microcystin-LR concentration in edible clams.

Accumulation of microcystin-LR (MC-LR) in edible aquatic organisms, particularly in bivalves, is widely documented. In this study, the effects of food storage and processing conditions on the free MC-LR concentration in clams (Corbicula fluminea) fed MC-LR-producing Microcystisaeruginosa (1×10(5) cell/mL) for four days, and the bioaccessibility of MC-LR after in vitro proteolytic digestion were investigated. The concentration of free MC-LR in clams decreased sequentially over the time with unrefrigerated and refrigerated storage and increased with freezing storage. Overall, cooking for short periods of time resulted in a significantly higher concentration (P<0.05) of free MC-LR in clams, specifically microwave (MW) radiation treatment for 0.5 (57.5%) and 1 min (59%) and boiling treatment for 5 (163.4%) and 15 min (213.4%). The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of MC-LR degradation by pancreatic enzymes. Our results suggest that risk assessment based on direct comparison between MC-LR concentrations determined in raw food products and the tolerable daily intake (TDI) value set for the MC-LR might not be representative of true human exposure.

Toxic effects of pure anatoxin-a on biomarkers of rainbow trout, Oncorhynchus mykiss.

Anatoxin-a is a neurotoxin produced by various bloom-forming cyanobacteria. Although it shows widespread occurrence and is highly toxic to rodents, its mechanisms of action and biotransformation, and effects in fish species are still poorly understood. The main aim of this study was, thus, to investigate sub-lethal effects of anatoxin-a on selected biochemical markers in rainbow trout fry in order to get information about the mechanisms of toxicity and biotransformation of this toxin in fish. Trout fry were administered sub-lethal doses of anatoxin-a (0.08-0.31 µg g⁻¹) intraperitoneally. Livers and muscle tissue were collected 72 h later for quantification of key enzyme activities as biochemical markers. Enzymes assessed in muscle tissues were related to cholinergic transmission (acetylcholinesterase [AChE]), energy metabolism (lactate dehydrogenase [LDH] and NADP⁺-dependent isocitrate dehydrogenase [IDH]). Enzymes assessed in the liver were involved in biotransformation (ethoxyresorufin-O-deethylase [EROD] and glutathione S-transferases [GST]). The results indicated a significant increasing trend for AChE activity with the dose of anatoxin-a, possibly representing an attempt to cope with overstimulation of muscle activity by the toxin, which competes with acetylcholine for nicotinic receptors binding. Anatoxin-a was also found to significantly induce the activities of liver EROD and GST, indicating the involvement of phase I and II biotransformation in its detoxification. Likewise, lactate dehydrogenase activity recorded in fry muscle increased significantly with the dose of anatoxin-a, suggesting an induction of the anaerobic pathway of energy production to deal with toxic stress induced by the exposure. Altogether, the results suggest that under continued exposure in the wild fish may experience motor difficulties, possibly becoming vulnerable to predators, and be at increased metabolic demand to cope with energetic requirements imposed by anatoxin-a biotransformation mechanisms.