Isolation and characterization of the morphology, size and particle number of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) cell line derived large and small extracellular vesicles.

Extracellular vesicles (EVs) are 50-1,000 nm lipid bilayer-bound vesicles, released into the extracellular environment by various cell types for intercellular communication purposes. The quantitative and qualitative characteristics of EVs can be affected by stress and pathological conditions. The majority of extracellular vesicle (EV) studies have been performed on mammalian cell lines or bodily fluids. EVs have been previously described from bodily fluids like plasma, serum or mucus in different fish species, however the available knowledge of fish cell line derived EVs is limited and in the vast majority of studies, the overall focus is on small EVs (< 200 nm). We isolated large and small extracellular vesicles from zebrafish (Danio rerio) liver (ZFL), rainbow trout (Oncorhynchus mykiss) liver (RTL-W1), gill (RTgill-W1) and intestinal epithelial (RTgutGC) cell lines using stepwise centrifugation and characterized the size and morphology of EVs. Here we demonstrated that large and small extracellular vesicles can be successfully isolated using stepwise centrifugation from the serum-free medium of the selected piscine cell lines after a 24-h incubation period. The size distribution of large and small EVs isolated from the piscine cell lines suggest that large and small EV groups show high diversity in size ranges, containing heterogenous subpopulations in sizes, and the results highly depend on the applied method and whether filtration steps were included following the isolation. The spherical morphology of EVs was verified by transmission electron microscopy.

A multi-biomarker study on Atlantic salmon (Salmo salar L.) affected by the emerging Red Skin Disease in the Baltic Sea.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so-called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Thyroid function and immune status in perch (Perca fluviatilis) from lakes contaminated with PFASs or PCBs.

The environment contains a multitude of man-made chemicals, some of which can act as endocrine disruptors (EDCs), while others can be immunotoxic. We evaluated thyroid disruption and immunotoxic effects in wild female perch (Perca fluviatilis) collected from two contaminated areas in Sweden; one site contaminated with per- and polyfluoroalkyl substances (PFASs) and two sites contaminated with polychlorinated biphenyls (PCBs), with one reference site included for each area. The hepatic mRNA expression of thyroid receptors α and ß, and the thyroid hormone metabolising iodothyronine deiodinases (dio1, dio2 and dio3) were measured using real-time PCR, while the levels of thyroid hormone T3 in plasma was analysed using a radioimmunoassay. In addition, lymphocytes, granulocytes, and thrombocytes were counted microscopically. Our results showed lower levels of T3 as well as lower amounts of lymphocytes and granulocytes in perch collected from the PFAS-contaminated site compared to reference sites. In addition, expressions of mRNA coding for thyroid hormone metabolising enzymes (dio2 and dio3) and thyroid receptor α (thra) were significantly different in these fish compared to their reference site. For perch collected at the two PCB-contaminated sites, there were no significant differences in T3 levels or in expression levels of the thyroid-related genes, compared to the reference fish. Fish from one of the PCB-contaminated sites had higher levels of thrombocytes compared with both the second PCB lake and their reference lake; hence PCBs are unlikely to be the cause of this effect. The current study suggests that lifelong exposure to PFASs could affect both the thyroid hormone status and immune defence of perch in the wild.

Assessing the effects of textile leachates in fish using multiple testing methods: From gene expression to behavior.

The textile industry, while of major importance in the world economy, is a toxic industry utilizing and emitting thousands of chemical substances into the aquatic environment. The aim of this project was to study the potentially harmful effects associated with the leaching of chemical residues from three different types of textiles: sportswear, children's bath towels, and denim using different fish models (cell lines, fish larvae and juvenile fish). A combination of in vitro and in vivo test systems was used. Numerous biomarkers, ranging from gene expression, cytotoxicity and biochemical analysis to behavior, were measured to detect effects of leached chemicals. Principle findings indicate that leachates from all three types of textiles induced cytotoxicity on fish cell lines (RTgill-W1). Leachates from sportswear and towels induced mortality in zebrafish embryos, and chemical residues from sportswear reduced locomotion responses in developing larval fish. Sportswear leachate increased Cyp1a mRNA expression and EROD activity in liver of exposed brown trout. Leachates from towels induced EROD activity and VTG in rainbow trout, and these effects were mitigated by the temperature of the extraction process. All indicators of toxicity tested showed that exposure to textile leachate can cause adverse reactions in fish. These findings suggested that chemical leaching from textiles from domestic households could pose an ecotoxicological threat to the health of the aquatic environment.

Development of three-dimensional (3D) spheroid cultures of the continuous rainbow trout liver cell line RTL-W1.

In vitro experimental systems based on continuous piscine cell lines can be used as an alternative to animal tests for obtaining qualitative and quantitative information on the possible fate and effect of chemicals in fish. However, their capability to reproduce complex metabolic processes and toxic responses as they occur in vivo is limited due to the lack of organ-specific tissue architecture and functions. Here we introduce a three-dimensional (3D) in vitro experimental system based on spheroidal aggregate cultures (spheroids) of the continuous rainbow trout liver cell line RTL-W1 and provide a first description of their structural and functional properties including growth, viability/longevity, metabolic activity, ultrastructure and cytochrome P450 1A (CYP1A) expression determined by bright-field, multi-photon fluorescence and transmission electron microscopy as well as RT-qPCR analysis. Our results show that RTL-W1 cells in 3D spheroids (ø ~ 150 µm) (including those in the interior) were viable, metabolically active and had higher basal and ß-naphthoflavone-induced CYP1A expression levels than conventional 2D cell cultures. Furthermore, they displayed ultrastructural characteristics similar to differentiated hepatocytes. The available evidence suggests that 3D RTL-W1 spheroids may have enhanced hepatotypic functions and be a superior in vitro model to assess hepatic biotransformation, bioaccumulation and chronic toxicity compared to conventional cell monolayer cultures.

Size Matters: Ingestion of Relatively Large Microplastics Contaminated with Environmental Pollutants Posed Little Risk for Fish Health and Fillet Quality.

In this study, we investigated biological effects associated with ingestion of polystyrene (PS) microplastic (MPs) in fish. We examined whether ingestion of contaminated PS MPs (100-400 µm) results in chemical stress in rainbow trout (Oncorhynchus mykiss) liver and we explored whether this exposure can affect the oxidative stability of the fillet during ice storage. Juvenile rainbow trout were fed for 4 weeks with four different experimental diets: control (1) and feeds containing virgin PS MPs (2) or PS MPs exposed to sewage (3) or harbor (4) effluent. A suite of ecotoxicological biomarkers for oxidative stress and xenobiotic-related pathways was investigated in the hepatic tissue, and included gene expression analyses and enzymatic measurements. The potential impact of MPs exposure on fillet quality was investigated in a storage trial where lipid hydroperoxides, loss of redness and development of rancid odor were assessed as indications of lipid peroxidation. Although, chemical analysis of PS MPs revealed that particles sorb environmental contaminants (e.g., PAHs, nonylphenol and alcohol ethoxylates and others), the ingestion of relatively high doses of these PS MPs did not induce adverse hepatic stress in fish liver. Apart from small effect on redness loss in fillets of fish exposed to PS MPs, the ingestion of these particles did not affect lipid peroxidation or rancid odor development, thus did not affect fillet's quality.

Exposure to a nicotinoid pesticide reduces defensive behaviors in a non-target organism, the rusty crayfish Orconectes rusticus.

Imidacloprid is the most widely used of the nicotinoid insecticides, the fastest growing class of pesticides on the global market. Although less toxic to mammals and birds compared to organophosphates, nicotinoids have the potential to impact non-target invertebrates, especially through sublehal effects on behavior, physiology, reproduction, and development. We investigated the impact of sublethal doses of imidacloprid on the defensive responses of rusty crayfish Orconectes rusticus exposed to 0, 1, 10, and 100 µg•L-1 of imidacloprid for 10 days (n = 7 crayfish per treatment). Defensive behaviors were examined with the rod test, in which a glass rod was jabbed into the crayfish's container at a 90 degree angle from the bottom and about 0.5 cm directly in front of the crayfish. Crayfish responded to the rod aggressively with claw raising and pinching, neutrally (no response), or by backing or tail-flipping away. The frequency of neutral responses more than doubled after four days in the high (100 µg•L-1) group and after eight days in the low (1 µg•L-1) exposure group. Furthermore, most crayfish in the 100 µg•L-1 treatment were not able to right themselves within 30 s when placed on their backs. Several studies have reported concentrations of imidacloprid contamination in freshwater ecosystems that exceed this study's lowest exposure scenario, 1 µg•L-1. We therefore conclude that imidacloprid contamination reduces the defensive behaviors of crayfish, impairing their ability to survive in habitats where they play important ecological roles.

Effects of Environmental Enrichment on Exposure to Human-Relevant Mixtures of Endocrine Disrupting Chemicals in Zebrafish.

Fish models used for chemical exposure in toxicological studies are normally kept in barren tanks without any structural environmental enrichment. Here, we tested the combined effects of environmental enrichment and exposure to two mixtures of endocrine disrupting chemicals (EDCs) in zebrafish. Firstly, we assessed whether developmental exposure to an EDC mixture (MIX G1) combined with rearing the fish in an enriched environment influenced behaviour later in life. This was evaluated using locomotion tracking one month after exposure, showing a significant interaction effect between enrichment and the MIX G1 exposure on the measured locomotion parameters. After three months, we assessed behaviour using custom-made behaviour tanks, and found that enrichment influenced swimming activity. Control fish from the enriched environment were more active than control fish from the barren environment. Secondly, we exposed adult zebrafish to a separate EDC mixture (MIX G0) after rearing them in a barren or enriched environment. Behaviour and hepatic mRNA expression for thyroid-related genes were assessed. There was a significant interaction effect between exposure and enrichment on swimming activity and an effect of environment on latency to approach the group of conspecifics, where enriched fish took more time to approach the group, possibly indicating that they were less anxious. Hepatic gene expression of a thyroid-related gene (thrb) was significantly affected by EDC exposure, while enrichment had no discernible impact on the expression of the measured genes. In conclusion, environmental enrichment is important to consider when studying the effects of EDCs in fish.

Immunomodulatory effects of single and combined exposure to ZnO and TiO2 nanoparticles on rainbow trout challenged with Aeromonas salmonicida achromogenes.

The increasing release of engineered nanoparticles (ENPs) in aquatic ecosystems stresses the need for stringent investigations of nanoparticle mixture toxicity towards aquatic organisms. Here, the individual and combined immunotoxicity of two of the most consumed ENPs, the ZnO and the TiO2 ones, was investigated on rainbow trout juveniles (Oncorhynchus mykiss). Fish were exposed to environmentally realistic concentrations (21 and 210 µg L-1 for the ZnO and 210 µg L-1 for the TiO2) for 28 days, and then challenged with the pathogenic bacterium, Aeromonas salmonicida achromogenes. Antioxidant and innate immune markers were assessed before and after the bacterial infection. None of the experimental conditions affected the basal activity of the studied innate immune markers and the redox balance. However, following the bacterial infection, the expression of genes coding for pro and anti-inflammatory cytokines (il1ß and il10), as well as innate immune compounds (mpo) were significantly reduced in fish exposed to the mixture. Conversely, exposure to ZnO NPs alone seemed to stimulate the immune response by enhancing the expression of the IgM and c3 genes for instance. Overall, our results suggest that even though the tested ENPs at their environmental concentration do not strongly affect basal immune functions, their mixture may alter the development of the immune response when the organism is exposed to a pathogen by interfering with the inflammatory response.

Physiological responses of Atlantic cod to climate change indicate that coastal ecotypes may be better adapted to tolerate ocean stressors.

Healthy ecosystems and species have some degree of resilience to changing conditions, however as the frequency and severity of environmental changes increase, resilience may be diminished or lost. In Sweden, one example of a species with reduced resilience is the Atlantic cod (Gadus morhua). This species has been subjected to overfishing, and with additional pressures such as habitat degradation and changing environmental conditions there has been little to no recovery, despite more than a decade of management actions. Given the historical ecological, economical, and cultural significance of cod, it is important to understand how Atlantic cod respond to global climate change to recover and sustainably manage this species in the future. A multi-stressor experiment was conducted to evaluate physiological responses of juvenile cod exposed to warming, ocean acidification, and freshening, changes expected to occur in their nursery habitat. The response to single drivers showed variable effects related to fish biometrics and increased levels of oxidative stress dependent parameters. Importantly, two separate responses were seen within a single treatment for the multi-stressor and freshening groups. These within-treatment differences were correlated to genotype, with the offshore ecotype having a heightened stress response compared to the coastal ecotype, which may be better adapted to tolerate future changes. These results demonstrate that, while Atlantic cod have some tolerance for future changes, ecotypes respond differently, and cumulative effects of multiple stressors may lead to deleterious effects for this important species.

Acoustic detection of melanosome transport in Xenopus laevis melanophores.

Organelle transport studies are often performed using melanophores from lower vertebrates due to the ease of inducing movements of pigment granules (melanosomes) and visualizing them by optical microscopy. Here, we present a novel methodology to monitor melanosome translocation (which is a light-sensitive process) in the dark using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. This acoustic sensing method was used to study dispersion and aggregation of melanosomes in Xenopus laevis melanophores. Reversible sensor responses, correlated to optical reflectance measurements, were obtained by alternating addition and removal of melatonin (leading to melanosome aggregation) and melanocyte-stimulating hormone (MSH) (leading to melanosome dispersion). By confocal microscopy, it was shown that a vertical redistribution of melanosomes occurred during the dispersion/aggregation processes. Furthermore, the transport process was studied in the presence of cytoskeleton-perturbing agents disrupting either actin filaments (latrunculin) or microtubules (nocodazole). Taken together, these experiments suggest that the acoustic responses mainly originate from melanosome transport along actin filaments (located close to the cell membrane), as expected based on the penetration depth of the QCM-D technique. The results clearly indicate the potential of QCM-D for studies of intracellular transport processes in melanophores.

Biliary PAH and alkylphenol metabolites, biomarker enzyme activities, and gene expression levels in the deep-sea fish Alepocephalus rostratus.

Biliary polycyclic aromatic hydrocarbon (PAH) and alkylphenol (AP) metabolites, hepatic gene expression, and corresponding enzyme activities were determined in the deep-sea fish Alepocephalus rostratus from two sites within the Mediterranean. Biliary metabolites included the hydroxylated PAH metabolites (OH-PAHs) 1-naphthol, 2-naphthol, 9-fluorenol, 9-phenanthrol, and 1-pyrenol and the APs 4-nonylphenol (NP) and 4-tert-octylphenol (OP). Five biomarker genes, namely, cytochrome P450 1A (CYP1A), vitellogenin (Vtg), catalase (CAT), Cu/Zn-superoxide-dismutase (Cu/Zn-SOD), and glutathione reductase (GR), were quantified using qRT-PCR. Moreover, corresponding enzyme activities (ethoxyresorufin-O-deethylase (EROD), CAT, SOD, and GR) were also determined. The ΣOH-PAHs detected ranged from 21.1 to 300.3 ng/g bile and were mainly composed of 1-naphthol. Both NP and OP metabolites were detected in all samples with concentrations ranging from 17.4 to 107.2 ng/g bile and 4.9 to 17.3 ng/g bile, respectively, and levels were significantly higher in samples from the western Mediterranean (WM) compared to those from the Catalan slope (CS). Accordingly, gene expression was significantly induced in male fish from the WM; however, these results were not reflected in enzyme activity levels. In particular, males caught at 2000 m in the WM exhibited 35-times higher Vtg levels compared to those from the CS, suggesting that endocrine-disrupting effects may potentially be occurring in such remote environments as the deep-sea.

Chronic poly(l-lactide) (PLA)- microplastic ingestion affects social behavior of juvenile European perch (Perca fluviatilis).

Juvenile perch were exposed to 2 % (w/w) poly(l-lactide) (PLA) microplastic particles (90-150 µm) in food pellets, or 2 % (w/w) kaolin particles, and a non-particle control food over 6 months. Chronic ingestion of PLA microplastics significantly affected the social behavior of juvenile perch, evident as a significantly increased reaction to the vision of conspecifics. PLA ingestion did not alter life cycle parameters, or gene expression levels. In addition to reactions to conspecifics, fish that ingested microplastic particles showed tendencies to decrease locomotion, internal schooling distance, and active predator responses. The ingestion of natural particles (kaolin) significantly downregulated the expression of genes related to oxidative stress and androgenesis in the liver of juvenile perch, and we found tendencies to downregulated expression of genes related to xenobiotic response, inflammatory response, and thyroid disruption. The present study demonstrated the importance of natural particle inclusion and the potential behavioral toxicity of one of the commercially available biobased and biodegradable polymers.

Characterization and Toxic Potency of Airborne Particles Formed upon Waste from Electrical and Electronic Equipment Waste Recycling: A Case Study.

Manual dismantling, shredding, and mechanical grinding of waste from electrical and electronic equipment (WEEE) at recycling facilities inevitably lead to the accidental formation and release of both coarse and fine particle aerosols, primarily into the ambient air. Since diffuse emissions to air of such WEEE particles are not regulated, their dispersion from the recycling plants into the adjacent environment is possible. The aim of this interdisciplinary project was to collect and characterize airborne WEEE particles smaller than 1 µm generated at a Nordic open waste recycling facility from a particle concentration, shape, and bulk and surface composition perspective. Since dispersed airborne particles eventually may reach rivers, lakes, and possibly oceans, the aim was also to assess whether such particles may pose any adverse effects on aquatic organisms. The results show that WEEE particles only exerted a weak tendency toward cytotoxic effects on fish gill cell lines, although the exposure resulted in ROS formation that may induce adverse effects. On the contrary, the WEEE particles were toxic toward the crustacean zooplankter Daphnia magna, showing strong effects on survival of the animals in a concentration-dependent way.

Influence of natural organic matter on the transformation of metal and metal oxide nanoparticles and their ecotoxic potency in vitro.

Increased use and production of engineered nanoparticles (NPs) lead to an elevated risk of their diffuse dispersion into the aquatic environment and increased concern on unknown effects induced by their release into the aquatic ecosystem. An improved understanding of the environmental transformation processes of NPs of various surface characteristics is hence imperative for risk assessment and management. This study presents results on effects of natural organic matter (NOM) on the environmental transformation and dissolution of metal and metal oxide NPs of different surface and solubility properties in synthetic freshwater (FW) with and without NOM. Adsorption of NOM was evident on most of the studied NPs, except Sb and Sb2O3, which resulted in the formation of negatively charged colloids of higher stability and smaller size distribution compared with the same NPs in FW only. The dissolution rate of the NPs in the presence of NOM correlated with the strength of interactions between the carboxylate group of NOM and the particle surface, and resulted in either no (Mn, Sb, ZnO NPs), increased (Co, Sn NPs) and decreased (Ni, NiO, Sb2O3, Y2O3 NPs) levels of dissolution. One type of metal NP from each group (Mn, Ni, Sn) were investigated to assess whether observed differences in adsorption of NOM and dissolution would influence their ecotoxic potency. The results showed Mn, Ni, and Sn NPs to generate intracellular reactive oxygen species (ROS) in a time and dose-dependent manner. The extent of ROS generation in FW was similar for both Mn and Ni NPs but higher for Sn NPs. These findings are possibly related to interactions and infiltration of the NPs with the cells, which lead to redox imbalances which could induce oxidative stress and cell damage. At the same time, the presence of NOM generally reduced the intracellular ROS generation by 20-40% for the investigated NPs and also reduced cytotoxicity of Sn NPs, which can be attributed to the stronger interaction of carboxylate groups of NOM with the surface of the NPs.

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures.

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.

Enzymatic, morphological, and genotoxic effects of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss).

Fish have defense systems that are capable of repairing damages caused by xenobiotics like benzo[a]pyrene (BaP), so the aims of this study were to identify BaP toxicity in melanomacrophages (MMs) cytoskeleton, evaluate the melanin area in MMs, and analyze genotoxicity. Rainbow trout juveniles (n = 24) were split in 48h and 7d treatments that received 2 mg/kg of BaP. After the experiment, blood samples were collected and liver was removed, to proceed with the analysis: EROD activity, MMs melanin area quantification, melanosomes movements, and a genotoxicity test. The results revealed increased in EROD activity after 48-h and 7-day BaP exposure. The group 7d displayed a reduction in MMs pigmented area, melanosomes aggregation, in addition to an increased frequency of micronucleus. By means of the EROD assay, it was possible to confirm the activation of BaP biotransformation system. The impairment of the melanosomes' movements possibly by an inactivation of the protein responsible for the pigment dispersion consequently affects the melanin area and thus might negatively impact the MMs detoxification capacity. In addition to this cytotoxicity, the increased frequency of micronucleus might also indicate the genotoxicity of BaP in this important fish species.

Exposure of Physalaemus cuvieri (Anura) to benzo[a]pyrene and α-naphthoflavone: Morphofunctional effects on hepatic melanomacrophages and erythrocytes abnormalities.

Benzo[a]pyrene (BaP) is a high-risk contaminant of elevated toxicity. Its biotransformation process occurs as the expression of CYP1A1 increases and produces toxic metabolites. In turn, α-naphthoflavone (aNF) represents an inhibitor of CYP1A1, preventing BaP metabolism. Toxicological studies in anurans show alterations in the melanomacrophage (MM) detoxification cell after exposure to xenobiotics. In this study, the production of melanin by MMs was evaluated, as were morphological alterations in the cytoskeleton, phagocytosis and the genotoxicity effects after exposure of an anuran species to BaP and aNF. Physalaemus cuvieri received subcutaneous injections of 2 mg/kg and/or 20 mg/kg aNF. For phagocytosis analyses, animals received an intraperitoneal injection with 0.4% trypan blue. The results revealed that melanin synthesis increased by 503.2% in animals exposed to BaP after 48 h, which was related to the antioxidant action of melanin, whereas the decreased in synthesis of 25.6% with the BaP + aNF interaction resulted in high toxicity to MMs and cell degeneration. The phagocytic activity reduced to 37.6% in animals exposed to BaP, characterizing a functional impairment; however, the BaP + aNF interaction led to the restoration of phagocytosis, reaching 419.23%. The decreased rate or absence of abnormalities may be explained by the fact that only the less damaged erythrocytes remained in the bloodstream, whereas the most damaged cells died. In conclusion, BaP and aNF are toxic to P. cuvieri, bringing risks to herpetofauna.

Effects of pesticides and metals on penaeid shrimps in Maputo Bay, Mozambique - A field study.

Estuaries are important nursery areas for many species and these habitats are often affected by anthropogenic activities. We investigate possible negative effects of pesticides and metals on penaeid shrimps in Maputo Bay, Mozambique. Shrimps and water samples were collected in three estuaries and one coastal area for biomarker and chemical analysis. Acetylcholinesterase (AChE) and glutathione-S transferase activities were analysed as biomarkers for pollutants. 37 different pesticides were analysed in water samples and shrimp muscle tissue was analysed for 10 metals. Risk assessment showed that the environmental thresholds were exceeded for several herbicides in three of four of the assessed nursery areas. Lower AChE activities were detected in shrimps captured close to an agriculture area and this location had the lowest shrimp densities. Metal analysis in shrimp showed low levels. Despite localized effects, results highlight the need to improve the regulation of pollutants in the Espírito Santo estuary in Maputo bay.

Influence of Aging on Bioaccumulation and Toxicity of Copper Oxide Nanoparticles and Dissolved Copper in the Sediment-Dwelling Oligochaete Tubifex tubifex: A Long-Term Study Using a Stable Copper Isotope.

For engineered metal nanoparticles (NPs), such as copper oxide (CuO) NPs, the sediment is recognized as a major compartment for NP accumulation. Sediment-dwelling organisms, such as the worm Tubifex tubifex, will be at particular risk of metal and metal NP exposure. However, a range of complex transformation processes in the sediment affects NP bioavailability and toxicity as the contamination ages. The objective of this study was to examine bioaccumulation and adverse effects of CuO NPs in T. tubifex compared to dissolved Cu (administered as CuCl2) and the influence of aging of spiked sediment. This was done in a 28-day exposure experiment with T. tubifex incubated in clean sediment or freshly spiked sediment with different concentrations of dissolved Cu (up to 230 µg g-1 dw) or CuO NPs (up to 40 µg g-1 dw). The experiment was repeated with the same sediments after it had been aged for 2 years. To obtain a distinct isotopic signature compared to background Cu, both Cu forms were based on the stable isotope 65Cu (>99%). The 28-day exposure to sediment-associated dissolved 65Cu and 65CuO NPs resulted in a clear concentration-dependent increase in the T. tubifex 65Cu body burden. However, despite the elevated 65Cu body burdens in exposed worms, limited adverse effects were observed in either of the two experiments (e.g., above 80% survival in all treatments, low or no effects on the growth rate, feeding rate, and reproduction). Organisms exposed to aged sediments had lower body burdens of 65Cu than those exposed to freshly spiked sediments and we suggest that aging decreases the bioavailability of both 65Cu forms. In this study, the use of a stable isotope made it possible to use environmentally realistic Cu concentrations and, at the same time, differentiate between newly accumulated 65Cu and background Cu in experimental samples despite the high background Cu concentrations in sediment and T. tubifex tissue. Realistic exposure concentrations and aging of NPs should preferably be included in future studies to increase environmental realism to accurately predict the environmental risk of metal NPs.