Glyphosate is Harmful to Early Life Stages of the Viviparous Fish Jenynsia Multidentata: Biochemical and Locomotor Effects.

Glyphosate is the most widely used herbicide worldwide due to its efficacy in weed control in agriculture. This herbicide has been consistently detected in the aquatic environment, causing harmful consequences to nontarget organisms residing in agricultural regions. In this study, we assessed the effects of environmentally relevant concentrations of glyphosate (30-100 µg/L) on the early life stages of the viviparous fish Jenynsia multidentata through biochemical and locomotor endpoints. At 96 h of exposure, 30 and 65 µg/L glyphosate caused an increase in acetylcholinesterase (AChE) activity, and 65 µg/L glyphosate also augmented the levels of lipid peroxidation. Glyphosate at 100 µg/L did not alter the activity of acetylcholinesterase or the levels of lipid peroxidation, but it stimulated the activity of the cellular detoxification enzyme glutathione S-transferase. In addition, all concentrations affected the swimming of the fish. Under light conditions, glyphosate caused hypolocomotion at all concentrations tested, whereas under dark conditions, this was observed at 30 and 100 µg/L. Hyperlocomotion was observed at 65 µg/L glyphosate. These findings are alarming for the health of fish, such as J. multidentata that inhabit streams that pass through agricultural areas, especially for the early life stages of these fish. Research studying the effects of pollutants on native species is relevant to improve regulation that protects aquatic ecosystems.

Biomineralization biomarkers to assess microplastics toxic effects in the freshwater snail Pomacea canaliculata.

Microplastics (MPs) pollution has increased the number of reports on the toxic effects on biota, especially aquatic organisms. Recently, studies highlighted changes in ion transport and concentration, especially Ca2+, in organisms exposed to MPs. For calcifying organisms, such as mollusks, Ca2+ homeostasis is critical for their shells construction. We investigated the effects of polyethylene (PE) MPs at 20 µg/L on biomineralization biomarkers (Ca2+ATPase, carbonic anhydrase, hemolymph [Ca2+], and shell regeneration) of the freshwater gastropod Pomacea canaliculata. Two experimental sets were performed: (1) animals in physiological condition and (2) animals with their shells excised. The results of the first set showed that within 24 h, the hemolymph [Ca2+] decreased, and the Ca2+ATPase activity increased in the mantle edge. For carbonic anhydrase (CA), the activity decreased in the gland and increased in the mantle. By 72 h, the hemolymph [Ca2+] had not changed, whereas both enzymes had increased in both tissues. In the second set, the hemolymph [Ca2+] increased after 72 h, whereas Ca2+ATPase activity decreased in both tissues. For AC, the opposite results were observed. At 120 h, calcium pumping was still reduced and CA values increased in the digestive gland. Additionally, MPs exposure increased the capacity of the gastropods to recover their shells. Based on this, our work provides novel data associating PE microplastic exposures (at 20 µg/L) and their potential to stimulate biomineralization enzymes of P. canaliculata, as well as increase shell regeneration in excised animal; a good prerogative for further investigations on both subjects that still lacks of more robust evidence.

Ecotoxicological impacts of the Fundão dam failure in freshwater fish community: Metal bioaccumulation, biochemical, genetic and histopathological effects.

This study investigates the ecotoxicological impacts of the Fundão dam rupture, one of the major environmental disaster that occurred in Brazil and in the world mining industry history, through multi-biomarkers responses and metals bioaccumulation in the fish community of different trophic levels. Specimens of the fishes (omnivorous/herbivorous and carnivorous) were collected along the Doce River channel and its affluent Guandú River, and in different lakes and coastal lagoons adjacent to the river channel, in the Espirito Santo State, Southeast of Brazil. Four sampling collections were carried out over two years (2018 to 2020, during dry and rainy seasons). For both trophic groups the biomarkers responses indicated physiological alterations related to metals exposure and showed strong seasonal variations. The principal component analysis and integrated biomarker response index showed that DNA damage and lipid peroxidation were more associated with dry season 2 (Sep/Oct 2019) and the oxidative damage in proteins, metallothioneins concentration and the activity of superoxide dismutase in the gills showed a greater association with rainy season 2 (Jan/Feb 2020). On the other hand, the enzymes of energy metabolism, catalase and histological damage in the liver and the gills, were more associated with the dry and rainy campaigns of the first year of monitoring. The multivariate approach also suggested a temporal intensification in the bioaccumulation of metals and biological effects in the lacustrine environments. Thus, these results demonstrate that the release of mineral residues from the rupture of the Fundão mine dam affects the health status of the fish from the Doce River basin, provoking metals bioaccumulation, hepatic and branchial damage in the fish besides inducing of enzyme activity related to metal contamination, even four years after the rupture.

Biochemical response and metals bioaccumulation in planktonic communities from marine areas impacted by the Fundão mine dam rupture (southeast Brazil).

The rupture of the Fundão dam (Mariana, MG, southeast Brazil) released a huge flood of mine tailings to Doce river basin and its adjacent coastal area, in November 2015. This catastrophic event exposed aquatic communities to metal contamination related to mine tailings, but its biological effects are still poorly understood. This study investigates how biochemical response related to metal exposure vary between locations and seasons during the years of 2018-2020, in planktonic communities (micro and mesoplankton). Marine microplankton collected in sectors in front and south of the Doce river mouth presented the highest lipid peroxidation (LPO) and induction of metallothioneins (MT). Mesoplankton collected in sectors in front and north of the Doce river mouth presented highest LPO, while MT in this size class did not respond to a clear spatial pattern. Our results showed that metals affected biomarkers in a non-linear pattern and highlighted the complex relationship between metals, biochemical parameters, and seasonality. The variation in biochemical biomarkers indicates physiological stress related to metals, once sectors contaminated by metals, especially Fe, Mn and Cd, presented stronger biochemical responses. Comparison of metal levels with bioaccumulation data collected before the impact indicates Fe, Cd, Cr and Cu more than 2-fold higher after disaster in sectors closer to the river. Literature showed that these sectors present zooplanktonic assemblages with lower biomass and biodiversity, suggesting that the opportunistic species that thrives in the area are also under biochemical stress, but possibly relies on repair or defense mechanisms. The physiological stress detected by this study is possibly related to the mine tailings, considering the metals that stood out and the proximity with the Doce river mouth. This suggests that the impacts related to the failure of Fundão dam are still affecting the marine planktonic community even three to four years after the environmental disaster.

Toxicity and sublethal effects of methylparaben on zebrafish (Danio rerio) larvae and adults.

Among the parabens, methylparaben (MeP) is the most commonly found in personal care products. Due to the continuous use of MeP and low removal efficiency by wastewater treatment plants (WWTPs), it reaches aquatic environments, where it is incorporated by organisms inhabiting these waters. The present study aimed to evaluate the effects of MeP on the zebrafish Danio rerio larvae and adults through toxicity tests and physiological and biochemical biomarkers in order to assess possible harmful effects of MeP. For biomarker measurements, fish were exposed to the environmental concentration of 30 µg/L of MeP and the non-effect concentration (NOEC) estimated for larvae (60 mg/L) and adult (50 mg/L) in toxicity tests. The median lethal concentration (LC50) of MeP was 105.09 mg/L for adults and 211.12 mg/L for larvae. These values unexpectedly indicated that adults were more sensitive to MeP compared to larvae. In adult fish, exposure to 50 mg/L MeP induced a significant decrease in phase 1 biotransformation (ethoxyresorufin O-deethylase activity) and an increase in lipoperoxidation (LPO) in gills, as well as an increase in frequency of micronuclei in erythrocytes of these fish. Biomarkers results were integrated (integrated biomarker response [IBR] index), and it observed lower IBR scores in tissues of fish exposed to MeP, suggesting a suppression of biological responses. In addition, LPO contributed mostly to the IBR score estimated for gills of fish exposed to 50 mg/L MeP. Based on LPO quantity, sublethal exposure of MeP (30 µg/L and 60 mg/L) did not cause toxicity to larvae. Hence, we investigated whether the difference in sensitivity between adults and larvae could be associated with the antimicrobial action of MeP that could affect the intestinal microbiota of adults. We only found an increase in the number of carbon sources consumed by them without effects on diversity and abundance. This outcome can be considered an adaptation to environmental stress, but not a negative effect. However, the LPO and genotoxicity caused by MeP to zebrafish adults call direct attention to the importance of regulating the presence of this compound in the environment and improve cleaning processes adopted by WWTP.

Effect of the UV filter, Benzophenone-3, on biomarkers of the yellow clam (Amarilladesma mactroides) under different pH conditions.

This work aimed to investigate effects of the ocean contamination by the sunscreen Benzophenone-3 (BP3) and acidification, caused by CO2 enrichment, to the yellow clam, Amarilladesma mactroides. Biochemical biomarkers were analyzed in tissues (gills, digestive gland, and mantle) of clams exposed to the environmental concentration of 1 µg/L BP3, at seawater natural pH (pH 8.1) and at lower pH (pH 7.6). The tissues responded in different ways considering their physiological roles. In general, BP3 altered activity of the enzymes, glutathione-S-transferase (GST) and glutathione cysteine ligase (GCL); but mostly increased the level of glutathione (GSH). These effects were enhanced by acidification, without augmenting lipid peroxidation (LPO). Carbonic anhydrase activity (CA) increased after BP3 exposure in the digestive gland and decreased in the gills at pH 7.6, while Ca2+-ATPase activity was affected by acidification only. Changing levels of these enzymes can alter shell formation and affect the bivalve maintenance in impacted environments.

Antifouling paint particles cause toxicity to benthic organisms: Effects on two species with different feeding modes.

Antifouling paint particles (APPs) are residues generated primarily during maintenance of vessels and marine structures, and usually occur in boat maintenance areas that are adjacent to aquatic environments, such as estuaries. APPs end up in sediment layers after their release into aquatic systems and represent a threat to benthic invertebrates, which have different habitat and feeding modes. Thus, the aim of the present study was to evaluate the toxicity of APPs-spiked sediment to the benthic microcrustaceans Monokalliapseudes schubarti (a tanaid) and Hyalella azteca (an amphipod), testing whole sediment and elutriate solutions under estuarine conditions. Whole sediment spiked with APPs was more toxic to these organisms than the elutriate solution. This toxicity was attributed to the high concentrations of Cu and Zn metals quantified in the APPs. During the whole sediment test, M. schubarti was more sensitive than H. azteca. M. schubarti is an infauna organism, and its interaction with sediments (e.g. by ingestion of sediment particles) makes it more susceptible to compounds released from APPs than H. azteca, which tends to interact with these compounds at the sediment-water interface. In addition, in tests with sediment elutriate and without sediment, M. schubarti was not affected, while elutriate with 1.50% APPs showed to be significantly toxic to H. azteca. Moreover, these results indicate that APPs act as continuous and localized sources of metals to benthic organisms, highlighting the importance of better APP management and disposal practices in boat maintenance areas to avoid local aquatic contamination.

Combined effects of waterborne copper exposure and salinity on enzymes related to osmoregulation and ammonia excretion by blue crab Callinectes sapidus.

Copper is essential, but can be toxic to aquatic organisms when present in high concentrations. In freshwater crustaceans, copper inhibits enzymes related to ionic and osmoregulation and to the ammonia efflux, that leads to Na+ imbalance and inhibition of ammonia excretion. In the animals inhabiting estuarine or seawater, mechanisms of copper toxicity is not clear, but had been described as disruption of ionregulation and metabolism. To clarify the mechanism of copper toxicity in crustaceans inhabiting variable salinity, this work investigated whether copper affects ammonia excretion and enzymes used for ammonia balance and osmoregulation in the blue crab Callintectes sapidus acclimated to salinity 2 and 30 ppt. To achieve this, juveniles of the blue crab were exposed to 63.5 µg/L of copper at both salinities for 96 h. This is an environmentally realistic copper concentration. Results of ammonia efflux, free amino acids and Na+ concentrations in hemolymph, Na+/K+-ATPase, H+-ATPase and, carbonic anhydrase (CA) activities in gills were consistent with the osmoregulatory pattern adopted by the blue crab, which hyperosmoregulates at salinity 2 ppt and osmoconforms at 30 ppt. At 30 ppt copper reduced free amino acid in hemolymph of crabs, suggesting an effect of the metal on osmotic performance. At 2 ppt, copper significantly increased the H+-ATPase activity involved in ammonia excretion. This may be a compensatory response of crabs to maintain low levels of ammonia in their hemolymph; which can be increased by copper exposure. Results presented here are useful for the improvement of the Biotic Ligand Model (BLM) to predict copper toxicity for saltwater environments.

Histological evaluation of vital organs of the livebearer Jenynsia multidentata (Jenyns, 1842) exposed to glyphosate: A comparative analysis of Roundup® formulations.

Roundup formulations are herbicides whose active principle is glyphosate. However, these formulations are potentially more toxic to non-target organisms than pure glyphosate. This study aimed to evaluate and compare the toxic potential of the Roundup formulations through histological alterations in fish. Thus, males and females of the neotropical fish species Jenynsia multidentata (Jenyns, 1842) were exposed for 24 or 96 h to the Roundup Original® (RO), Roundup Transorb® (RT) or Roundup WG® (RWG) formulations, at a fixed concentration of 0.5 mg/L of glyphosate. This concentration is close to the maximum glyphosate limits found in the environment and is non-lethal to J. multidentata. The three formulations caused histological damage to the liver, gills and brain of J. multidentata, which increased over the exposure time. Differences in the histological alterations between females and males were observed in the liver and brain. Females were more tolerant to RO and RT than RWG. Males did not exhibit these differences in sensitiveness with formulations. The RWG caused more damage in the liver and gills and RT in the brain. Overall, there were differences in the toxicity of RO, RT and RWG and the toxic effect was presented through histological damage, reinforcing the usefulness of histological biomarkers for Roundup® toxicity. The comparison of the toxic potential of glyphosate-based herbicides is important because it could give support to the governmental organizations to set protective rules for the water ecosystems and human health, as well as to reduce the use of highly toxic formulations in agriculture.

A vortex-assisted MSPD method for the extraction of pesticide residues from fish liver and crab hepatopancreas with determination by GC-MS.

A method based on matrix solid-phase dispersion (MSPD) and gas chromatography-mass spectrometry to determine pesticides in fish liver and crab hepatopancreas was optimized. Ethyl acetate and acetonitrile were evaluated as elution solvents and their volumes were also checked. The best results were obtained with 1.0 g reused C18 as sorbent, using 5 mL acetonitrile as the elution solvent. Analytical recoveries ranged between 57 and 107% with RSD lower than 26% in fish liver and between 56 and 122% with RSD lower than 21% in crab hepatopancreas. The LOQ values for these compounds ranged from 0.05 to 0.5 mg kg(-1) for crab hepatopancreas and from 0.125 to 1.25 mg kg(-1) for fish liver. MPSD was shown to be easy and fast to use, with a clear advantage regarding costs because it does not need any expensive instrument. The proposed method was successfully applied to determine dimethoate, atrazine, clomazone, fenitrothion, malathion, fipronil and tebuconazole in fish liver and crab hepatopancreas samples.

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio).

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na(+), K(+)-ATPase, H(+)-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na(+), K(+)-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na(+). Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na(+) import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.

Assessing the effects of Cu, Cd, and exposure period on metallothionein production in gills of the Brazilian brown mussel Perna perna by using factorial design.

Factorial design plan was experimented in Perna perna in order to find out the contribution of day exposure, Cd and Cu concentrations in water, and their interactions on metallothioneins (MT), Cd, and Cu (the dependent measured variables) in the gills. The picture obtained is more adequate than by studying the factor effect separately. Compared with the control group, the MT concentration after 22 days exposure period in the mixture of 100 µg/mL Cu and Cd is increased almost two times, showing that P. perna might be used as a biomonitor. Cd showed stronger effect than Cu on MT inducing.

Metallothionein-like proteins in the blue crab Callinectes sapidus: effect of water salinity and ions.

The effect of water salinity and ions on metallothionein-like proteins (MTLP) concentration was evaluated in the blue crab Callinectes sapidus. MTLP concentration was measured in tissues (hepatopancreas and gills) of crabs acclimated to salinity 30 ppt and abruptly subjected to a hypo-osmotic shock (salinity 2 ppt). It was also measured in isolated gills (anterior and posterior) of crabs acclimated to salinity 30 ppt. Gills were perfused with and incubated in an isosmotic saline solution (ISS) or perfused with ISS and incubated in a hypo-osmotic saline solution (HSS). The effect of each single water ion on gill MTLP concentration was also analyzed in isolated and perfused gills through experiments of ion substitution in the incubation medium. In vivo, MTLP concentration was higher in hepatopancreas than in gills, being not affected by the hypo-osmotic shock. However, MTLP concentration in posterior and anterior gills significantly increased after 2 and 24 h of hypo-osmotic shock, respectively. In vitro, it was also increased when anterior and posterior gills were perfused with ISS and incubated in HSS. In isolated and perfused posterior gills, MTLP concentration was inversely correlated with the calcium concentration in the ISS used to incubate gills. Together, these findings indicate that an increased gill MTLP concentration in low salinity is an adaptive response of the blue crab C. sapidus to the hypo-osmotic stress. This response is mediated, at least in part, by the calcium concentration in the gill bath medium. The data also suggest that the trigger for this increase is purely branchial and not systemic.