Short-term effects of glyphosate and Roundup Transorb® formulation on the cyanobacteria Synechococcus elongatus

Glyphosate is an active ingredient used in herbicide formulations worldwide. However, besides glyphosate, these formulations have other components to facilitate glyphosate absorption by plants. These include the surfactants such as polyoxyethylene amine (POEA) present in the Roundup Transorb® (RT) formulation. Glyphosate formulations are potentially more toxic to non-target organisms than the pure active ingredient. In this work, we evaluated the toxicity (72 h) of pure glyphosate and RT for the cyanobacterium Synechococcus elongatus, based on biomass growth and cell viability. The formulation proved more toxic than pure glyphosate for both parameters analysed, with an IC50 (Inhibition concentration mean) based on biomass measured by optical density (750 nm) that was sixty times lower. Cell viability was not as sensitive as the biomass because, of the few cells left in the culture, most were viable. This indicates that there is a variation in tolerance between the cyanobacteria present in the inoculum. Thus, cell viability may underestimate the results of glyphosate and RT toxicity and be useful only in low concentrations of exposure

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 hypoosmotic 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

mRNA expression and activity of ion-transporting proteins in gills of the blue crab callinectes sapidus: effects of waterborne copper

Waterborne Cu effects on the transcription of genes encoding ion-transporting proteins and the activities of these proteins were evaluated in gills of the blue crab Callinectes sapidus acclimated to diluted (2%) and full (30%) seawater. Crabs were exposed (96 h) to an environmentally relevant concentration of dissolved Cu (0.78 mM) and had their posterior (osmoregulating) gills dissected for enzymatic and molecular analysis. Endpoints analyzed were the activity of key enzymes involved in crab osmoregulation (sodiumpotassium adenosine triphosphatase [Naþ/Kþ-ATPase], hydrogen adenosine triphosphatase [Hþ-ATPase], and carbonic anhydrase [CA]) and the mRNA expression of genes encoding these enzymes and the sodium-potassium-chloride (Naþ/Kþ/2Cl_) cotransporter. Copper effects were observed only in crabs acclimated to diluted seawater (hyperosmoregulating crabs) and were associated with an inhibition of the expression of mRNA of genes encoding the Naþ/Kþ-ATPase and the Naþ/Kþ/2Cl_ cotransporter. However, Cu did not affect Naþ/Kþ-ATPase activity, indicating that the gene transcription is downregulated before a significant inhibition of the enzyme activity can be observed. This also suggests the existence of a compensatory response of this enzyme to prevent osmoregulatory disturbances after short-term exposure to environmentally relevant Cu concentrations. These findings suggest that Cu is a potential ionoregulatory toxicant in blue crabs C. sapidus acclimated to low salinity. The lack of Cu effect on blue crabs acclimated to full seawater would be due to the reduced ion uptake needed for the regulation of the hemolymph osmotic concentration in full seawater (30%). Also, this could be explained considering the lower bioavailability of toxic Cu (free ion) associated with the higher ionic content and dissolved organic matter concentration in high salinity (30%) than in diluted seawater (2%)

Acute toxicity, accumulation and tissue distribution of copper in the blue crab Callinectes sapidus acclimated to different salinities: In vivo and in vitro studies

In vivo and in vitro studies were performed to evaluate acute toxicity, organ-specific distribution, and tissue accumulation of copper in Callinectes sapidus acclimated to two different experimental salinities (2 and 30 ppt). Blue crabs were quite tolerant to copper. Acute dissolved copper toxicity (96-h LC50 and its corresponding 95% confident interval) was higher at salinity 2 ppt (5.3 (3.50–8.05)_M Cu) than at 30 ppt (53.0 (27.39–102.52)_M Cu). The difference between salinities can be completely explained based on the water chemistry because it disappeared when 96-h LC50 values were expressed as the free Cu2+ ion (3.1 (1.93–4.95)_M free Cu at 2 ppt versus 5.6 (2.33–13.37)_M free Cu at 30 ppt) or the Cu2+ activity (1.4 (0.88–2.26)_M Cu activity at 2 ppt versus 1.7 (0.71–4.07)_M Cu activity at 30 ppt). The relationships between gill Cu burden and % mortality were very similar at 2 and 30 ppt, in accord with the Biotic Ligand Model. In vivo experiments showed that copper concentration in the hemolymph is not dependent on metal concentration in the surrounding medium at either experimental salinity. They also showed that copper flux into the gills is higher than into other tissues analyzed, and that anterior and posterior gills are similarly important sites of copper accumulation at both experimental salinities. In vitro experiments with isolated-perfused gills showed that there is a positive relationship between copper accumulation in this tissue and the metal concentration in the incubation media for both anterior and posterior gills. A similar result was observed at both low and high salinities. Furthermore, in vitro experiments showed that copper accumulation in posterior gills is also positively and strongly dependent on the incubation time with copper. Gill copper accumulation occurred at a lower rate in the first 2 h of metal exposure, increasing markedly after this “steady-state” period. This finding was corroborated by a significant increase in copper influx to the gill perfusate (corresponding to crab hemolymph) after this time, measured using 64Cu. In vivo, after uptake from solution, 64Cu was primarily accumulated in the gills and the rest of the body rather than in the hemolymph, hepatopancreas, or other internal tissues. Overall, the present findings indicate that gills are a key target organ for copper accumulation, as well as an important biological barrier against the excessive uptake of copper into the hemolymph and the subsequent distribution of this metal to internal organs of the blue crab

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

Glyphosate-based herbicides affect behavioural patterns of the livebearer Jenynsia multidentata

Roundup® is one of the most widely marketed glyphosate-based herbicides in the world. There are many different formulations of this brand that differ from each other in glyphosate concentration, salts and adjuvants, including surfactants, which are labelled as “inert” compounds. Several studies have shown that these formulations are highly toxic to fish, even compared with pure glyphosate. However, mechanisms underlying this toxicity are not fully understood. In this context, this study evaluated the effects of exposure to Roundup Original® (RO), Roundup Transorb® (RT), and Roundup WG® (RWG) on the behavioural patterns of the livebearer Jenynsia multidentata. This fish naturally inhabits agricultural areas in southern Brazil and Argentina where glyphosate is used extensively. In the experiment, animals were exposed to the herbicides for 96 h, at the environmentally relevant concentration of 0.5 mg/L of glyphosate. Swimming performance, anxiety, aggressiveness, long-term memory and male sexual activity were recorded. The formulation RWG negatively affected swimming performance, thigmotaxia and long-term memory consolidation. Conversely, RT reduced the sexual performance of males. These results confirm that Roundup® formulations are extremely harmful and also that they have different targets of toxicity, affecting behaviours that are essential for fish survival.Fil: Albañil Sánchez, Jessica Andrea. Universidade Federal do Rio Grande; BrasilFil: Barros, Daniela Marti. Universidade Federal do Rio Grande; BrasilFil: Bistoni, Maria de Los Angeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Ballesteros, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Roggio, María Angelina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: De Gaspar Martinez Martins, Camila. Universidade Federal do Rio Grande; Brasi

Pollution biomarkers in estuarine animals: Critical review and new perspectives

In this review, recent developments in monitoring toxicological responses in estuarine animals are analyzed, considering the biomarker responses to different classes of pollutants. The estuarine environment imposes stressful conditions to the organisms that inhabit it, and this situation can alter their sensitivity to many pollutants. The specificity of some biomarkers like metallothionein tissue concentration is discussed in virtue of its dependence on salinity, which is highly variable in estuaries. Examples of cholinesterase activity measurements are also provided and criteria to select sensitive enzymes to detect pesticides and toxins are discussed. Regarding non-specific biomarkers, toxic responses in terms of antioxidant defenses and/or oxidative damage are also considered in this review, focusing on invertebrate species. In addition, the presence of an antioxidant gradient along the body of the estuarine polychaete Laeonereis acuta (Nereididae) and its relationship to different strategies, which deal with the generation of oxidative stress, is reviewed. Also, unusual antioxidant defenses against environmental pro-oxidants are discussed, including the mucus secreted by L. acuta. Disruption of osmoregulation by pollutants is of paramount importance in several estuarine species. In some cases such as in the estuarine crab Chasmagnathus granulatus, there is a trade off between bioavailability of toxicants (e.g. metals) and their interaction with key enzymes such as Na+–K+-ATPase and carbonic anhydrase. Thus, the metal effect on osmoregulation is also discussed in the present review. Finally, field case studies with fish species like the croaker Micropogonias furnieri (Scianidae) are used to illustrate the application of DNA damage and immunosuppressive responses as potential biomarkers of complex mixture of pollutants

Biomarkers in croakers Micropogonias furnieri (Teleostei: Sciaenidae) from polluted and non-polluted areas from the Patos Lagoon estuary (Southern Brazil): Evidences of genotoxic and immunological effects

Biomarkers of exposure and effect of pollutants were analyzed in croakers Micropogonias furnieri (Teleostei: Sciaenidae) captured in winter and summer in a polluted and in a non-polluted site at the Patos Lagoon estuary (Southern Brazil). Catalase and glutathione Stransferase activities (exposure biomarkers) and lipid peroxidation (effect biomarker) were analyzed in liver samples. Other two effect biomarkers were also studied: blood cells DNA damage (through comet assay and micronucleus test) and respiratory burst measurements. In a broad view, results point to an important seasonal variation of the biochemical biomarkers analyzed. However, data obtained clearly indicate that croakers collected in winter at the polluted site were subjected to a level of clastogenic agents sufficient to generate irreversible genetic damages (mutations) and impair the fish immune system