Biomarkers of waterborne copper exposure in the Neotropical fish Prochilodus lineatus.

The main goal of the present study was to investigate the effects of acute exposure to copper (Cu) using a Neotropical freshwater fish as sentinel species through multi biomarkers analysis at different biological levels. Juveniles of Prochilodus lineatus were kept under control condition (no Cu addition in the water) or exposed to environmentally relevant concentrations of waterborne Cu (5, 9 and 20µgL(-1)) for 96h. These concentrations were selected to bracket the current Brazilian water quality criteria for Cu in fresh water (9 and 13µgL(-1) dissolved copper). Endpoints analyzed included ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity, reduced glutathione (GSH) and metallothionein-like protein (MT) concentration, lipid peroxidation (LPO) level, tissue damage index, and incidence of free melano-macrophages (FMM) and melano-macrophage centers (MMC) in the liver. They also included DNA damage (frequency of nucleoids per comet class, number of damaged nucleoids per fish and DNA damage score) in erythrocytes, as well as muscle and brain acetylcholinesterase (AChE) activity and behavioral parameters (swimming distance and velocity, time spent swimming and swimming activity in the upper and lower layers of the water column). Fish exposed to any of the Cu concentrations tested showed increased liver MT concentration and LPO level, higher number of damaged nucleoids in erythrocytes per fish, and inhibited muscle AChE activity. Also, increased liver SOD activity was observed in fish exposed to 9 and 20µgL(-1) Cu. Fish exposed to 5 and 9µgL(-1) Cu spent lower amount of time swimming. Fish exposed to 9µgL(-1) Cu showed increased swimming distance and velocity while those exposed to 20µgL(-1) Cu had lower swimming distance and velocity, as well as, spent less time swimming in the lower layer of the water column when compared to those kept under control condition. These findings indicate that Cu exposure at environmentally relevant concentrations (below or close to the current Brazilian water quality criteria) induced significant biological (histological, biochemical and genetic) and ecological (swimming and exploratory abilities) damages in the Neotropical fish P. lineatus. They also suggest that MT concentration, DNA damage (comet assay), LPO (TBARS method), SOD and AChE activity, together with swimming behavior analyses are potential biomarkers to assess and monitor areas impacted by Cu in fresh water.

Mixtures of benzo(a)pyrene, dichlorodiphenyltrichloroethane and tributyltin are more toxic to neotropical fish Rhamdia quelen than isolated exposures.

The effects of benzo(a)pyrene (BaP), dichlorodiphenyltrichloroethane (DDT) and tributyltin (TBT) association were investigated through a multi-biomarker approach. Ten Rhamdia quelen fish per group were exposed through intraperitoneal injections either to BaP (0.3; 3 or 30 mg kg(-1)), DDT or TBT (0.03; 0.3 or 3 mg kg(-1)) or BaP/DDT, BaP/TBT, DDT/TBT or BaP/DDT/TBT on their lowest doses. The experiments were divided in acute (one dose, 5-day) and sub-chronic (3 doses, 15-day). Control groups received an equal volume of PBS or canola oil (1 ml kg(-1)). The three tested contaminants altered AChE activity in brain and muscle in similar ways; the mixtures antagonized the increase evoked by the contaminants alone. BaP and TBT increased GSH content and mixtures reduced it. GPx activity was increased by DDT and TBT in the 15-day experiment and reduced by the mixtures. BaP increased GST activity in sub-chronic experiment while TBT reduced it in the acute experiment. BaP/TBT increased GST activity compared to all groups; the other mixtures reduced it compared to BaP or DDT in the 5-day experiment. BaP, DDT and TBT increased δ-ALAd activity mainly in acute exposure; the mixtures also increased δ-ALAd compared to DDT or TBT in 5 and 15-day. BaP, TBT and BaP/DDT decreased LPO in the acute experiment. In the sub-chronic experiment DDT/TBT increased LPO when compared to TBT. None of the contaminants alone altered PCO, but all mixtures increased it compared to one or another contaminant. Contaminants isolated had a more acute effect in ALT plasma level; their lowest dose, which had no effect alone, in combination has led to an increase of this enzyme, especially after 15 days. DDT increased AST in the acute and sub-chronic experiments, while TBT did the same in the latter. DDT/TBT decreased AST opposing the effect of the contaminants alone in the 5-day experiment. Hepatic lesions index could be explained by a more acute effect of the contaminants alone or combined and by activation of cell defenses after the sub-chronic exposure. TBT increased melanomacrophages counting in the 5-day experiment and the mixtures increased it in the 5 and 15-day experiments. Overall, the majority of the biomarkers pointed to a more toxic effect when these contaminants were combined, leading to unexpected toxicities compared to individual exposure scenarios. These findings are relevant considering environmental exposure conditions, since organisms are often exposed to different combinations of contaminants.

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil.

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Complex metabolic interactions between benzo(a)pyrene and tributyltin in presence of dichlorodiphenyltrichloroethane in South American catfish Rhamdia quelen.

In an attempt to explore complex metabolic interactions between toxicants present in polluted freshwater, hepatic metabolism of benzo(a)pyrene (BaP) and tributyltin (TBT) in fish was investigated when these compounds were administrated alone, mixed together and along with dichlorodiphenyltrichloroethane (DDT). Ten Rhamdia quelen per group were treated with a single intra-peritoneal (IP) dose (5-day experiment) or three successive doses (15-day experiment) either containing BaP (0.3; 3 or 30mgkg(-1)) or TBT (0.03; 0.3 or 3mgkg(-1)) or a combination of BaP+TBT, BaP+DDT, TBT+DDT and BaP+TBT+DDT under their respective lower doses, with DDT dose kept at 0.03mgkg(-1). Tetrahydroxy-benzo(a)pyrene (BaP-tetrol-I), and dibutyltin (DBT) and monobutyltin (MBT) were analyzed to assess BaP and TBT hepatic metabolism, respectively. A significant difference in BaP-tetrol-I concentration was observed in liver and bile between the lowest and the highest doses of BaP in both 5 and 15-day experiments. In the 15-day experiment, the presence of TBT with BaP reduced the amount of BaP-tetrol-I in bile compared to the BaP alone. The time of exposure and the number of doses affected BaP-tetrol-I concentration in the bile of fish exposed to BaP 0.3mgkg(-1) and BaP+DDT. TBT and its metabolites concentrations showed a dose-dependent increase in the liver in both experiments and in the bile in the 5-day experiment. TBT at its lowest dose was completely metabolized into DBT and MBT in the liver in the 15-day experiment. No TBT metabolites were detected in the bile of fish exposed to the mixtures in the 5-day experiment, except for a small MBT amount found in BaP+TBT+DDT. This study strengthens the hypothesis of a metabolic interaction between BaP and TBT in fish and suggests DDT as an important third player when present in the mixture.

Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)

The bacterium Herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. However, the mechanisms involved in the interaction between H. seropedicae and these plants are not completely characterized. We investigated the attachment of Herbaspirillum to maize roots and the invasion of the roots by this bacterium using H. seropedicae strain SMR1 transformed with the suicide plasmid pUTKandsRed, which carries a mini-Tn5 transposon containing the gene for the Discosoma red fluorescent protein (Dsred) constitutively expressed together with the kanamycin resistance gene. Integration of the mini-Tn5 into the bacterial chromosome yielded the mutant H. seropedicae strain RAM4 which was capable of expressing Dsred and could be observed on and inside fresh maize root samples. Confocal microscopy of maize roots inoculated with H. seropedicae three days after germination showed that H. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.