Valéria F. Magalhães

Biomonitoring of cyanotoxins in two tropical reservoirs by cladoceran toxicity bioassays

This study evaluates the potential for the use of cladocerans in biomonitoring of cyanobacterial toxins. Two zooplankton species (Daphnia gessneri and Moina micrura) were cultivated in the laboratory for use in acute (48 h) and chronic (10 days) bioassays. Water samples were collected from two reservoirs and diluted in mineral water at four concentrations. Survivorship in the acute bioassays was used to calculate LC50, and survivorship and fecundity in chronic bioassays were used to calculate the intrinsic population growth rate (r) and the EC50. Analysis of phytoplankton in the water samples from one reservoir revealed that cyanobacteria were the dominant group, represented by the genera Anabaena, Cylindrospermopsis, and Microcystis. Results of bioassays showed adverse effects including death, paralysis, and reduced population growth rate, generally proportional to the reservoir water concentration. These effects may be related to the presence of cyanobacteria toxins (microcystins or saxitoxins) in the water.

Analyses of paralytic shellfish toxins and biomarkers in a southern Brazilian reservoir

The Alagados Reservoir (Brazil) is an important source for the supply of water, recreation and fishery. Since 2002, the occurrence of cyanobacterial blooms (paralytic shellfish toxins - PST producers) have been noted. This study was aimed at the monitoring of PST occurrence in the Reservoirs water and fish. Biomarkers such as ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), and acetylcholinesterase (AchE) activities, lipoperoxidation (LPO), histopathology, and comet assay were analyzed in fish. Water and fish were sampled in spring, summer and autumn. The PST concentrations in water were 5.15, 43.84, and 50.78 ng equiv Saxitoxin/L in the spring, summer and autumn, respectively. The PST muscle concentration was below the limit for shellfish. Gonyautoxins (GTX) were found in water samples and fish muscle, and GTX 5 was the major analogous found in muscle. In the summer samples, the LPO, genetic damage, and the GST and AchE activities increased while in the autumn an increase in EROD activity and genetic damage were observed. In all samplings, histopathological alterations in the fish gills and liver were found. The results showed a seasonal variation in the fishes health, which could be related also to farming activities and to the contaminants bioavailability during the year.

Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii

The cyanobacterium Cylindrospermopsis raciborskii is a widespread species increasingly being recorded in freshwater systems around the world. It is of particular concern because strains in some geographic areas are capable of producing toxins with implications for human and animal health. Studies of this species have increased rapidly in the last two decades, especially in the southern hemisphere where toxic strains are prevalent. A clearer picture is emerging of the strategies adopted by this species to bloom and out-compete other species. This species has a high level of flexibility with respect to light and nutrients, with higher temperatures and carbon dioxide also promoting growth. There are two types of toxins produced by C. raciborskii: cylindrospermopsins (CYNs) and saxitoxins (STXs). The toxins CYNs are constitutively produced irrespective of environmental conditions and the ecological or physiological role is unclear, while STXs appear to serve as protection against high salinity and/or water hardness. It is also apparent that strains of this species can vary substantially in their physiological responses to environmental conditions, including CYNs production, and this may explain discrepancies in findings from studies in different geographical areas. The combination of a flexible strategy with respect to environmental conditions, and variability in strain response makes it a challenging species to manage. Our ability to improve bloom prediction will rely on a more detailed understanding of the complex physiology of this species.

First report about saxitoxins in freshwater fish Hoplias malabaricus through trophic exposure.

Cyanobacterial waterblooms, such as the saxitoxin (STX) producer Cylindrospermopsis raciborskii, have been a worldwide concern in environmental health. However, the bioaccumulation of this neurotoxin in the trophic chain is not completely known. The aim of the present work was to evaluate STX bioaccumulation through chemical analyses and the toxic and trophic effects using biomarkers in the tropical freshwater fish Hoplias malabaricus. They were fed once every five days with Astyanax sp. before being subjected to intraperitoneal inoculation with STX extract (0.08 μg/100 g) obtained by lysis of toxic C. raciborskii strain (T3). After 20 days the brain was collected for acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione (GSH), lipoperoxidation (LPO), protein carbonylation (PCO), and comet assay analysis. The muscle was collected for STX chemical analysis. The activities of SOD and concentrations of PCO and LPO increased. The CAT, GST, and GPx activities decreased. Genotoxicity was observed in the experimental group. STX was not detected in muscle samples. Thus, an oxidative stress was observed in the brain, leading to the damage of lipids, proteins, and DNA. The mechanism of action of the neurotoxin in this subchronic exposure suggests an apoptotic cellular process.

Sublethal Microcystin Exposure and Biochemical Outcomes among Hemodialysis Patients

Cyanobacteria are commonly-occurring contaminants of surface waters worldwide. Microcystins, potent hepatotoxins, are among the best characterized cyanotoxins. During November, 2001, a group of 44 hemodialysis patients were exposed to microcystins via contaminated dialysate. Serum microcystin concentrations were quantified with enzyme-linked immunosorbent assay which measures free serum microcystin LR equivalents (ME). We describe serum ME concentrations and biochemical outcomes among a subset of patients during 8 weeks following exposure. Thirteen patients were included; 6 were males, patients’ median age was 45 years (range 16–80), one was seropositive for hepatitis B surface antigen. The median serum ME concentration was 0.33 ng/mL (range: <0.16–0.96). One hundred thirty nine blood samples were collected following exposure. Patients’ biochemical outcomes varied, but overall indicated a mixed liver injury. Linear regression evaluated each patient’s weekly mean biochemical outcome with their maximum serum ME concentration; a measure of the extrinsic pathway of clotting function, prothrombin time, was negatively and significantly associated with serum ME concentrations. This group of exposed patients’ biochemical outcomes display evidence of a mixed liver injury temporally associated with microcystin exposure. Interpretation of biochemical outcomes are complicated by the study population’s underlying chronic disease status. It is clear that dialysis patients are a distinct ‘at risk’ group for cyanotoxin exposures due to direct intravenous exposure to dialysate prepared from surface drinking water supplies. Careful monitoring and treatment of water supplies used to prepare dialysate is required to prevent future cyanotoxin exposure events.

Saxitoxins induce cytotoxicity, genotoxicity and oxidative stress in teleost neurons in vitro.

The aim of this study was establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of neurotoxic substances as saxitoxins (STXs). Cells from brain of H. malabaricus were treated with different concentrations of trypsin, dispase and papain for tissue dissociation. Cells type was separated by cellular gradient and basic fibroblast growth factor (bFGF) supplement nutrition media were added. The dissociated cells were plated with medium and different STXs concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity and apoptosis process were evaluated. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STXs increased specific activity of glutathione peroxidase and lipoperoxidation levels, were cytotoxic and genotoxic indicated by the comet assay. Although the STXs effects due the blockage of sodium channels is reported to be reversible, the time exposure and concentration of STXs suggested cellular injuries which can lead to neuropathology. The establishment of primary neuronal culture protocol enables new applications for neurotoxicological assessments.

Effects of Cylindrospermopsis raciborskii (cyanobacteria) on the swimming behavior of Daphnia (cladocera)

The present study aimed to test the effects of raw water samples from a eutrophic reservoir and of a saxitoxin-producing strain of Cylindrospermopsis raciborskii on the swimming behavior of 2 key herbivore species of Daphnia. Two complementary approaches were used, acute bioassays and behavioral assays using an automated movement tracking system for measuring the following activity parameters: swimming time, resting time, distance traveled, and mean velocity. In both assays, animals were exposed to field samples or to toxic filaments in different concentrations and observed for 2u2009h to 3u2009h. In the acute bioassays, there was a decrease in the number of swimming individuals during the exposure period and a recovery following removal from toxic algae. A significant relationship was found between median effective concentration and the saxitoxin content of seston (r(2) u2009=u20090.998; pu2009=u20090.025) in the acute bioassays with raw water samples. Behavioral assays also showed significant effects in the activity parameters with both field samples and the strain of C. raciborskii, with some recovery during the exposure period. Both approaches corroborated previous research on the effects of neurotoxic C. raciborskii on the swimming activity of Daphnia, and these effects are compatible with the mechanism of action of saxitoxins. The present study showed that activity parameters of aquatic organisms may be a useful tool in the evaluation of sublethal toxicity and detection of neurotoxins in raw water.