Ecotoxicological assessment of guanitoxin-producing cyanobacteria in Danio rerio and Daphnia similis.

Anthropogenic activity has dramatically deteriorated aquatic ecosystems in recent years. Such environmental alterations could change the primary producers' composition, exacerbating the proliferation of harmful microorganisms such as cyanobacteria. Cyanobacteria can produce several secondary metabolites, including guanitoxin, a potent neurotoxin and the only naturally occurring anticholinesterase organophosphate ever reported in the literature. Therefore, this study investigated the acute toxicity of guanitoxin-producing cyanobacteria Sphaerospermopsis torques-reginae (ITEP-024 strain) aqueous and 50% methanolic extracts in zebrafish (Danio rerio) hepatocytes (ZF-L cell line), zebrafish embryos (fish embryo toxicity - FET) and specimens of the microcrustacean Daphnia similis. For this, hepatocytes were exposed to 1-500 mg/L of the ITEP-024 extracts for 24 h, the embryos to 31.25-500 mg/L for 96 h, and D. similis to 10-3000 mg/L for 48 h. Non-target metabolomics was also performed to analyze secondary metabolites produced by the ITEP-024 using LC-MS/MS. Metabolomics indicated the guanitoxin presence just in the aqueous extract of the ITEP-024 and the presence of the cyanopeptides namalides, spumigins, and anabaenopeptins in the methanolic extract. The aqueous extract decreased the viability of zebrafish hepatocytes (EC(I)50(24h) = 366.46 mg/L), and the methanolic extract was not toxic. FET showed that the aqueous extract (LC50(96) = 353.55 mg/L) was more toxic than the methanolic extract (LC50(96) = 617.91 mg/L). However, the methanolic extract had more sublethal effects, such as abdominal and cardiac (cardiotoxicity) edema and deformation (spinal curvature of the larvae). Both extracts immobilized daphnids at the highest concentration analyzed. However, the aqueous extract was nine times more lethal (EC(I)50(48h) = 108.2 mg/L) than the methanolic extract (EC(I)50(48h) = 980.65 mg/L). Our results showed an imminent biological risk for aquatic fauna living in an ecosystem surrounded by ITEP-024 metabolites. Our findings thus highlight the urgency of understanding the effects of guanitoxin and cyanopeptides in aquatic animals.

Cyanotoxins and water quality parameters as risk assessment indicators for aquatic life in reservoirs.

We assessed the extent of pollution in an essential public water supply reservoir (southeastern Brazil). An environmental monitoring study was performed at the Billings Reservoir (at the water catchment site) to assess the water quality in 2017, 2018, and 2019. Physicochemical parameters were analyzed, quantifying the total cyanobacteria and the cyanotoxins microcystins (MCs) and saxitoxins (SXTs), as well as their possible ecological risk to the aquatic environment. We also determined metals and metalloids (As, Ba, Cd, Pb, Cu, Cr, Fe, Mn, Ni, Zn, and Sb) and fecal bacteria (Escherichia coli). Monthly samplings were performed for 2017, 2018, and 2019 (totaling 36 sampling campaigns). Metals, metalloids, and E. coli values were below the maximum limit allowed by the Brazilian legislation. High concentrations of total cyanobacteria (3.07 × 104 - 3.23 × 105 cells/mL), microcystin variants MC-LR (0.67-23.63 µg/L), MC-LA (0.03-8.66 µg/L), MC-RR (0.56-7.92 µg/L), and MC-YR (0.04-1.24 µg/L), as well as the saxitoxins GTX2 (0.18-5.37 µg/L), GTX3 (0.13-4.40 µg/L), and STX (0.12-2.92 µg/L) were detected. From an ecotoxicological point of view, the estimated values for the risk quotient (RQ) for microcystins and saxitoxins were largely greater than 1, indicating a high risk to aquatic life. Therefore, further efforts need to be made to delay the eutrophication of the reservoir.

Ecological risk of imidacloprid on the Brazilian non-target freshwater organisms Chironomus sancticaroli and Poecilia reticulata.

Imidacloprid (IMI) is a neonicotinoid insecticide widely used in agriculture worldwide. This pesticide has been found in freshwater ecosystems, including Brazilian freshwaters. For this reason, studies are being conducted to detect the presence of IMI in freshwater and understand its effects on the aquatic biota. In the present study, the acute toxic effect of the imidacloprid commercial formulation (ICF) Galeão® on the Brazilian non-target aquatic organisms Chironomus sancticaroli and Poecilia reticulata was evaluated. Enzymatic activities (glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX)) were also determined. Moreover, we considered 11 studies that detected IMI concentrations up to 3.65 µg.L-1 in 28 different Brazilian freshwaters to evaluate the acute ecological risk of IMI in these environments. From the ecotoxicological assays, we determined the LC50 values for C. sancticaroli (LC50-48 h 1.52 µg.L-1) and P. reticulata (LC50-96 h 122.65 mg.L-1). The high sensitivity of C. sancticaroli demonstrates that this species could be used as a bioindicator in studies investigating the contamination of freshwater by IMI. Enzymatic activity changes were observed in both organisms and offered sublethal responses to the effects of the pollution by IMI on aquatic biota. Our results suggest that the presence of IMI in Brazilian aquatic ecosystems can represent a potential ecological risk for the aquatic insect populations and, consequently, cause an imbalance in these ecosystems. The present study provides relevant and comparable toxicity information that may be useful to develop public policies to protect the Brazilian aquatic ecosystem from IMI contamination.

Effects of different cultivation conditions on the production of ß-cyclocitral and ß-ionone in Microcystis aeruginosa.

BACKGROUND: Cyanobacteria blooms have become a major environmental problem and concern because of secondary metabolites produced by cyanobacteria released into the water. Cyanobacteria produce volatile organic compounds (VOCs), such as the compounds ß-cyclocitral and ß-ionone, which comprise odors, off-flavors, defense compounds, as well as growth regulators. Therefore, the general objective of this work was to evaluate the VOCs produced by two strains of Microcystis aeruginosa, differing in their ability to produce microcystins (LTPNA 01-non-producing and LTPNA 08-toxin-producing). The analysis of VOC production was carried out in (1) normal culture conditions, (2) under different light intensities (LI), and (3) after the external application of ß-ionone in both cultures. RESULTS: The results showed that ß-cyclocitral and ß-ionone are produced in all growth phases of LTPNA 01 and LTPNA 08. Both strains were producers of ß-cyclocitral and ß-ionone in normal culture conditions. It was observed that the ß-cyclocitral concentration was higher than ß-ionone in all light intensities investigated in this study. Additionally, the strain LTPNA 01 produced more ß-cyclocitral than LTPNA 08 at almost all times and LIs analyzed. However, the strain LTPNA 08 produced more ß-ionone, mainly at the initial times. In addition, the experiment results with the external addition of ß-ionone in the cultures showed that the strain LTPNA 01 produced more ß-cyclocitral in control conditions than in treatment. Nonetheless, ß-ionone production was higher in treatment conditions in LTPNA 08, indicating that the addition of ß-ionone may favor the production of these compounds and inhibit the production of ß-cyclocitral. CONCLUSION: Our results showed that some abiotic factors, such as different light intensities and external application of ß-ionone, can be triggers that lead to the production of VOCs.

'Floc and Sink' Technique Removes Cyanobacteria and Microcystins from Tropical Reservoir Water.

Combining coagulants with ballast (natural soil or modified clay) to remove cyanobacteria from the water column is a promising tool to mitigate nuisance blooms. Nevertheless, the possible effects of this technique on different toxin-producing cyanobacteria species have not been thoroughly investigated. This laboratory study evaluated the potential effects of the "Floc and Sink" technique on releasing microcystins (MC) from the precipitated biomass. A combined treatment of polyaluminium chloride (PAC) with lanthanum modified bentonite (LMB) and/or local red soil (LRS) was applied to the bloom material (mainly Dolichospermum circinalis and Microcystis aeruginosa) of a tropical reservoir. Intra and extracellular MC and biomass removal were evaluated. PAC alone was not efficient to remove the biomass, while PAC + LMB + LRS was the most efficient and removed 4.3-7.5 times more biomass than other treatments. Intracellular MC concentrations ranged between 12 and 2.180 µg L-1 independent from the biomass. PAC treatment increased extracellular MC concentrations from 3.5 to 6 times. However, when combined with ballast, extracellular MC was up to 4.2 times lower in the top of the test tubes. Nevertheless, PAC + LRS and PAC + LMB + LRS treatments showed extracellular MC concentration eight times higher than controls in the bottom. Our results showed that Floc and Sink appears to be more promising in removing cyanobacteria and extracellular MC from the water column than a sole coagulant (PAC).

Vinasse from sugarcane bagasse (hemicellulose) acid hydrolysate and molasses supplemented: biodegradability and toxicity.

Vinasse sugarcane is a valuable byproduct of the ethanol production process, presenting a perspective of volume increase with the development of second generation ethanol (2GE). However, this byproduct needs new methods of treatment and management for sustainability. Besides that, 2GE vinasse can be associated with some compounds (such as furan derivatives, phenolic compounds and organic acids), depending on the process used to solubilize hemicellulose, which could compromise vinasse destination or utilization. For this reason, detoxification methods of the hemicellulosic hydrolysates, from which vinasse is obtained in subsequent steps, are crucial. This study aimed to investigate whether the biological detoxification of vinasse from 2GE presents a difference concerning the microbial activity of biodegradation and toxicity when compared to vinasse without the detoxification process. Two vinasses (1, before; and 2, after detoxification) from fermented sugarcane bagasse (hemicellulose fraction) acid hydrolysate (supplemented with its molasses), under different concentrations: 2.5; 5 e 10% were evaluated. Their physicochemical characterization, biodegradation microbial activity (through Bartha and Pramer respirometric method, with total count of heterotrophic bacteria and fungi), and toxicity evaluation (through bioassays with Lactuca sativa at concentraction: 2.5; 5 e 10% and Daphnia similis to 1.5; 2.5; 3.5; 4.5; 5 and 10%) were performed. The results indicated high mineral and organic matter, which under a specific circumstance (2.5% of soil conditioning), enabled high efficiency in biodegradation (>80%). The bioassays with L. sativa signaled negative effect for radicular growth when the vinasses were applied at a concentration of 5 and 10% (sublethal effect and delayed root growth). Acute effects were observed in D. similis, with 50% of immobilization, at concentrations of 4.13% and 4.74% for vinasses 1 and 2, respectively. These results indicate that the biodegradation of vinasse from sugarcane bagasse acid hydrolysate occurs at relatively low levels (up to 5%) and suggests that higher concentrations (≥10%) may impair the growth of soil-associated microorganisms.

Can the insecticide Imidacloprid affect the health of the Neotropical freshwater fish Astyanax altiparanae (Teleostei: Characidae)?

Female juveniles of the Neotropical fish Astyanax altiparanae were exposed for 96 h to four treatments containing the active ingredient from Imidacloprid® commercial formulation (IMI 1, IMI 2, IMI 3, and IMI 4) and to a control treatment (only dechlorinated tap water). Glutathione content, glutathione S-transferase activity, lipid peroxidation (LPO) and protein carbonylation levels, acetylcholinesterase (AChE) activity, and frequency of micronuclei and erythrocyte nuclear abnormalities (ENA) were measured in the fish. The muscle and gills were the most affected organs; their antioxidant defense was not enough to prevent oxidative damage (LPO) in the IMI 2 and IMI 4 treatment fish. IMI also inhibited AChE activity in the muscle (IMI 3 and IMI 4) and increased ENA frequency (IMI 4). IMI can affect the health of A. altiparanae in environmentally relevant concentrations, causing oxidative damage in different organs, neurotoxic effects in the muscle, and genotoxicity.

Responses of Aquatic Nontarget Organisms in Experiments Simulating a Scenario of Contamination by Imidacloprid in a Freshwater Environment.

Several studies have indicated the presence of the neonicotinoid insecticide imidacloprid (IMI) in aquatic ecosystems in concentrations up to 320.0 µg L-1. In the present study, we evaluated the effects of the highest IMI concentration detected in surface water (320.0 µg L-1) on the survival of Chironomus sancticaroli, Daphnia similis, and Danio rerio in three different scenarios of water contamination. The enzymatic activities of glutathione S-transferase (GST), catalase (CAT), and ascorbate peroxidase (APX) in D. rerio also were determined. For this evaluation, we have simulated a lotic environment using an indoor system of artificial channels developed for the present study. In this system, three scenarios of contamination by IMI (320.0 µg L-1) were reproduced: one using reconstituted water (RW) and the other two using water samples collected in unpolluted (UW) and polluted (DW) areas of a river. The results indicated that the tested concentration was not able to cause mortality in D. similis and D. rerio in any proposed treatment (RW, UW, and DW). However, C. sancticaroli showed 100% of mortality in the presence of IMI in the three proposed treatments, demonstrating its potential to impact the community of aquatic nontarget insects negatively. Low IMI concentrations did not offer risks to D. rerio survival. However, we observed alterations in GST, CAT, and APX activities in treatments that used IMI and water with no evidence of pollution (i.e., RW and UW). These last results demonstrated that fish are more susceptible to the effects of IMI in unpolluted environments.

Inhibition of Porcine Aminopeptidase M (pAMP) by the Pentapeptide Microginins.

Aminopeptidase M (AMP) inhibition is of interest for several diseases, such as highly vascularized cancer types. AMP can be inhibited by linear pentapeptides isolated from Microcystis aeruginosa LTPNA08 (MG7XX). Porcine AMP inhibition-a model for human AMP-activity was spectrophotometrically measured by the formation of p-nitroanilide from L-leucine-p-nitroanilide substrate by AMP. AMP inhibition by MG770 exhibited comparable inhibition levels to amastatin (IC50 values: 1.20 ± 0.1 µM and 0.98 ± 0.1 µM, respectively), while MG756 was slightly less potent (with IC50 values of 3.26 ± 0.5 µM). Molecular modelling suggests a potential binding mode, based on the interaction with the Zn2+ cofactor, where MG770's extra methyl group contributes to the disturbance of the Zn2+ cofactor complex and highlights the importance of hydrophobicity for the site.