Environmental Homeopathy: Homeopathic Potencies Regulate the Toxicity and Growth of Raphidiopsis raciborskii (cyanobacteria) and can be Tracked Physico-Chemically. Part 1: Biological Results.

INTRODUCTION: Cyanobacteria are microorganisms found in many parts of the world and several genera, such as Raphidiopsis raciborskii, are producers of cyanotoxins. Homeopathic potencies have been found to modulate toxicity in different biological models, and the present study endeavors to discover whether this might also be the case with cyanobacteria. OBJECTIVES: Our objective was to investigate the possible effects of homeopathic potencies on the resilience of Artemia franciscana (brine shrimp) embryos to saxitoxin (STX; cyanotoxin) and on controlling the growth of R. raciborskii in vitro. METHOD: A. franciscana cysts were cultivated in seawater in 96-well plates to evaluate the hatching rate and vitality, plus the gene expression of heat shock proteins (HSPs), after being challenged with R. raciborskii extract containing 2.5 µg/L of STX and treated with different homeopathic potencies. Untreated wells were used as controls ("base-line"). Potencies were chosen from a screening process based on seven selected homeopathic preparations according to the similitude of STX symptoms (Sulphur, Zincum metallicum, Nitric acidum, Plumbum metallicum, Mercurius solubilis, Phosphoric acidum, Isotherapic from R. raciborskii extract; all at 6cH, 30cH and 200cH). Cultures of R. raciborskii maintained in an artificial seawater medium were equally treated with screened homeopathic potencies selected from the same list but specifically for their growth control as a function of time. RESULTS: A 15% lower rate of hatching of A. franciscana cysts was observed after treatment with Nitric acidum 6cH in comparison with baseline (p = 0.05). A complete toxicity reversal was seen after treatment with Isotherapic 200cH, with a 23-fold increase of Hsp 26 gene expression (p = 0.023) and a 24-fold increase of p26 gene expression (p ≤ 0.001) in relation to baseline. Nitric acidum 200cH and Mercurius solubilis 30cH limited the exponential growth of cyanobacteria up to 95% and 85% respectively (p ≤ 0.003) in relation to baseline. Succussed water presented only a transitory 50% inhibition effect. CONCLUSION: Isotherapic 200cH improved A. franciscana bioresilience to STX; Nitric acidum 200cH and Mercurius solubilis 30cH showed the optimal performance on limiting R. raciborskii growth. The results point to the potential of homeopathic potencies to mitigate environmental problems related to water quality.

Environmental Homeopathy: Homeopathic Potencies Regulate the Growth and Toxicity of Raphidiopsis raciborskii (cyanobacteria) and Can be Tracked Physico-Chemically. Part 2: Physico-chemical Results.

INTRODUCTION: The control of cyanobacterial toxicity and growth by homeopathic potencies was described in Part 1 of this two-part report. Here, a parallel approach characterized the physico-chemical features of the potencies used and the liquid media treated with them, correlating these results with their respective biological effects. OBJECTIVES: Our objective was to establish if physico-chemical parameters can track homeopathic potencies in seawater or artificial seawater medium (ASM)-1 and to discover whether these parameters correlate with previously described biological effects. METHOD: Artemia franciscana (brine shrimp) cysts were cultivated in seawater challenged with Raphidiopsis raciborskii extract and treated with different homeopathic potencies chosen from a screening process. Cultures of R. raciborskii maintained in ASM-1 were also treated with previously screened homeopathic potencies, and their growth was monitored as a function of time. The physico-chemical properties of the treated media (seawater or ASM-1) were evaluated by their interaction with solvatochromic dyes and changes in pH, conductivity and temperature. RESULTS: Coumarin 7 was found to be a marker for Nitric acidum 6cH and Isotherapic (R. raciborskii extract) 200cH in seawater (analysis of variance [ANOVA], p = 0.0015). Nile red was found to be a marker for Nitric acidum 200cH and Mercurius solubilis 30cH in ASM-1 (ANOVA, p ≤ 0.001). An increase in pH of ASM-1 and endothermic effects were observed after these treatments (two-way ANOVA, p = 0.0001). Seawater and ASM-1 to which potencies had been added were also subjected to a constant unidirectional 2,400 Gauss static magnetic field and found to have enhanced effects on the solvatochromic dyes tested. CONCLUSION: Homeopathic potencies were specifically traceable in aqueous media using solvatochromic dyes, especially when the samples were subjected to a magnetic field. Results from monitoring other physical parameters, such as pH and temperature, were less specific in relation to potency tracking. However, potency-induced endothermic effects might provide valuable thermodynamic data relating to the nature of potencies.

In vitro and in silico assessment of cytotoxicity and chromosome instability induced by saxitoxin in human derived neural cell line.

In freshwater, saxitoxins (STX) are produced by different cyanobacteria genera, including Raphidiopsis. Data regarding cytogenotoxicity effects of STX on human cells are scarse, this merit further studies of its toxicology. This study assessed the cytotoxicity and the chromosome instability of STX on SHSY-5Y human cell line. The CBMN assay allows the detection of chromosome breaks and abnormal chromosomal segregation. Additionally, in silico systems biology approach, used to search for known and predicted interaction networks, was applied to study the interactions between STX and SHSY-5Y cellular components. The results of the CBMN assay demonstrated that STX concentrations of 2.5 - 10 µg/L induced cytostasis and chromosome instability in a dose-response relationship. Apoptosis was detected after exposure of SHSY-5Y cultured cells to STX concentration of 10 µg/L. The results of the systems biology analysis revealed the interaction of STX with proteins related with acetylcoline pathway, cell cycle regulation and apoptosis. Furthermore, combining the in vitro and in silico approachs, it was possible to suggest a mechanism of action of STX in SHSY-5Y cells. Overall, the data demonstrated the cytotoxicity and mutagenicity of environmentally relevant concentrations of STX. These results should be considered when setting up guidelines for monitoring STX in water supply.

Effects of homeopathic preparations on raphidiopsis raciborskii (Cyanobacteria) toxicity in artemia salina mode

Cyanobacteria are microorganisms found in different parts of the world. Some genera are cyanotoxins producers a sodium channel blockingneurotoxin (saxitoxins). Some homeopathic preparations have been identified as remedial action on toxicity models in Artemia salina. This study aimed to observe whether homeopathic products influence the toxicity ofR.raciborskiiextract onA.salinaby inducing cyst hatching arrest, anembryo bioresiliencemodel previously developed in our laboratory (Pinto et al., 2021; Mohammad et al., 2022). Thus, previous toxicity testswere carried out on cysts in 96-well plates, using different concentrations of the extract obtained from regular cultivation of R. raciborskii in HCl 0.05M, whose strain, named T3, is kept in the laboratory of Cyanobacteria at FURGS, Brazil.The standardization of toxin concentration was based on an established scale developed at FURGS, in which the number of T3 filaments is associated withspecific saxitoxin concentrationsdefined by chromatography. The concentration of 2.6 µg/L was chosen since it reducedthe cysthatching rate by 30%, the ideal level to observe embryo bioresilience. Then, a screeningstudy with 22 homeopathic preparations was tested blind in three experimental series, in duplicate,against threecontrols (unchallenged, water,and succussed water)for possible toxicity attenuationon Artemia salinacysts hatching rate. Homeopathic medicines were prepared in pure,sterile water from a stock homeopathic solution, one potency below the working potency. After the 1:100 dilution, 100 succussions were made using a robotic arm (Denise, Autic). The medicines were inserted into the seawater on a 10% basis. Due to the high sensitivity of A. salinato the circalunar variations, all experiments were performed during the first quarter moon. Statistical analysis was performed by two-way ANOVA followed by Tukey, with α=0.05. The most significant results indicative of bioresilience improvement were seen after the treatment with Nitric acidum6 cH, Plumbum metallicum6 cH, isotherapic 200 cH, and hydrochloric acid 1 cH being the last one used as a vehicle of the extracts. Thus, these preparations were chosen to be used in further experiments. In conclusion, the Artemia salinamodel has also beenuseful to study bioresilienceimprovement by homeopathic medicines after intoxication with saxitoxin.

Chemoprotection mediated by açaí berry (Euterpe oleracea) in white shrimp Litopenaeus vannamei exposed to the cyanotoxin saxitoxin analyzed by in vivo assays and docking modeling.

Saxitoxin (STX) is a neurotoxic cyanotoxin that also generate reactive oxygen species, leading to a situation of oxidative stress and altered metabolism. The Amazonian fruit açaí Euterpe oleracea possesses a high concentration of antioxidant molecules, a fact that prompted us to evaluate its chemoprotection activity against STX toxicity (obtained from samples of Trichodesmium sp. collected in the environment) in the shrimp Litopenaeus vannamei. For 30 days, shrimps were maintained in 16 aquaria containing 10 shrimps (15% salinity, pH 8.0, 24 °C, 12C/12D photoperiod) and fed twice daily with a diet supplemented with lyophilized açaí pulp (10%), in addition to the control diet. After, shrimps (7.21 ± 0.04 g) were exposed to the toxin added to the feed for 96 h. Four treatments were defined: CTR (control diet), T (lyophilized powder of Trichodesmium sp. 0.8 µg/g), A (10% of açaí) and the combination T + A. HPLC analysis showed predominance of gonyautoxin-1 concentrations (GTX-1) and gonyautoxin-4 concentrations (GTX-4). The results of molecular docking simulations indicated that all variants of STX, including GTX-1, can be a substrate of isoform mu of the glutathione-S-transferase (GST) enzyme since these molecules obtained similar values of estimated Free Energy of Binding (FEB), as well as similar final positions on the binding site. GSH levels were reduced in muscle tissues of shrimp in the T, A, and T + A treatments. Increased GST activity was observed in shrimp hepatopancreas of the T treatment and the gills of the A and T + A treatments. A decrease of protein sulfhydryl groups (P-SH) was observed in gills of shrimps from T + A treatment. A reduction in malondialdehyde (MDA) levels was registered in the hepatopancreas of the T + A treatment in respect to the Control, T, and A treatments. The use of açaí supplements in L. vannamei feed was able to partially mitigate the toxic effects caused by Trichodesmium sp. extracts, and points to mu GST isoform as a key enzyme for saxitoxin detoxification in L. vannamei, an issue that deserves further investigation.

Saxitoxins from the freshwater cyanobacterium Raphidiopsis raciborskii can contaminate marine mussels.

Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii) is a freshwater cyanobacterium potentially producing saxitoxins (STX) and cylindrospermopsin. Its ecophysiological versatility enables it to form blooms in the most diverse types of environments, from tropical to temperate, and from relatively pristine to polluted. In Peri Lake, located in the subtropical south of Brazil, growing populations of STX-producing R. raciborskii have been detected since 1994, posing risks to the use of its waters that supply a population of about 100,000 inhabitants. Despite the existence of a monitoring system for the presence and toxicity of cyanobacteria in Peri Lake water, no assessment has been made in the coastal region, downstream of outflowing lake water, thereby potentially making available a toxic biomass to natural and cultivated shellfish populations in the salt water ecosystem. To address this problem, the present study evaluated environmental variables and STX concentration by profiling the outflowing waters between Peri Lake and the adjacent coastal zone. Laboratory experiments were carried out with three strains of R. raciborskii in order to confirm the effect of salinity on STX production and verify if Perna Perna mussels fed with R. raciborskii cultures would absorb and accumulate STX. Results showed that environmental concentrations of STX reach high levels (up to 6.31 µg L-1 STX eq.), especially in the warmer months, reaching the coastal zone. In laboratory tests, it was found that the strains tolerate salinities between 4 and 6 and that salinity influences the production of STX. In addition, mussels fed with R. raciborskii effectively absorb and accumulate STX, even in typically marine salinities (22 to 30), suggesting that R. raciborskii biomass remains available and toxic despite salinity shock. These results draw attention to the ecological and health risk associated with R. raciborskii blooms, both in the lake environment and in the adjacent marine environment, calling attention to the need to improve the monitoring and management systems for water and shellfish toxicity in the region of interest, as well as other places where toxic cyanobacteria of limnic origin can reach the coastal zone.

Toxigenic phytoplankton groups and neurotoxin levels related to two contrasting environmental conditions at the coastal area of Rio de Janeiro (west of South Atlantic).

An assessment of the major pigments and neurotoxins and a description of the phytoplankton community were carried out within the coastal region of Rio de Janeiro State (Brazil), during winter and the following spring of 2018. Overall, six stations were investigated for oceanographic conditions (with CTD casts). Filtered water samples were used to estimate the chlorophyll a (CHL-a), carotenoids (CAR), and phycobiliproteins (PHY) using UV-Vis spectrophotometry, as well as the quantification of saxitoxins (STX) and domoic acid (DA), through High Performance Liquid Chromatography (HPLC). Planktonic organisms were counted using sedimentation chambers of different volumes and an inverted microscope. A cluster analysis, SIMPER, and ANOSIM were applied to the phytoplankton data along with diversity indexes, and non-parametric statistics to phycotoxins and pigments. There was a significant difference between the winter and spring phytoplankton community, associated with the mixed layer depth (r2 = -0.626, p < 0.05) and temperature (r2 = 0.641, p < 0.05). Phytoplankton biomass and C:CHL-a indicated a higher production during the winter than in spring, with the potentially toxic genus Pseudo-nitzschia responsible for 12.79% of autotrophic abundance (SIMPER output). Pigments showed a slight increase in CAR during spring, while PHY remained at trace concentrations. Both the DA and STX were quantified in winter and spring, but with significant differences only for STX between the sampling periods. Among the 71 taxa, 11 were identified as potentially toxic with an emphasis on STX-producing dinoflagellates and cyanobacteria, such as Alexandrium sp., Gymnodinium spp. along with Trichodesmium spp. Season-related environmental variability may be the major driving force modulating the mixed assemblage of species that support different levels of phycotoxins.

Modulation of nodularin toxicity in shrimp Litopenaeus vannamei (BOONE, 1931) fed with dietary açai (Euterpe oleracea) inclusion.

This study evaluated the effect of dietary inclusion of lyophilized açaí Euterpe oleracea (LEO) on redox status of shrimp Litopenaeus vannamei (initial weight 1.5 ± 0.39 g) upon exposure to cyanotoxin nodularin (NOD) in bioflocs system. Three hundred juvenile shrimps were randomly divided into two groups and fed twice a day with two diets: one containing 0.00 (control diet) and the other 10.0% LEO (w/w) for 30-days. After the feeding period, both shrimp groups were submitted to three treatments (14 L; 7 shrimp/tank) with different concentrations of cyanotoxin NOD (0.00; 0.25; and 1.00 µg/L) dissolved in water with 96 h of exposure. Then, the shrimps were sampled (n = 15/treatment) for the determination of reduced glutathione (GSH), the activity of glutathione-S-transferase (GST), sulfhydryl groups associated to proteins (P-SH), and lipid peroxidation (TBARS) in the hepatopancreas, gills and muscle. The NOD accumulation was measured in the muscle. The results revealed that dietary LEO significantly increased GSH levels in the hepatopancreas and gills of the shrimps exposed to NOD. Toxin exposure did not modify GST activity in all organs. Muscle TBARS levels were lower in the shrimp fed with the LEO diet and exposed to NOD. The NOD toxin did not accumulate in the muscle but notably was detected in the control groups fed or not with dietary LEO. Açaí was able to induce the antioxidant system of L. vannamei, as well as lowered the oxidative damage in shrimps exposed to NOD, suggesting its use as a chemoprotectant against cyanotoxins.

The effects of Microcystis aeruginosa cells lysate containing microcystins on physiological and molecular responses in the nematode Caenorhabditis elegans.

Microcystins (MCs) are potent toxins produced by environmental cyanobacterial blooms. The present study evaluated the effects of a Microcystis aeruginosa cyanobacterial lysate containing 0.1, 1, and 10 µg L-1 MC-LR equivalent in the C. elegans Bristol N2 wild-type and the effects caused by equivalent concentrations of a MC-LR standard. The lysate was prepared from a culture of toxic strain (RST9501) originated from the Patos Lagoon Estuary (RS, Brazil). The minimal concentration necessary to cause significant effects in C. elegans under exposure to M. aeruginosa lysate or to MC-LR standard were, respectively, 10 and 0.1 µg L-1 MC-LR equivalent for growth and 10 and 1 µg L-1 MC-LR equivalent for fertility. Reproduction (ie, brood size) was only affected by the exposure to 10 µg L-1 MC-LR standard and was not affected by the lysate. The nematodes that were exposed to lysate containing 1 µg L-1 MC-LR equivalent or MC-LR were also analyzed for pharyngeal pumping and gene expression using RT-qPCR. The worms' rhythmic contractions of the pharynx were similarly affected by the lysate containing 1 µg L-1 of MC-LR equivalent and the MC-LR standard. The MC-LR standard caused down-regulation of genes related to growth (daf-16), fertility (spe-10), and biotransformation (gst-2). This is the first study to evaluate the effects of a toxic cyanobacterial lysate using the C. elegans model. This study suggests the organism as a potential biotest to evaluate toxicity of natural waters containing M. aeruginosa cells and to environmental risk assessment associated to cyanobacterial bloom events.

Co-inoculation of rhizobia, azospirilla and cyanobacteria for increasing common bean production / Co-inoculação de rizobio, azospirillum e cianobactérias no aumento da produção de feijão comum

The combined inoculation of Rhizobium (R. tropici+R. freirei), Azospirillum brasilense, and Anabaena cylindrica, a diazotrophic cyanobacterium, is a technology that has not yet been tested and established in the production of the common bean (Phaseolus vulgaris). The inoculation may be a promising strategy for increasing crop productivity by combining the benefits of biological nitrogen fixation with the production of plant growth phytohormones. Therefore, the objective of this study was to evaluate the co-inoculation of Rhizobium, Azospirillum brasilense, and Anabaena cylindrica as an alternative method for optimizing the symbiotic performance and development of the common bean at greenhouse conditions. The treatments were as follows: (T1) control without N and inoculation, (T2) N addition (100kg N ha-¹), (T3) Riz (addition of R. tropici+R. freirei), (T4) Azo (Azospirillum brasilense addition), (T5)Ana (Anabaena cylindrical addition), (T6) Riz+Azo, (T7) Riz+Ana, (T8) Azo+Ana, (T9) Riz+Azo+Ana. We used a completely randomized experimental design with four replications. The co-inoculation of Riz+Azo+Ana promoted plant height, root length and volume, shoot dry matter, accumulated shoot N, number and dry matter of nodules at flowering, number of grains per pod, hundred seed weight, andgrain production of the common bean, contributing to increased yield per plant. We observed an increase in common bean grain yield ranging from 62 to 84% after double and triple co-inoculation of rhizobia with azospirilla and/or cyanobacteria, with the highest yield observed in the plants inoculated with Riz+Azo+Ana (84%), similar to those observed in plants after N addition. However, field experiments are necessary to elucidate the performances of the inoculated beneficial microorganisms.(AU)

A inoculação associada de Rhizobium, Azospirillum e uma cianobactéria diazotrófica Anabaena cylindrica é uma tecnologia que ainda não foi testada e estabelecida para o feijão comum (Phaseolus vulgaris) e pode ser uma estratégia promissora para aumentar a produtividade das culturas, combinando os benefícios da fixação biológica de nitrogênio com a produção de fitohormônios de crescimento vegetal. Portanto, o objetivo deste estudo foi avaliar a co-inoculação de Rizobium, Azospirilum brasilense e Anabaena cylindrica, como uma alternativa para otimizar o desempenho simbiótico e o desenvolvimento do feijoeiro, em casa de vegetação. Os tratamentos foram: T1-control, T2-N-mineral (100 kg N ha-¹), T3- Rhizobium (R. tropici+R. freirei), T4- A. brasilense, T5- A. cylindrica, T6- Rhizobium + A. brasilense, T7- Riz + Ana, T8- Azo + Ana, T9- Riz + Azo + Ana. O delineamento experimental foi inteiramente casualizado, com quatro repetições. A co-inoculação de Rhizobium (R.tropici+R. freirei) + Azo + Ana promoveu aumento do feijão (IPR Campos Gerais) em altura das plantas, comprimento e volume de raiz, massa seca da parte aérea, N acumulado da parte aérea, número e matéria seca dos nódulos no florescimento, número de grãos por vagem, peso de cem grãos e de grãos por planta, o que contribuiu para incremento da produção. Houve um aumento na produção de grãos de feijão com co-inoculação dupla e tripla de rizobia com azospirila e / ou cianobactérias, variando de 62a 84%, com o maior aumento de produção observado no tratamento inoculado com Riz + Azo + Ana(84%), semelhante ao tratamento com nitrogênio mineral. No entanto, experimentos de campo podem contribuir para elucidar o desempenho de microrganismos benéficos inoculados.(AU)

Co-inoculation of rhizobia, azospirilla and cyanobacteria for increasing common bean production / Co-inoculação de rizobio, azospirillum e cianobactérias no aumento da produção de feijão comum

The combined inoculation of Rhizobium (R. tropici+R. freirei), Azospirillum brasilense, and Anabaena cylindrica, a diazotrophic cyanobacterium, is a technology that has not yet been tested and established in the production of the common bean (Phaseolus vulgaris). The inoculation may be a promising strategy for increasing crop productivity by combining the benefits of biological nitrogen fixation with the production of plant growth phytohormones. Therefore, the objective of this study was to evaluate the co-inoculation of Rhizobium, Azospirillum brasilense, and Anabaena cylindrica as an alternative method for optimizing the symbiotic performance and development of the common bean at greenhouse conditions. The treatments were as follows: (T1) control without N and inoculation, (T2) N addition (100kg N ha-¹), (T3) Riz (addition of R. tropici+R. freirei), (T4) Azo (Azospirillum brasilense addition), (T5)Ana (Anabaena cylindrical addition), (T6) Riz+Azo, (T7) Riz+Ana, (T8) Azo+Ana, (T9) Riz+Azo+Ana. We used a completely randomized experimental design with four replications. The co-inoculation of Riz+Azo+Ana promoted plant height, root length and volume, shoot dry matter, accumulated shoot N, number and dry matter of nodules at flowering, number of grains per pod, hundred seed weight, andgrain production of the common bean, contributing to increased yield per plant. We observed an increase in common bean grain yield ranging from 62 to 84% after double and triple co-inoculation of rhizobia with azospirilla and/or cyanobacteria, with the highest yield observed in the plants inoculated with Riz+Azo+Ana (84%), similar to those observed in plants after N addition. However, field experiments are necessary to elucidate the performances of the inoculated beneficial microorganisms.

A inoculação associada de Rhizobium, Azospirillum e uma cianobactéria diazotrófica Anabaena cylindrica é uma tecnologia que ainda não foi testada e estabelecida para o feijão comum (Phaseolus vulgaris) e pode ser uma estratégia promissora para aumentar a produtividade das culturas, combinando os benefícios da fixação biológica de nitrogênio com a produção de fitohormônios de crescimento vegetal. Portanto, o objetivo deste estudo foi avaliar a co-inoculação de Rizobium, Azospirilum brasilense e Anabaena cylindrica, como uma alternativa para otimizar o desempenho simbiótico e o desenvolvimento do feijoeiro, em casa de vegetação. Os tratamentos foram: T1-control, T2-N-mineral (100 kg N ha-¹), T3- Rhizobium (R. tropici+R. freirei), T4- A. brasilense, T5- A. cylindrica, T6- Rhizobium + A. brasilense, T7- Riz + Ana, T8- Azo + Ana, T9- Riz + Azo + Ana. O delineamento experimental foi inteiramente casualizado, com quatro repetições. A co-inoculação de Rhizobium (R.tropici+R. freirei) + Azo + Ana promoveu aumento do feijão (IPR Campos Gerais) em altura das plantas, comprimento e volume de raiz, massa seca da parte aérea, N acumulado da parte aérea, número e matéria seca dos nódulos no florescimento, número de grãos por vagem, peso de cem grãos e de grãos por planta, o que contribuiu para incremento da produção. Houve um aumento na produção de grãos de feijão com co-inoculação dupla e tripla de rizobia com azospirila e / ou cianobactérias, variando de 62a 84%, com o maior aumento de produção observado no tratamento inoculado com Riz + Azo + Ana(84%), semelhante ao tratamento com nitrogênio mineral. No entanto, experimentos de campo podem contribuir para elucidar o desempenho de microrganismos benéficos inoculados.

Domoic acid in the tropical South Atlantic Ocean - An environment case study.

Domoic acid (DA) or Amnesic Shellfish Poisoning (ASP) produced by the genus Pseudo-nitzschia diatom was investigated in two seasonal periods in fishing areas of Katsuwonus pelamis in the South Atlantic Ocean. Higher DA concentrations were found in spring compared to winter. Pseudo-nitzschia spp. more quantified in winter than in spring, while P. pungens, a species among the most reported for an AD toxic potential, was only found in spring.

Environmental Variability and Cyanobacterial Blooms in a Subtropical Coastal Lagoon: Searching for a Sign of Climate Change Effects.

Cyanobacterial blooms in marine and freshwater environments may be favored by shifts in physical water column parameters due to warming under climate change. The Patos Lagoon (PL), a subtropical coastal environment in southern Brazil, is known for recurrent blooms of Microcystis aeruginosa complex (MAC). Here, we analyze the variability of these blooms and their relation to changes in wind direction and speed, rainfall and freshwater run-off from 2000 to 2017. Also, we discuss both longer time-series of air temperature and rainfall and a review of local studies with microcystins produced by these noxious species. Since the 1980s, MAC blooms were associated to negative anomalies in annual precipitation that occur during La Niña periods and, in the last years (2001-2014), accompanied by a trend in low river discharge. MAC blooms were conspicuous from December to March, i.e., austral summer, with massive patches seen in satellite images as for 2017. We suggest that low rainfall and run-off years under NE wind-driven hydrodynamics might accumulate MAC biomass in the west margin of the PL system. In contrast, a positive, long-term trend in precipitation (from 1950 to 2016; slope = 3.9868 mm/yr, p < 0.05) should imply in high river discharge and, consequently, advection of this biomass to the adjacent coastal region. Due to the proximity to urban areas, the blooms can represent recreational and economic hazards to the region.

Validation of Housekeeping Genes as Internal Controls for the Study of the Effects of Microcystin-LR in Zebrafish by Real-Time PCR.

Microcystin-leucine arginine (MC-LR) is a natural toxin produced by cyanobacterial blooms that causes severe liver damage in fish. It is crucial to investigate if housekeeping genes are affected by MC-LR in zebrafish, to permit the adequate evaluation of gene expression by RT-qPCR. We evaluated the gene expression stability (M value) and regulation by chemical treatment (using E-Ct and E-ΔCt) to validate the use of eight housekeeping genes in fish exposed to 0, 0.31, and 6.10 µg L-1 MC-LR for 24 h. We suggest the use of the combination of ß-actin1, b2m, and arnt2 in the liver and ß-actin1, 18S rRNA, and arnt2 in gills as housekeeping genes. The evaluation of gene regulation following MC-LR exposure denoted a strong repression of 18S rRNA (17- and 10-fold decrease) and tbp (10- and 2-fold decrease) and induction of ef-1α (8- and 14-fold increase) in the liver of zebrafish exposed to 0.31 and 6.10 µg L-1 MC-LR, respectively. This is the first study showing that housekeeping genes commonly used in gene expression could be affected in the liver by environmentally relevant concentrations of MC-LR. The study validates adequate housekeeping genes that could be used in toxicological studies with MC-LR in zebrafish.

Effect of a toxic Microcystis aeruginosa lysate on the mRNA expression of proto-oncogenes and tumor suppressor genes in zebrafish.

Cyanobacterial blooms of Microcystis aeruginosa represent a significant risk to the environment and have become a worldwide concern. M. aeruginosa can produce the hepatotoxins microcystins (MCs) with potential for tumor promotion. The present study evaluated the time-dependent effects in the transcription of tumor-related genes in the zebrafish, Danio rerio, exposed to dilutions of a M. aeruginosa lysate containing 3.5 and 54.6 µg L-1 MCs. We used a cultured M. aeruginosa strain, RST 9501, which contains mainly the variant [D-Leu1] MC-LR and originated from the Patos Lagoon Estuary (RS, Brazil). The exposure caused short-term repression of tumor suppressor genes and long-term repression of proto-oncogenes. These responses were more evident for p53 that was repressed with exposure for 6, 24 and 96 h, and fosab and myca that were consistently repressed with exposure for 384 h, when fish were exposed to both M. aeruginosa lysate dilutions, compared to controls (p < 0.05). The suppressor genes, baxa and gadd45α, and the proto-oncogene, junba, were suppressed mainly at 96 h, where both dilutions of the lysate caused repression compared to controls (p < 0.05). The p53 gene was the only gene to be induced; this occurred in fish exposed to lysate containing 3.5 µg L-1 for 384 h. This is the first study to show that M. aeruginosa containing an environmentally relevant concentration of [D-Leu1] MC-LR could cause time-dependent repression of proto-oncogenes and tumor suppressor genes in fish. The results suggest that short-term repression of tumor suppressor genes could participate in the mechanism of tumor promotion caused by M. aeruginosa in fish.

Microcystin-LR leads to oxidative damage and alterations in antioxidant defense system in liver and gills of Brycon amazonicus (SPIX & AGASSIZ, 1829).

Microcystin's (MCs) are toxins produced by several groups of cyanobacteria, in water bodies throughout the world, in a process which is being intensified by human action. Among the variants of MCs, MC-LR stands out for its distribution and toxicity. MCs are potent inhibitors of protein phosphatases 1 and 2 A, which causes disruption of the cytoskeleton and consequent cell death. They can also alter the antioxidant system and induce oxidative stress in various organs of many species. There is, however, a lack of information about the effects of MCs on the antioxidant system and oxidative damage in Brazilian fishes. This study evaluated the effect of microcystin-LR on the antioxidant system in liver and gills of the Brazilian fish Brycon amazonicus, after 48 h of i.p injection of 100 µg MC-LR.kg-1 body mass. The liver exhibited increases in the activity of GST (74%) and GPx (217%), and a 47% decrease in SOD activity, with no changes in CAT values. In the gills of fish exposed to MC-LR, CAT and GPx activities did not show significant changes, while SOD and GST activity decreased by 66% and 37%, respectively. The GSH content did not change significantly in the liver, however, a decrease of 43% was observed in the gills. Oxidative damage measured by protein oxidation (PC) and lipoperoxidation (LPO) showed significant effects in both tissues. In hepatic tissue, there was no change in PC levels but LPO increased by 116%. Conversely, in the gills LPO levels did not change but PC increased by 317%. In conclusion, these data show that MC-LR induces oxidative damage in both tissues but in different ways, with being liver most sensitive to LPO and gills to PC. This also suggests that the gills are most sensitive to oxidative stress than liver, due to the inhibition of its antioxidant responses following MC-LR exposure.

Interaction of single-walled carbon nanotubes and saxitoxin: Ab initio simulations and biological responses in hippocampal cell line HT-22.

Saxitoxins (STXs) are potent neurotoxins that also induce cytotoxicity through the generation of reactive oxygen species. Carbon nanotubes (CNTs) are nanomaterials that can promote a Trojan horse effect, facilitating the entry of toxic molecules to cells when adsorbed to nanomaterials. The interaction of pristine single-walled (SW)CNTs and carboxylated (SWCNT-COOH) nanotubes with STX was evaluated by ab initio simulation and bioassays using the cell line HT-22. Cells (5 × 104 cells/mL) were exposed to SWCNT and SWCNT-COOH (5 µg mL-1 ), STX (200 µg L-1 ), SWCNT+STX, and SWCNT-COOH+STX for 30 min or 24 h. Results of ab initio simulation showed that the interaction between SWCNT and SWCNT-COOH with STX occurs in a physisorption. The interaction of SWCNT+STX induced a decrease in cell viability. Cell proliferation was not affected in any treatment after 30 min or 24 h of exposure (p > 0.05). Treatment with SWCNT-COOH induced high reactive oxygen species levels, an effect attenuated in SWCNT-COOH+STX treatment. In terms of cellular oxygen consumption, both CNTs when coexposed with STX antagonize the toxin effect. Based on these results, it can be concluded that the results obtained in vitro corroborate the semiempirical evidence found using density functional theory ab initio simulation. Environ Toxicol Chem 2017;36:1728-1737. © 2016 SETAC.

Behavioral alterations induced by repeated saxitoxin exposure in drinking water.

BACKGROUND: Blooms of the saxitoxin-producing cyanobacterium Cylindrospermopsis raciborskii have been contaminating drinking water reservoirs in Brazil for many years. Although acute effects of saxitoxin intoxication are well known, chronic deleterious outcomes caused by repeated saxitoxin exposure still require further investigation. The aim of the present work is to investigate the effects of consumption of drinking water contaminated with C. raciborskii for 30 days on learning and memory processes in rats. METHODS: The effects of saxitoxin (3 or 9 µg/L STX equivalents) or cyanobacteria on behavior was determined using the open field habituation task, elevated plus maze anxiety model task, inhibitory avoidance task, and referential Morris water maze task. RESULTS: No effects of saxitoxin consumption was observed on anxiety and motor exploratory parameters in the elevated plus maze and open field habituation tasks, respectively. However, groups treated with 9 µg/L STX equivalents displayed a decreased memory performance in the inhibitory avoidance and Morris water maze tasks. CONCLUSIONS: These results suggest an amnesic effect of saxitoxin on aversive and spatial memories.

Toxic Trichodesmium bloom occurrence in the southwestern South Atlantic Ocean.

Harmful Trichodesmium blooms have been reported on the continental slope of the southwestern South Atlantic Ocean; we sampled six such blooms. The highest saxitoxin concentration was observed where the number of colonies was proportionally greater relative to the total density of trichomes. Trichodesmium blooms are harmful to shrimp larvae and may lead to plankton community mortality. This study is the first record of neurotoxic blooms in the open waters of the South Atlantic.

Behavioral alterations induced by repeated saxitoxin exposure in drinking water

Background: Blooms of the saxitoxin-producing cyanobacterium Cylindrospermopsis raciborskii have been contaminating drinking water reservoirs in Brazil for many years. Although acute effects of saxitoxin intoxication are well known, chronic deleterious outcomes caused by repeated saxitoxin exposure still require further investigation. The aim of the present work is to investigate the effects of consumption of drinking water contaminated with C. raciborskii for 30 days on learning and memory processes in rats. Methods: The effects of saxitoxin (3 or 9 µg/L STX equivalents) or cyanobacteria on behavior was determined using the open field habituation task, elevated plus maze anxiety model task, inhibitory avoidance task, and referential Morris water maze task. Results: No effects of saxitoxin consumption was observed on anxiety and motor exploratory parameters in the elevated plus maze and open field habituation tasks, respectively. However, groups treated with 9 µg/L STX equivalents displayed a decreased memory performance in the inhibitory avoidance and Morris water maze tasks. Conclusions: These results suggest an amnesic effect of saxitoxin on aversive and spatial memories.(AU)