Unlocking Biological Activity and Metabolomics Insights: Primary Screening of Cyanobacterial Biomass from a Tropical Reservoir.

Cyanobacterial harmful algal blooms can pose risks to ecosystems and human health worldwide due to their capacity to produce natural toxins. The potential dangers associated with numerous metabolites produced by cyanobacteria remain unknown. Only select classes of cyanopeptides have been extensively studied with the aim of yielding substantial evidence regarding their toxicity, resulting in their inclusion in risk management and water quality regulations. Information about exposure concentrations, co-occurrence, and toxic impacts of several cyanopeptides remains largely unexplored. We used liquid chromatography-mass spectrometry (LC-MS)-based metabolomic methods associated with chemometric tools (NP Analyst and Data Fusion-based Discovery), as well as an acute toxicity essay, in an innovative approach to evaluate the association of spectral signatures and biological activity from natural cyanobacterial biomass collected in a eutrophic reservoir in southeastern Brazil. Four classes of cyanopeptides were revealed through metabolomics: microcystins, microginins, aeruginosins, and cyanopeptolins. The bioinformatics tools showed high bioactivity correlation scores for compounds of the cyanopeptolin class (0.54), in addition to microcystins (0.54-0.58). These results emphasize the pressing need for a comprehensive evaluation of the (eco)toxicological risks associated with different cyanopeptides, considering their potential for exposure. Our study also demonstrated that the combined use of LC-MS/MS-based metabolomics and chemometric techniques for ecotoxicological research can offer a time-efficient strategy for mapping compounds with potential toxicological risk. Environ Toxicol Chem 2024;43:2222-2231. © 2024 SETAC.

Cyanopeptides occurrence and diversity in a Brazilian tropical reservoir: Exploring relationships with water quality.

Microcystins (MCs) are a class of toxic secondary metabolites produced by some cyanobacteria strains that endanger aquatic and terrestrial organisms in various freshwater systems. Although patterns in MC occurrence are being recognized, divergences in the global data still hamper our ability to predict the toxicity of cyanobacterial blooms. This study aimed (i) to determine the dynamics of MCs and other cyanopeptides in a tropical reservoir, (ii) to investigate the correlation between peptides and potential cyanotoxin producers (iii) identifying the possible abiotic factors that influence the peptides. We analyzed, monthly, eight MC variants (MC-RR, -LA, -LF, -LR, -LW, -YR, [D-Asp3]-RR and [D-Asp3]-LR) and other peptides in 47 water samples collected monthly, all season long, from two sampling sites in a tropical eutrophic freshwater reservoir, in southeastern Brazil. The cyanopeptides were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The biomass of potential cyanobacterial producers and water quality variables were measured. MCs were detected in both sampling sites year-round; the total MC concentration varied from 0.21 to 4.04 µg L-1, and three MC variants were identified and quantified (MC-RR, [D-Asp3]-RR, -LR). Additionally, we identified 28 compounds belonging to three other cyanopeptide classes: aeruginosin, microginin, and cyanopeptolin. As potential MC producers, Microcystis spp. and Dolichospermum circinalis were dominant during the study, representing up to 75% of the total phytoplankton. Correlational and redundancy analysis suggested positive effects of dissolved oxygen, nitrate, and total phosphorus on MC and microginins concentration, while water temperature appeared to favor aeruginosins. A comparison between our results and historical data showed a reduction in total phosphorus and cyanobacteria, suggesting increased water quality in the reservoir. However, the current MC concentrations indicate a rise in cyanobacterial toxicity over the last eight years. Moreover, our study underscores the pressing need to explore cyanopeptides other than MCs in tropical aquatic systems.

Temporal and spatial variation in the efficiency of a Floc & Sink technique for controlling cyanobacterial blooms in a tropical reservoir.

One of the main symptoms of eutrophication is the proliferation of phytoplankton biomass, including nuisance cyanobacteria. Reduction of the external nutrient load is essential to control eutrophication, and in-lake interventions are suggested for mitigating cyanobacterial blooms to accelerate ecosystem recovery. Floc & Sink (F&S) is one such intervention technique that consists of applying a low dose of coagulants in combination with ballasts for removing cyanobacteria biomass. It is especially suitable for deep lakes with an external nutrient load that is higher than the internal load and suffers from perennial cyanobacterial bloom events. Studies showing the efficacy of the F&S technique have been published, but those testing its variation in efficacy with changes in the environmental conditions are still scarce. Therefore, we evaluated the efficiency of the F&S technique to remove cyanobacteria from water samples collected monthly from two different sites in a deep tropical reservoir (Funil Reservoir, Brazil) in the laboratory. We tested the efficacy of two coagulants, chitosan (CHI) and poly-aluminum chloride (PAC), alone and in combination with lanthanum-modified bentonite (LMB) in settling phytoplankton biomass. We hypothesized that: ⅰ) the combined treatments are more effective in removing the algal biomass and ⅱ) the efficiency of F&S treatments varies spatially and monthly due to changes in environmental conditions. The combined treatments (PAC + LMB or CHI + LMB) removed up to seven times more biomass than single treatments (PAC, CHI, or LMB). Only the treatments CHI and LMB + CHI differed in efficiency between the sites, although all treatments showed significant variation in efficiency over the months at both the sampling sites. The combined treatments exhibited lower removal efficacy during the warm-rainy months (October-March) than during the mild-cold dry months (April-September). At high pH (pH > 10), the efficiency of the CHI and LMB + CHI treatments decreased. CHI had lower removal efficiency when single-cell cyanobacteria were abundant, while the combined treatments were equally efficient regardless of the morphology of the cyanobacteria. Hence, the combination of PAC as a coagulant with a ballast LMB is the most effective technique to precipitate cyanobacteria under the conditions that are encountered around the year in this tropical reservoir.

'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).

Increasing Temperature Counteracts the Negative Effect of UV Radiation on Growth and Photosynthetic Efficiency of Microcystis aeruginosa and Raphidiopsis raciborskii.

High temperature can promote cyanobacterial blooms, whereas ultraviolet radiation (UVR) can potentially depress cyanobacterial growth by damaging their photosynthetic apparatus. Although the damaging effect of UVR has been well documented, reports on the interactive effects of UV radiation exposure and warming on cyanobacteria remain scarce. To better understand the combined effects of temperature and UVR on cyanobacteria, two strains of nuisance species, Microcystis aeruginosa (MIRF) and Raphidiopsis raciborskii (formerly Cylindrospermopsis raciborskii, CYRF), were grown at 24°C and 28°C and were daily exposed to UVA + UVB (PAR + UVA+UVB) or only UVA (PAR + UVA) radiation. MIRF and CYRF growth rates were most affected by PAR + UVA+UVB treatment and to a lesser extent by the PAR + UVA treatment. Negative UVR effects on growth, Photosystem II (PSII) efficiency and photosynthesis were pronounced at 24°C when compared to that at 28°C. Our results showed a cumulative negative effect on PSII efficiency in MIRF, but not in CYRF. Hence, although higher temperature ameliorates UVR damage, interspecific differences may lead to deviating impacts on different species, and combined elevated temperature and UVR stress could influence species competition.

Assessment of possible solid-phase phosphate sorbents to mitigate eutrophication: Influence of pH and anoxia.

Managing eutrophication remains a challenge to water managers. Currently, the manipulation of biogeochemical processes (i.e., geo-engineering) by using phosphorus-adsorptive techniques has been recognized as an appropriate tool to manage the problem. The first step in finding potential mitigating materials is conducting a sequence of upscaling studies that commence with controlled laboratory experiments. Here, the abilities of 10 possible solid-phase-sorbents (SPS) to adsorb P were examined. Four materials adsorbed P, and two of these materials were modified, i.e., a lanthanum-modified-bentonite (LMB) and an aluminum-modified-zeolite (AMZ), and had the highest adsorption capacities of 11.4 and 8.9mgPg-1, respectively. Two natural materials, a red soil (RS) and a bauxite (BAU), were less efficient with adsorption capacities of 2.9 and 3.4mgPg-1, respectively. Elemental composition was not related to P adsorption. Since SPS might be affected by pH and redox status, we also tested these materials at pH values of 6, 7, 8 and 9 and under anoxic condition. All tested materials experienced decreased adsorption capacities under anoxic condition, with maximum adsorptions of 5.3mgPg-1 for LMB, 5.9mgPg-1 for AMZ, 0.2mgPg-1 for RS and 0.2mgPg-1 for BAU. All materials were able to adsorb P across the range of pH values that were tested. The maximum adsorption capacities of LMB and RS were highest at pH6, AMZ was higher at a pH of 9 and BAU at a pH of 8. Thus, pH influenced P adsorption differently. Given the effects of pH and anoxia, other abiotic variables should also be considered. Considering the criteria that classify a useful SPS (i.e., effective, easy to produce, cheap and safe), only the two modified materials that were tested seem to be suitable for upscaling to enclosure studies with anoxic sediments.

Critical assessment of chitosan as coagulant to remove cyanobacteria.

Removal of cyanobacteria from the water column using a coagulant and a ballast compound is a promising technique to mitigate nuisance. As coagulant the organic, biodegradable polymer chitosan has been promoted. Results in this study show that elevated pH, as may be common during cyanobacterial blooms, as well as high alkalinity may hamper the coagulation of chitosan and thus impair its ability to effectively remove positively buoyant cyanobacteria from the water column. The underlying mechanism is likely a shielding of the protonated groups by anions. Inasmuch as there are many chitosan formulations, thorough testing of each chitosan prior to its application is essential. Results obtained in glass tubes were similar to those from standard jar tests demonstrating that glass tube tests can be used for testing effects of coagulants and ballasts in cyanobacteria removal whilst allowing far more replicates. There was no relation between zeta potential and precipitated cyanobacteria. Given the well-known antibacterial activity of chitosan and recent findings of anti-cyanobacterial effects, pre-application tests are needed to decipher if chitosan may cause cell leakage of cyanotoxins. Efficiency- and side-effect testing are crucial for water managers to determine if the selected approach can be used in tailor-made interventions to control cyanobacterial blooms and to mitigate eutrophication.

Chitosan as coagulant on cyanobacteria in lake restoration management may cause rapid cell lysis.

Combining coagulant and ballast to remove cyanobacteria from the water column is a promising restoration technique to mitigate cyanobacterial nuisance in surface waters. The organic, biodegradable polymer chitosan has been promoted as a coagulant and is viewed as non-toxic. In this study, we show that chitosan may rapidly compromise membrane integrity and kill certain cyanobacteria leading to release of cell contents in the water. A strain of Cylindrospermopsis raciborskii and one strain of Planktothrix agardhii were most sensitive. A 1.3 h exposure to a low dose of 0.5 mg l-1 chitosan already almost completely killed these cultures resulting in release of cell contents. After 24 h, reductions in PSII efficiencies of all cyanobacteria tested were observed. EC50 values varied from around 0.5 mg l-1 chitosan for the two sensitive strains, via about 5 mg l-1 chitosan for an Aphanizomenon flos-aquae strain, a toxic P. agardhii strain and two Anabaena cylindrica cultures, to more than 8 mg l-1 chitosan for a Microcystis aeruginosa strain and another A. flos-aquae strain. Differences in sensitivity to chitosan might be related to polymeric substances that surround cyanobacteria. Rapid lysis of toxic strains is likely and when chitosan flocking and sinking of cyanobacteria is considered in lake restoration, flocculation efficacy studies should be complemented with investigation on the effects of chitosan on the cyanobacteria assemblage being targeted.

Controlling cyanobacterial blooms through effective flocculation and sedimentation with combined use of flocculants and phosphorus adsorbing natural soil and modified clay.

Eutrophication often results in blooms of toxic cyanobacteria that hamper the use of lakes and reservoirs. In this paper, we experimentally evaluated the efficacy of a metal salt (poly-aluminium chloride, PAC) and chitosan, alone and combined with different doses of the lanthanum modified bentonite Phoslock(®) (LMB) or local red soil (LRS) to sediment positively buoyant cyanobacteria from Funil Reservoir, Brazil, (22°30'S, 44°45'W). We also tested the effect of calcium peroxide (CaO2) on suspended and settled cyanobacterial photosystem efficiency, and evaluated the soluble reactive P (SRP) adsorbing capacity of both LMB and LRS under oxic and anoxic conditions. Our data showed that buoyant cyanobacteria could be flocked and effectively precipitated using a combination of PAC or chitosan with LMB or LRS. The SRP sorption capacity of LMB was higher than that of LRS. The maximum P adsorption was lowered under anoxic conditions especially for LRS ballast. CaO2 addition impaired photosystem efficiency at 1 mg L(-1) or higher and killed precipitated cyanobacteria at 4 mg L(-1) or higher. A drawback was that oxygen production from the peroxide gave positive buoyancy again to the settled flocs. Therefore, further experimentations with slow release pellets are recommended.