Elsevier

Toxicon

Volume 195, May 2021, Pages 69-77
Toxicon

The sensitivity of multiple ecotoxicological assays for evaluating Microcystis aeruginosa cellular algal organic matter and contribution of cyanotoxins to the toxicity

https://doi.org/10.1016/j.toxicon.2021.03.003Get rights and content

Highlights

  • Multiple ecotoxicological assays were compared for their sensitivity to Microcystis aeruginosa cellular organic matter.

  • Cytotoxicity assay with rainbow trout cell lines (RTgill-W1, RTG-2, and RTL-W1) was evaluated as the most sensitive.

  • Microcystins and anatoxin-a found in the cellular organic matter by LC–MS/MS were not responsible for the toxic response.

Abstract

Secondary metabolites of cyanobacteria and algae released during algal blooms often exhibit toxic effects, but only a small number of the metabolites are the subject of routine analytical screenings. Alternatively, ecotoxicological assays offer a better representation of the overall negative effects. The aim of this work was to compare multiple assays in their sensitivity towards cellular algal organic matter (COM) of the toxin-producing cyanobacterium Microcystis aeruginosa. Multiple endpoints were investigated: mortality, growth inhibition, bioluminescence inhibition, genotoxicity, endocrine-disrupting effects, oxidative stress, and the induction of ethoxyresorufin-O-deethylase (EROD). Three rainbow trout (Oncorhynchus mykiss) cell lines as well as representatives of bacteria, yeasts, algae, vascular plants, and crustaceans were employed, and the results were expressed per mg of dissolved organic carbon (DOC) in the COM. M. aeruginosa COM was toxic to the RTgill-W1, RTG-2, and RTL-W1 cell lines (EC50 values ranging from 0.48 ± 0.02 to 1.9 ± 0.1 mgDOC/L), to the crustacean Thamnocephalus platyurus (LC50 = 20 ± 1 mgDOC/L), and to Lepidium sativum (IC50 = 241 ± 13 mgDOC/L). In contrast, no effect was observed for bacteria and yeasts, and the growth of the alga Desmodesmus subspicatus was even stimulated. No genotoxicity, endocrine-disrupting effects or increase in oxidative stress or EROD activity was detected. The content of six microcystins (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the M. aeruginosa COM was determined by liquid chromatography–tandem mass spectrometry. An artificially prepared mixture of the detected cyanotoxins in the corresponding concentrations did not induce response in the O. mykiss cell lines and T. platyurus, suggesting that other cyanobacterial metabolites are responsible for the toxicity of M. aeruginosa.

Introduction

The occurrence of algal blooms continues to increase due to factors such as the eutrophication of water reservoirs and climate change (Gobler, 2020). When cyanobacteria and algae proliferate and their biomass eventually decomposes, light penetration is hindered, and oxygen is depleted. In reservoirs utilized as sources of drinking water, the presence and characteristics of algal organic matter (AOM) can result in a decline in water quality by influencing its organoleptic properties, interfering with the treatment processes and contributing to the formation of disinfection by-products (Pivokonsky et al., 2016). Moreover, cyanobacteria produce over 1000 known secondary metabolites (peptides, lipids, alkaloids, polyketides, terpenes), some of which exhibit irritating and toxic effects (Dittmann et al., 2015). The production of toxins by cyanobacteria is species-specific and even varies between strains (D'ors et al., 2012; Gkelis et al., 2019).

One of the most frequent contributors to the development of freshwater blooms and toxin production is Microcystis aeruginosa. Other genera commonly associated with the production of cyanotoxins include Anabaena, Cylindrospermopsis, Nodularia, Nostoc, Oscillatoria, Anabaenopsis, Hapalosiphon, Lyngbya, Synechococcus, Aphanizomenon, and Planktothrix (Rastogi et al., 2014). The toxic compounds produced by cyanobacteria vastly differ in their structure and are often categorized according to their mechanism of toxicity into hepatotoxins (microcystins, MCs; nodularins), neurotoxins (anatoxins, saxitoxins, β‑methylamino-L-alanine), cytotoxins (cylindrospermopsin), and dermatotoxins such as lipopolysaccharides (Corbel et al., 2014).

The most commonly encountered (and therefore studied) cyanotoxins are MCs, which are cyclic heptapeptides that are distinguished by their amino acid composition, in which positions 2 and 4 are the most variable (Bouaïcha et al., 2019). For instance, the most frequently detected MC-LR contains leucine (L) and arginine (R). In some countries, MC-LR is subject to regulations since the World Health Organization set a provisional guideline value of 1 μg/L within the guidelines for drinking water quality. Nevertheless, more than 250 different MCs have been identified (Bouaïcha et al., 2019), some of which can also dominate in water blooms, as can cyanotoxins from other classes (Blom et al., 2001; Gurbuz et al., 2016; Loftin et al., 2016). Less common MCs can significantly contribute to the overall negative effects because of their increased toxicity (Faassen and Lürling, 2013). In addition, numerous studies pointed out that the most commonly detected cyanotoxins may not necessarily constitute the main causes of the toxicity of water blooms (Keil et al., 2002; Pichardo et al., 2006; Smutná et al., 2014; Sorichetti et al., 2014; Teneva et al., 2013).

The employment of toxicological tests therefore has many advantages for water quality assessments as opposed to methods of targeted analytical chemistry. Crustaceans such as Thamnocephalus platyurus and mammalian or fish cell lines have often been used for such evaluations (Ács et al., 2013; Bober and Bialczyk, 2017; Kohler et al., 2014; Sierosławska et al., 2014), although half maximal effective concentrations (EC50) obtained for single MCs are relatively high, usually 1–100 mg/L (Blom et al., 2001; Pichardo et al, 2005, 2007; Štěpánková et al., 2011). Apart from the ethical standpoint, the advantages of the use of cell lines over tests employing whole organisms or primary cultures include higher sample throughput, better reproducibility, easier manipulation, and time- and cost-effectiveness. Fish cell lines have been found to show lower response to MCs and AOM than mammalian cell lines (Boaru et al., 2006a; Teneva et al., 2013; Zhou et al., 2018); nevertheless, they better represent organisms acutely exposed in the environment from an ecological perspective. Moreover, toxicity results obtained with the RTgill-W1 cell line derived from the gills of rainbow trout (Oncorhynchus mykiss) were found to greatly correspond with in vivo fish acute toxicity tests (Tanneberger et al., 2013).

When assessing the responses to the whole AOM, a comparison of the assays is far from straightforward on account of the differences in sample collection, the cultivation of the cyanobacteria, and AOM preparation protocols. The objective of this study was to compare commonly used bioassays in terms of their sensitivity towards a single cellular AOM (COM) sample. The effects of COM originating from M. aeruginosa, one of the main cyanotoxin producers, were evaluated by ecotoxicological assays comprising multiple trophic levels and endpoints. The assays focused primarily on acute toxicity because our goal was to select an assay that would be the most suitable for rapid toxicity determination of COM. Additionally, the content of six MCs (MC-LR, MC-RR, MC-YR, MC-LY, MC-LW, and MC-LF), anatoxin-a, cylindrospermopsin, and nodularin in the COM was determined by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS), and their contribution to the toxic effects was investigated.

Section snippets

Chemicals

Analytical standards of MC-LR, MC-RR, MC-YR, MC-LW, MC-LY, MC-LF, (±)anatoxin-a, cylindrospermopsin, and nodularin were purchased from Cyano Biotech (Germany). For LC–MS/MS analysis, acetonitrile (≥99.9%), ultrapure water (CHROMASOLV™ LC-MS), ammonium fluoride (≥98.0%), and formic acid (≥97.5%) were obtained from Honeywell (USA).

Leibovitz's L-15 medium (no phenol red, Gibco), Foetal Bovine Serum (FBS; Gibco), Penicillin-Streptomycin (Gibco), and alamarBlue™ Cell Viability Reagent (AB;

Cellular algal organic matter characterization

The COM of M. aeruginosa is composed of protein and non-protein organic matter. The protein portion accounted for approximately 63% of the total DOC, and the non-protein portion constituted the remaining 37% of the organic matter. Peptides and proteins of molecular weights of approximately 1, 2.8, 4, 4.5, 5, 5.7, 6, 6.8, 8, 8.5, 12, 30, 40, 52, 106, 266, 470, and 1077 kDa were identified by HPSEC (Supplementary Fig. A1). The pI values of the peptides/proteins determined by isoelectric focusing

Discussion

In this study, we compared the sensitivities of multiple toxicological assays on the COM of M. aeruginosa. All results of the toxicological assays were expressed per mg of DOC, which is a parameter frequently employed in water quality assessments. The COM of M. aeruginosa contained approximately 87% of hydrophilic compounds (Pivokonsky et al., 2014). The proteins constituted approximately 63% of the DOC content, which is in agreement with the literature where the protein portion comprised

Conclusions

As has been concluded in a number of studies, the small fraction of routinely detected cyanobacterial and algal secondary metabolites are not the sole cause of algal bloom toxicity. Relying on analytical methods targeted towards toxins such as MCs may therefore easily result in a substantial underestimation of the true environmental risks, and the use of ecotoxicological assays is vital. However, due to inconsistencies in COM extraction and the numerous other parameters that influence the

Statement of Contributions

Kamila Šrédlová; – Investigation, Writing – original draft, Data curation, Simona Šilhavecká; – Investigation, Data curation, Lucie Linhartová; – Methodology, Data curation, Jaroslav Semerád – Methodology, Data curation, Klára Michalíková; – Data curation, Martin Pivokonský; – Resources, Conceptualization, Tomáš Cajthaml – Funding acquisition, Supervision, Methodology, Conceptualization, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This study was supported by the Czech Science Foundation [Grant No. GA18-14445 S]. Institutional support was provided by the Center for Geosphere Dynamics [UNCE/SCI/006] and by the Czech Academy of Sciences [RVO: 67985874]. We acknowledge the Cytometry and Microscopy Facility at the Institute of Microbiology of the Czech Academy of Sciences for the use of cytometry equipment.

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