Effect of pretreatment approach on the ELISA-based detection of cyanotoxins in water: Analysis and application.

Common cyanotoxins, such as microcystins and nodularins, are produced by frequently occurring harmful cyanobacterial algal blooms in freshwater systems. The required routine monitoring of microcystins and nodularins in drinking water and ambient water demands cost-efficient and reliable enzyme-linked immunosorbent assay kits. We validated the performance of a self-produced broad-spectrum enzyme-linked immunosorbent assay kit and investigated two different methods of mitigating the matrix effects to elucidate the effect of the respective pretreatment approaches recommended by China and the United States on the quantitative detection of cyanotoxins in surface water. We found that the enzyme-linked immunosorbent assay kit achieved a detection limit of 0.15 µg/L with a linear detection range from 0.27 µg/L to 1.87 µg/L for microcystin-LR (the most studied and widely distributed cyanotoxin). The matrix effects could be mitigated both by dilution of water samples with an optimal dilution ratio and dilution of antibody with the buffer containing phosphate buffer solution (10×), bovine serum albumin (1 %) and ethylene diamine tetraacetic acid (0.5 %). In terms of the surface water samples being tested, the concentrations of microcystins and nodularins measured based on pretreatment approach recommended by the United States were 1- 5 times that measured based on pretreatment approach recommended by China, indicating that the pretreatment approach of China overlooks cyanotoxins. In addition, all the measured total microcystins and nodularins of the surface water samples were below the health advisory limit (1.6 µg/L) for microcystins in drinking water proposed by the United States Environmental Protection Agency for school-age children and adults. Our research could provide significant information for outbreak warnings and risk management of harmful cyanobacterial algal blooms.

Interspecific Interactions Drive Nonribosomal Peptide Production in Nodularia spumigena.

Nodularia spumigena is a bloom-forming cyanobacterium that produces several classes of nonribosomal peptides (NRPs) that are biologically active; however, the ecological roles of specific NRPs remain largely unknown. Here, we explored the involvement of NRPs produced by N. spumigena in interspecific interactions by coculturing the cyanobacterium and its algal competitors, the diatom Phaeodactylum tricornutum and the cryptomonad Rhodomonas salina, and measuring NRP levels and growth responses in all three species. Contrary to the expected growth suppression in the algae, it was N. spumigena that was adversely affected by the diatom, while the cryptomonad had no effect. Reciprocal effects of N. spumigena on the algae were manifested as the prolonged lag phase in R. salina and growth stimulation in P. tricornutum; however, these responses were largely attributed to elevated pH and not to specific NRPs. Nevertheless, the NRP levels in the cocultures were significantly higher than in the monocultures, with an up to 5-fold upregulation of cell-bound nodularins and exudation of nodularin and anabaenopeptin. Thus, chemically mediated interspecific interactions can promote NRP production and release by cyanobacteria, resulting in increased input of these compounds into the water. IMPORTANCE NRPs were involved in growth responses of both cyanobacteria and algae; however, the primary driver of the growth trajectories was high pH induced by N. spumigena. Thus, the pH-mediated inhibition of eukaryotic phytoplankton may be involved in the bloom formation of N. spumigena. We also report, for the first time, the reciprocal growth inhibition of N. spumigena by diatoms resistant to alkaline conditions. As all species in this study can co-occur in the Baltic Sea during summer, these findings are highly relevant for understanding ecological interactions in planktonic communities in this and other systems experiencing regular cyanobacteria blooms.

A Feasibility Study into the Production of a Mussel Matrix Reference Material for the Cyanobacterial Toxins Microcystins and Nodularins.

Microcystins and nodularins, produced naturally by certain species of cyanobacteria, have been found to accumulate in aquatic foodstuffs such as fish and shellfish, resulting in a risk to the health of the seafood consumer. Monitoring of toxins in such organisms for risk management purposes requires the availability of certified matrix reference materials to aid method development, validation and routine quality assurance. This study consequently targeted the preparation of a mussel tissue reference material incurred with a range of microcystin analogues and nodularins. Nine targeted analogues were incorporated into the material as confirmed through liquid chromatography with tandem mass spectrometry (LC-MS/MS), with an additional 15 analogues detected using LC coupled to non-targeted high resolution mass spectrometry (LC-HRMS). Toxins in the reference material and additional source tissues were quantified using LC-MS/MS, two different enzyme-linked immunosorbent assay (ELISA) methods and with an oxidative-cleavage method quantifying 3-methoxy-2-methyl-4-phenylbutyric acid (MMPB). Correlations between the concentrations quantified using the different methods were variable, likely relating to differences in assay cross-reactivities and differences in the abilities of each method to detect bound toxins. A consensus concentration of total soluble toxins determined from the four independent test methods was 2425 ± 575 µg/kg wet weight. A mean 43 ± 9% of bound toxins were present in addition to the freely extractable soluble form (57 ± 9%). The reference material produced was homogenous and stable when stored in the freezer for six months without any post-production stabilization applied. Consequently, a cyanotoxin shellfish reference material has been produced which demonstrates the feasibility of developing certified seafood matrix reference materials for a large range of cyanotoxins and could provide a valuable future resource for cyanotoxin risk monitoring, management and mitigation.

Cyanotoxins and Food Contamination in Developing Countries: Review of Their Types, Toxicity, Analysis, Occurrence and Mitigation Strategies.

Cyanotoxins have gained global public interest due to their potential to bioaccumulate in food, which threatens human health. Bloom formation is usually enhanced under Mediterranean, subtropical and tropical climates which are the dominant climate types in developing countries. In this context, we present an up-to-date overview of cyanotoxins (types, toxic effects, analysis, occurrence, and mitigation) with a special focus on their contamination in (sea)food from all the developing countries in Africa, Asia, and Latin America as this has received less attention. A total of 65 publications have been found (from 2000 until October 2021) reporting the contamination by one or more cyanotoxins in seafood and edible plants (five papers). Only Brazil and China conducted more research on cyanotoxin contamination in food in comparison to other countries. The majority of research focused on the detection of microcystins using different analytical methods. The detected levels mostly surpassed the provisional tolerable daily intake limit set by the World Health Organization, indicating a real risk to the exposed population. Assessment of cyanotoxin contamination in foods from developing countries still requires further investigations by conducting more survey studies, especially the simultaneous detection of multiple categories of cyanotoxins in food.

ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 µg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Comprehensive insights into the occurrence and toxicological issues of nodularins.

The occurrence of cyanobacterial toxins is being increasingly reported. Nodularins (NODs) are one of the cyanotoxins group mainly produced by Nodularia spumigena throughout the world. NODs may exert adverse effects on animal and human health, and NOD-R variant is the most widely investigated. However, research focused on them is still limited. In order to understand the realistic risk well, the aim of this review is to compile the available information in the scientific literature regarding NODs, including their sources, distribution, structural characteristics, physicochemical properties, biosynthesis and degradation, adverse effects in vitro and vivo, and toxicokinetics. More data is urgently needed to integrate the cumulative or synergistic effects of NODs on different species and various cells to better understand, anticipate and aggressively manage their potential toxicity after both short- and long-term exposure in ecosystem, and to minimize or prevent the adverse effects on human health, environment and the economy.

Morphological and Molecular Identification of Microcystin-Producing Cyanobacteria in Nine Shallow Bulgarian Water Bodies.

The paper presents results from the first application of polyphasic approach in studies of field samples from Bulgaria. This approach, which combined the conventional light microscopy (LM) and molecular-genetic methods (based on PCR amplified fragments of microcystin synthetase gene mcyE), revealed that almost all microcystin-producers in the studied eutrophic waterbodies belong to the genus Microcystis. During the molecular identification of toxin-producing strains by use of HEPF × HEPR pair of primers, we obtained 57 sequences, 56 of which formed 28 strains of Microcystis, spread in six clusters of the phylogenetic tree. By LM, seven Microcystis morphospecies were identified (M. aeruginosa, M. botrys, M. flos-aquae, M. natans, M. novacekii, M. smithii, and M. wesenbergii). They showed significant morphological variability and contributed from <1% to 98% to the total biomass. All data support the earlier opinions that taxonomic revision of Microcystis is needed, proved the presence of toxigenic strains in M. aeruginosa and M. wesenbergii, and suppose their existence in M. natans. Our results demonstrated also that genetic sequencing, and the use of HEPF × HEPR pair in particular, can efficiently serve in water quality monitoring for identifying the potential risk from microcystins, even in cases of low amounts of Microcystis in the water.

Nodularins in poisoning.

Nodularins are an important class of hepatotoxic cyclic pentapeptides that are produced by the cyanobacteria Nodularia spumigena. These peptides have been found worldwide and have been implicated in the deaths of animals as well as a potent cyanotoxin in humans. To date, approximately 10 variants have been discovered, among which nodularin-R is the most abundant. Though the mechanisms of their potential hepatotoxicity and carcinogenicity are not well understood, the most frequently proposed mechanisms are described here. Most importantly, a comprehensive review of nodularins in poisoning is presented, including their bioaccumulation in water, cyanobacterial blooms and aquatic animals, the IC50, LC50 and LD50 values determined in laboratories, and wild, domestic and laboratory animal cases. However, the hazard of these toxins to humans has not been fully elucidated, predominantly due to the lack of exposure data. One of reasons underlying is that most current methods are ill suited for clinical monitoring. Thus, the available assays for the detection and quantification of nodularins are described with an emphasis on the problems encountered with each assay. Our ultimate aim is to demonstrate the urgency of better understanding the toxicity of nodularins, especially in humans, and thus effectively protecting ourselves from their poisoning.