Investigation of the Occurrence of Cyanotoxins in Lake Karaoun (Lebanon) by Mass Spectrometry, Bioassays and Molecular Methods

Lake Karaoun is the largest artificial lake in Lebanon and serves multiple purposes. Recently, intensive cyanobacterial blooms have been reported in the lake, raising safety and aesthetic concerns related to the presence of cyanotoxins and cyanobacterial taste and odor (T&O) compounds, respectively. Here, we communicate for the first time results from a recent investigation by LC-MS/MS covering multiple cyanotoxins (microcystins (MCs), anatoxin-a, cylindrospermopsin, nodularin) in water and fish collected between 2019 and 2020. Eleven MCs were identified reaching concentrations of 211 and 199 μg/L for MC-LR and MC-YR, respectively. Cylindrospermopsin, anatoxin-a and nodularin were not detected. The determination of the total MCs was also carried out by ELISA and Protein Phosphatase Inhibition Assay yielding comparable results. Molecular detection of cyanobacteria (16S rRNA) and biosynthetic genes of toxins were carried out by qPCR. Untargeted screening analysis by GC-MS showed the presence of T&O compounds, such as β-cyclocitral, β-ionone, nonanal and dimethylsulfides that contribute to unpleasant odors in water. The determination of volatile organic compounds (VOCs) showed the presence of anthropogenic pollutants, mostly dichloromethane and toluene. The findings are important to develop future monitoring schemes in order to assess the risks from cyanobacterial blooms with regard to the lake’s ecosystem and its uses.

Evaluation of laboratory methods for the analysis of microcystins

Finding low cost, rapid tests to monitor microcystins in water is paramount to protect environmental and public health worldwide. Bioassays like Protein Phosphatase Inhibition Assay (PPIA) and Enzyme-linked Immunosorbent Assay (ELISA) have many advantages over liquid chromatography–tandem mass spectrometry (LC-MS/MS). Analytical cost per sample was found to be $136 by ELISA and $365 by LC-MS/MS. An agreement of 76% was found between ELISA and LC-MS/MS results from 2010 to 2012 (n=854) on the basis of Method Detection Limits (MDLs). Among samples with LC-MS/MS results >1.5μg/L, ELISA missed 3 samples in 2010, 1 in 2011 and none in 2012. Correlation between PP2A and ELISA was strong (R2=0.8155, p=0.8054, n=27) in surface water samples but non-existent in drinking water (R2=0.0366, p=0.0665, n=38). PP2A was found useful for monitoring non-coloured surface water but not for drinking water. A 2-tier test system is proposed: tier-1 ELISA and tier-2 PP2A for surface water samples.

Evaluation of laboratory methods for the analysis of microcystins

Finding low cost, rapid tests to monitor microcystins in water is paramount to protect environmental and public health worldwide. Bioassays like Protein Phosphatase Inhibition Assay (PPIA) and Enzyme-linked Immunosorbent Assay (ELISA) have many advantages over liquid chromatography–tandem mass spectrometry (LC-MS/MS). Analytical cost per sample was found to be $136 by ELISA and $365 by LC-MS/MS. An agreement of 76% was found between ELISA and LC-MS/MS results from 2010 to 2012 (n=854) on the basis of Method Detection Limits (MDLs). Among samples with LC-MS/MS results >1.5μg/L, ELISA missed 3 samples in 2010, 1 in 2011 and none in 2012. Correlation between PP2A and ELISA was strong (R2=0.8155, p=0.8054, n=27) in surface water samples but non-existent in drinking water (R2=0.0366, p=0.0665, n=38). PP2A was found useful for monitoring non-coloured surface water but not for drinking water. A 2-tier test system is proposed: tier-1 ELISA and tier-2 PP2A for surface water samples.

A Mini-Review on Detection Methods of Microcystins

Cyanobacterial harmful algal blooms (CyanoHABs) produce microcystins (MCs) which are associated with animal and human hepatotoxicity. Over 270 variants of MC exist. MCs have been continually studied due of their toxic consequences. Monitoring water quality to assess the presence of MCs is of utmost importance although it is often difficult because CyanoHABs may generate multiple MC variants, and their low concentration in water. To effectively manage and control these toxins and prevent their health risks, sensitive, fast, and reliable methods capable of detecting MCs are required. This paper aims to review the three main analytical methods used to detect MCs ranging from biological (mouse bioassay), biochemical (protein phosphatase inhibition assay and enzyme linked immunosorbent assay), and chemical (high performance liquid chromatography, liquid chromatography-mass spectrometry, high performance capillary electrophoresis, and gas chromatography), as well as the newly emerging biosensor methods. In addition, the current state of these methods regarding their novel development and usage, as well as merits and limitations are presented. Finally, this paper also provides recommendations and future research directions towards method application and improvement.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)−m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)−m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)−m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)−m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Microcystin Content in Phytoplankton and in Small Fish from Eutrophic Nyanza Gulf, Lake Victoria, Kenya

The human health risks posed by exposure to cyanobacterial toxins such as microcystin (MC) through water and fish consumption remain poorly described. During the last two decades, coastal regions of Lake Victoria such as Nyanza Gulf (Kisumu Bay) have shown severe signs of eutrophication with blooms formed by Microcystis producing MC. In this study, the spatial variability in MC concentration in Kisumu Bay was investigated which was mostly caused by Microcystis buoyancy and wind drifting. Small fish (<6 cm) mainly composed of Rastrineobola argentea were examined for MC content by means of biological methods such as ELISA and protein phosphatase inhibition assay (PPIA) and partly by chemical-analytical methods such as LC-MS/MS. Overall, the MC content in small fish was related to the MC content observed in the seston. When comparing the MC content in the seston in relation to dry weight with the MC content in small fish the latter was found three orders of magnitude decreased. On average, the ELISA-determined MC contents exceeded the PPIA-determined MC contents by a factor of 8.2 ± 0.5 (SE) while the MC contents as determined by LC-MS/MS were close to the detection limit. Using PPIA, the MC content varied from 25–109 (mean 62 ± 7) ng/g fish dry weight in Kisumu Bay vs. 14 ± 0.8 ng MC/g in the more open water of L. Victoria at Rusinga channel. Drying the fish under the sun showed little effect on MC content, although increased humidity might indirectly favor photocatalyzed MC degradation.

Evaluation of the Direct and Indirect Regulation Pathways of Glutathione Target to the Hepatotoxicity of Microcystin-LR

Glutathione (GSH) plays crucial roles in regulating the hepatotoxicity of Microcystin-LR (MCLR) by inhibiting oxidative stress or by toxin conjugation. Based on MCLR conjugation product preparation and purification, the direct and indirect regulation pathways for GSH were fully evaluated. Protein phosphatase inhibition analysis verified that GSH conjugation was an effective pathway to regulate the inhibition effect of MCLR, while GSH had slight influence on the toxicity of MCLR. Research on oxidative stress showed that both regulation pathways could reduce the formation of reactive oxygen species (stimulated by MCLR and regulated by NADH oxidase) and regulate the adverse effects on antioxidant enzymes. By evaluating the contributions for both pathways, it could be found that the indirect pathway had significant contribution to eliminating cellular reactive oxygen species and regulating protein phosphatases inhibition, while the direct regulation pathway had moderate influence. As glutathione transferases facilitated the transformation of MCLR, the hepatotoxicity of MCLR could be effectively regulated by GSH conjugation pathway, especially with abundant exogenous GSH.