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.

Influence of refrigeration and freezing in Microcystins and Cylindrospermopsin concentrations on fish muscle of tilapia (Oreochromis niloticus) and tench (Tinca tinca).

The consumption of fish contaminated with cyanotoxins is an important public health issue due to their potential adverse effects. The aim of this study was to assess the influence of refrigeration (4 °C) and freezing (-20 °C) on the concentration of Cylindrospermopsin (CYN), Microcystins (MCs) and their combination in tilapia (Oreochromis niloticus) and tench (Tinca tinca). Fish muscle were spiked with a stock solution of each toxin to reach 750 µg/g dry weight (d.w.). Three different periods of time were investigated for each treatment: 24 h, 48 h and 7 days for refrigeration, and 24 h, 7 days and 1 month for freezing. Samples were extracted and quantified by Ultra Performance Liquid Chromatography - Tandem Mass Spectrometry (UPLC-MS/MS). The results showed that freezing for 1 month produced highest decreases of these toxins in both species in comparison to refrigeration, being CYN the most stable cyanotoxin. Moreover, MCs are more stable to storage processes in the mixtures than alone, and fish species is a factor to take into account in their stability. These findings highlight the need to assess the influence of food storage processes on the presence of cyanotoxins in fish species for a more realistic human health risk assessment.

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 ß-cyclocitral, ß-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.

Removal of Cyanobacteria and Cyanotoxins in Waters.

Harmful cyanobacterial algal blooms and cyanotoxins currently pose a major threat to global society, one that exceeds local and national interests due to their extremely destructive effects on the environment and human health [...].

Cyanotoxin Analysis and Amino Acid Profiles of Cyanobacterial Food Items from Chad.

In some parts of the world, cyanobacteria are used as a food in the human diet, due to their ready availability. Lake Chad, has long been a traditional site for the collection of Arthrospira fusiformis which is dried and processed at the lake into thin wafers called Dihé for later consumption or is transported to market for sale. However, Dihé purchased from markets in Chad has not been analyzed for known cyanobacterial toxins or assessed for total amino acid content. Since BMAA in traditional foodstuffs of the indigenous Chamorro people of Guam causes neurodegenerative illness, it is important that Dihé from Chad be analyzed for this neurotoxin. BMAA and its isomer AEG were not detected in our analyses, but a further isomer DAB was detected as both a free and bound amino acid, with an increase in the free concentration after acid hydrolysis of this fraction. Microcystins were present in 6 samples at up to 20 µg/g according to UPLC-PDA, although their presence could not be confirmed using PCR for known microcystin synthetic genes. Amino acid analysis of the cyanobacterial material from Chad showed the presence of large amounts of canonical amino acids, suggesting that this may supplement indigenous people on low protein diets, although regular monitoring of the foodstuffs for the presence of cyanotoxins should be performed.

Second Laboratory Validation of ß-N-Methylamino-L-Alanine, N-(2aminoethyl)Glycine, and 2,4-Diaminobuytric Acid by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry.

The analysis of ß-N-methylamino-L-alanine (BMAA) has been validated according to AOAC international standards by a single laboratory (Glover et al. 2015). Using the same validated method, we add a second laboratory validation optimizing for different equipment. Given publicized concerns about standardizing methods across laboratories and recent reviews indicating superior results using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatization for the separation of BMAA and its isomers N-(2aminoethyl)glycine (AEG), and 2,4-diaminobuytric acid (DAB) (Bishop and Murch 2019), we add a second laboratory validation to this method demonstrating that the method is robust across laboratories using different equipment. Using the US Food and Drug Administration (FDA 2018) method for evaluating instrument parameters, we calculated a limit of detection (LOD) of 10 pg/ml for BMAA, AEG, and DAB and lower limits of quantification (LLOQ) of 37 pg/ml based on reagent blanks. In biological matrices, a higher LLOQ may be warranted for AEG and DAB. We demonstrate that the endogenous BMAA in mussel tissue can be lost by drying the hydrolyzed preparation and suggest sample preparation parameters be evaluated for robustness.

A Single Laboratory Validation for the Analysis of Underivatized ß-N-Methylamino-L-Alanine (BMAA).

ß-N-Methylamino-L-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria that can accumulate in ecosystems and food webs. Human exposure to cyanobacterial and algal blooms may be a risk factor for neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Analytical chemists have struggled to find reliable methods for BMAA analysis in complex sample matrices. Analysis of BMAA is complicated by at least 3 naturally occurring isomers: N-(2-aminoethyl)glycine (AEG), 2,4-diaminobutyric acid (DAB), and ß-aminomethyl-L-alanine (BAMA). More than 350 publications have reported detection and quantification of BMAA and its isomers, but varying results have led to controversy in the literature. The objective of this study was to perform a single laboratory validation (SLV) of a frequently published method for BMAA analysis using a ZIC-HILIC column. We investigated the selectivity, linearity, accuracy, precision, and sensitivity of the method and our data show that this HILIC method fails many of the criteria for a validated method. The method fails the criterion for selectivity as the chromatography does not separate BMAA from its isomer BAMA. Sensitivity of the method greatly decreased over the experimental period and it demonstrated a higher limit of detection (LOD) (7.5 pg on column) and a higher lower limit of quantification (LLOQ) (30 pg on column) than other published validated methods. The method demonstrated poor precision of repeated injections of standards of BMAA with % relative standard deviation (%RSD) values that ranged from 37 to 107% while HorRat values for BMAA had a fail rate of 80% and BAMA had a fail rate of 73%. No HorRat values between 0.5 and 2 were found for repeated injections of standards of AEG and DAB. Recovery of 13C3,15N2-BMAA in a cyanobacterial matrix was < 10% in experiments and we were also unable to accurately detect other protein amino acids including methionine, cysteine, or alanine, indicating matrix effects. The results of this study demonstrate that the ZIC-HILIC column is not fit for purpose for the analysis of BMAA in cyanobacterial matrices and further provides explanations for the high level of negative results reported by researchers using this method.

Toxin Analysis of Freshwater Cyanobacterial and Marine Harmful Algal Blooms on the West Coast of Florida and Implications for Estuarine Environments.

Recent marine and freshwater algal and cyanobacterial blooms in Florida have increased public concern and awareness of the risks posed by exposure to these organisms. In 2018, Lake Okeechobee and the Caloosahatchee river, on the west coast of Florida, experienced an extended bloom of Microcystis spp. and a bloom of Karenia brevis in the coastal waters of the Gulf of Mexico that coincided in the Fort Myers area. Samples from the Caloosahatchee at Fort Myers into Pine Island Sound and up to Boca Grande were collected by boat. High concentrations of microcystin-LR were detected in the cyanobacterial bloom along with brevetoxins in the marine samples. Furthermore, ß-N-methylamino-L-alanine (BMAA) and isomers N-(2-aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) were detected in marine diatoms and dinoflagellates, and cyanobacteria of freshwater origin. High freshwater flows pushed the cyanobacterial bloom to barrier island beaches and Microcystis and microcystins could be detected into the marine environment at a salinity of 41 mS/cm. For comparison, in 2019 collections of Dapis (a new generic segregate from Lyngbya) mats from Sarasota showed high concentrations of BMAA, suggesting the possibility of long-term exposure of residents to BMAA. The findings highlight the potential for multiple, potentially toxic blooms to co-exist and the possible implications for human and animal health.

Biocrust-Produced Cyanotoxins Are Found Vertically in the Desert Soil Profile.

The fate and persistence of the neurotoxin ß-N-methylamino-L-alanine (BMAA) and its isomers N-(2aminoethyl)glycine (AEG) and 2,4-diaminobuytric acid (DAB) in soil profiles is poorly understood. In desert environments, these cyanotoxins are commonly found in both terrestrial and adjacent marine ecosystems; they accumulate in biocrusts and groundwater catchments, and have been previously shown to persist in soil as deep as 25 cm. To determine the depth that BMAA and its isomers can be found, samples were incrementally collected every 5 cm from bedrock to surface in triplicate soil cores in a biocrust field in the terrestrial desert of Qatar. Biocrust surface samples were also collected from each core priorly. Toxins were extracted from soil sub-samples, derivatized, and analyzed with UPLC-MS/MS. All toxins were detected in all soil cores at all depths. AEG and DAB were within a quantifiable concentration threshold; however, the low concentration of BMAA was considered below the threshold for quantification. This may have environmental health implications if these toxins are able to infiltrate and contaminate the bedrock aquifer, as well as the sand and gravel aquifers. Human and animal health may also be impacted through exposure to contaminated groundwater wells or through inhalation of aerosolized particles of soil, resuspended during construction or recreational activities.