Studies of naturally occurring algal toxins like domoic acid (DA) and saxitoxins (STXs) in tropical developing countries are still very rare despite their harmful effect on marine life, human health, and seafood safety. We conducted a rapid assessment of DA, which is responsible for Amnestic Shellfish poisoning (ASP), and STXs and its congeners that are known to cause a foodborne illness called Paralytic Shellfish poisoning (PSP) between the 15th and 30th of July 2022. The assessment was based on earlier studies that recorded the presence of potentially toxic species that are causative agents of ASP and PSP in Kenya's coastal waters. In these geo-referenced areas, site characteristics including environmental variables such as temperature, salinity, dissolved oxygen (DO), and turbidity were recorded and surface seawater samples were collected for laboratory analysis of dissolved inorganic nutrients (nitrogen, phosphorus, and silicates), and chlorophyll a. Additionally, the abundance of potential PSP toxin-producing species Alexandrium spp., Aphanizomenon spp., Brachydinium capitatum, Gonyaulax spp., and Gymnodinium spp., and potential ASP toxin-producing species Amphora spp., Nitzschia acicularis, Nitzschia closterium, Nitzschia longissimi, Nitzschia reversa, Nitzschia sigma, Nitzschia spp., and Pseudo-nitzschia spp. were assessed alongside the toxins at each sampling location.
The phytotoxin samples were collected through horizontal net hauls at the water surface (depth of 0.5 m) using a 20 µm mesh size plankton net (Reguera et al., 2016). The haul contents at the net cod end were transferred into a container and sieved through 100 µm mesh to remove larger microzooplankton organisms that consume phytoplankton. The samples were transferred into glass containers and then transported to the laboratory in an icebox for further filtration. In the laboratory, 50 ml of the concentrated phytoplankton sample was filtered through a glass-fiber filter (25 mm) with a vacuum pump (e.g. Millipore), making sure that all the water was filtered through the filtration unit. The filter was then removed, and freeze-dried in cryogenic vials for analysis of PSP-saxitoxin and ASP-domoic acid. The PSP toxins in the filters were determined by LC-FLD using LC-FLP: LC1100 series liquid chromatography system consisting of a G1379A degasser, a G1311A quaternary pump, a G1229A autosampler, and a G1321A fluorescence detector (Agilent Technologies, Waldbronn, Germany) coupled to PCX 2500 post-column derivatization system (Pickering Laboratories, Mountain View, CA, USA, Reeves et al., 2021). The domoic acid was established by LC-MS/MS technique using LC1100 System (Agilent Technologies, Waldbronn, Germany) coupled to a 4000 Q-Trap triple quadrupole mass spectrometer (SCIEX, Darmstadt, Germany, Thomas et al., 2022).
Qualitative concentrated samples were collected by filtering 20 l of water (collected from just below the water surface) through a 20 µm phytoplankton net. For numerical analysis and potentially toxic species identification, 100 ml of water samples were fixed in 5% Lugol's solution. The samples were further concentrated to a volume of 50 ml and 1 ml (in triplicate) of the concentrated sample was transferred into a Sedgewick-Rafter chamber mounted on an inverted compound microscope (Leica DM IL) and counting of phytoplankton cells carried out in 100 squares of the cell chosen randomly. The results were expressed as the number of cells per liter. For chlorophyll determination, 1 l of seawater was taken from a depth of 50 cm, filtered onto a microfiber filter (Whatman GF/F), wrapped in tinfoil and stored at -20°C. In the laboratory, the filters were digested and chlorophyll a extracted using 96% Ethanol. The chlorophyll a/ethanol solutions were measured using a fluorometer (TD10AU Flouru, Ex436, Em680) according to (Welschmeyer, 1994).
Nutrient samples were filtered through disposable syringe filters (Sartorius Minisart ® - 0.45 µm pore size) immediately after sampling and filled into pre-rinsed polyethylene bottles, preserved with a mercury chloride solution (50 µl of a 20 g/l HgCl2-solution added to 100 ml sample) and stored cool until analysis. In the laboratory, nutrient concentrations were determined using standard methods in Grasshoff et al., (2007) by spectrophotometry (Infinite® M200 NanoQuant, Tecan Group Ltd., Switzerland).
Additionally, temperature, salinity, DO and turbidity were measured in situ using a handheld probe YSI Professional Plus (SKU6050000) Water Quality Meter, and Portable TN100 turbidity meter (Thermo Scientific™ ) respectively. All the field samplings and data collection were done in duplicates. All geo-references and field data were collected using KoboCollect v2022.2.3. Toxin-producing phytoplankton species identification was done at Kenya Marine and Fisheries Research Institute (KMFRI) laboratories.
