Effects of Refrigeration and Freezing in Cylindrospermopsin and Microcystin Concentrations on Leaves of Lettuce (Lactuca sativa) and Spinach (Spinacia oleracea) ^† †

Abstract

As a consequence of climate change, an increase in the occurrence of cyanotoxins is happening. These toxins are a large group of secondary metabolites with different chemical structures, mechanisms of action and worldwide distribution, and can cause harmful effects in humans and animals. Humans can come in contact with these toxins through the oral route through the consumption of contaminated water and food. Frequently, in order to improve their shelf life, many foods, such as vegetables, are refrigerated or frozen before being consumed. Therefore, the aim of this work was to assess the potential effect of refrigeration (4 °C for 24 h, 48 h and 7 days) and freezing (−20 °C for 7 days, 1 month and 3 months) on the concentration of cylindrospermopsin (CYN) and microcystins (MCs) (MC-LR, MC-RR and MC-YR) in lettuce and spinach leaves. Vegetable samples were spiked with a stock solution of each toxin containing 0.75 µg toxin/mL (equivalent to 0.75 µg/g fresh weight) and subjected to the corresponding conservation process. Samples were extracted and quantified with ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). In the case of CYN, only in spinach samples the refrigeration process caused a significant time-dependent decreased in its concentration (48 h and 7 days). However, this preservation process was not shown to be effective in reducing the concentration of this toxin in lettuce. Moreover, the freezing process in spinach didn´t show differences in the CYN content between the control and the experimental groups. In general, results obtained in MC concentrations showed that the refrigeration process in both lettuce and spinach caused a significant decrease in toxin concentrations, and this decrease was higher in lettuce. Moreover, freezing caused a significant reduction in MC concentrations in spinach after 3 months. These results showed the differences in the toxin content depending on the type of process selected (refrigeration versus freezing) and vegetable species. Furthermore, more studies are needed to study the influence of different storage processes on cyanotoxin concentrations in vegetables humans consume to assess the risk of human exposure to these cyanotoxins in a more realistic way.