Introduction: The Stevia plant, recognized as a natural sweetener with proven antioxidant and insulinotropic properties, has seen increased consumption, raising concerns about its safety at various doses. This study assessed the toxicity of three commercially available Stevia products (leaves, powder, and tea bags) by preparing solutions at concentrations ranging from 100 to 20,000 mg/L using E3 solution. Zebrafish eggs were exposed to these concentrations in 24-well ELISA plates, with E3 solution serving as a negative control. Over 96 hours, larvae were monitored for morphological changes and mortality. Consistent with OECD 236 guidelines, over 80% of control larvae remained healthy. All substances at 20,000 mg/L resulted in complete mortality, while significant morphological changes were observed at concentrations of 10,000 mg/L and above. No mortality was recorded at 100 mg/L across all groups. The median lethal concentration (LC50) was determined using SPSS software.
Methods: During the 96-hour examination of zebrafish larvae, it was found that in the groups exposed to concentrations equal to and higher than 5000 mg/L of the tested compounds, there are significant differences with the control group in terms of mortality and morphological abnormalities. Also, studies show that the lethal concentration (LC50) in stevia leaf extract is higher than other groups (Table 1) and therefore, stevia leaf extract has lower acute toxicity than other studied groups. Also, two groups of stevia industrial products (powder and tea sweetened with stevia), although according to statistical analysis, they did not show any significant difference from each other, but the LC50 tables of each and the graphs show the relatively higher toxicity of the stevia powder solution compared to the sweetened tea solution. Also, the average lethal concentration in these two groups (according to Table 2) was not higher than 5000 mg/liter, which was similar to the results of the experiments
Results: In terms of acute and chronic toxicity in two groups of stevia industrial products at high concentrations, it was determined that at a concentration of 15000 mg/liter in larvae exposed to tea solution sweetened with stevia at 96hpf, none of the surviving larvae did not hatched, but the heart rate was normal (75-85 times per minute), while in the stevia powder solution under the same conditions, the surviving larvae had a very low heart rate and irregular blood flow. Also, at the highest concentration tested in this study (20000 mg/liter), all the larvae exposed to the stevia powder solution had coagulated by the third day (72 hours after fertilization), while a number of larvae exposed to stevia-sweetened tea solution were alive until the fourth day (96 hours after fertilization) and died after hatching and in direct exposure to the substance, which probably indicates a relatively higher toxicity of stevia powder solution compared to stevia-sweetened tea solution.
Conclusion: After 72 hours of exposure of mice to different concentrations of pure stevioside, it was found that the number of CD4 and CD8 positive lymphocytes decreased by almost 48%.[14] Since CD4 and CD8 positive lymphocyte cells are young cells that have not yet gone through the cell maturation process, this process can cause a significant decrease in mature lymphocytes, which are important cells of the immune system. Also, the presence of inflammation in some organs of rats exposed to stevia extracts in pathological investigations can be justified. Although the joint committee of FAO and WHO considers steviol glycosides as permitted food additives, there are still discussions about the safety and toxicity of other glycosidic diterpenes in stevia plant.[18] In addition, the amount of acetylene used in industrial products as well as their degree of purity may lead to complications that indicate the need to check these products. For example, it has been found that sugar alcohols, which are polymer alcohols, are added to some industrial products containing stevia in stores, which may cause digestive side effects for people who are allergic to chemicals. However, nutrition and health centers have expressed their concerns about the toxicity of consuming high doses of stevia, which seems to require further studies and research. In this research, it was concluded that in the group of larvae exposed to stevia extract and tea solution sweetened with stevia at a high dose (20,000 mg/L) during 96 hours after fertilization and in the larvae exposed to stevia powder solution in 72 hours after fertilization had 100% mortality (acute toxicity), although at doses higher than 1000 mg/L, some deaths were reported in all study groups during each day. The most morphological changes during the test period related to the larvae exposed to stevia powder solution were reported, in other studied groups at doses higher than 1000 mg/liter, different morphological changes were also observed, which indicates the chronic toxicity of stevia products on zebrafish larvae. In this experiment, urban tap water was used instead of distilled water for extraction because this research is a simulation of the conditions that people use in their food. However, in order to form different dilutions in each of the tested materials (due to the fact that the solutions are in contact with the larvae for 96 hours and there is a possibility of contamination during the experiment), in order to prevent contamination, solution was made using E3 solution. Also, after being separated from the egg-laying trap, the larvae were washed using E3 solution to prevent the transfer of contamination with the eggs to the test plate, which may affect the test process. This experiment was repeated three times for all three studied groups in the six desired concentrations, and the resulting data showed good reproducibility.