The toxicity assessment of phosmet on development, reproduction, and gene expression in Daphnia magna

Abstract

The use of pesticides to control pests, weeds, and diseases or to regulate plant growth is indispensable in agricultural production. However, the excessive use of these chemicals has led to significant concern about their potential negative impacts on health and the environment. Phosmet is one such pesticide that is commonly used on plants and animals against cold moth, aphids, mites, suckers, and fruit flies. Here, we investigated the effects of phosmet on a model organism, Daphnia magna using acute and chronic toxicity endpoints such as lethality, mobility, genotoxicity, reproduction, and gene expression. We performed survival experiments in six-well plates at seven different concentrations (0.01, 0.1, 1, 10, 25, 50, 100 μM) as well as the control in three replicates. We observed statistically significant mortality rates at 25 µM and above upon 24 h of exposure, and at 1 µM and above following 48 h of exposure. Genotoxicity analysis, reproduction assay and qPCR analysis were carried out at concentrations of 0.01 and 0.1 μM phosmet as these concentrations did not show any lethality. Comet assay showed that exposure to phosmet resulted in significant DNA damage in the cells. Interestingly, 0.1 μM phosmet produced more offspring per adult compared to the control group indicating a hormetic response. Gene expression profiles demonstrated several genes involved in different physiological pathways, including oxidative stress, detoxification, immune system, hypoxia and iron homeostasis. Taken together, our results indicate that phosmet has negative effects on Daphnia magna in a dose- and time-dependent manner and could also induce lethal and physiological toxicities to other aquatic organisms.

Keywords: Comet assay; Daphnia magna; Gene expression; Phosmet; Reproduction.

Figure 1. Phosmet resulted in mortality.

Daphina magna neonates (<24 h old) were exposed to phosmet (0.01, 0.1, 1, 10, 25, 50, 100 µM) in six-well plates with 10 organisms in each well in triplicates. Mortality rates were recorded at 24 h (A) and 48 h (B). LC50 of phosmet was determined at 24 h (C) and 48 h (D). Statistical analyses were performed using one-way ANOVA followed by Dunnett post-test and nonlinear regression. The difference was accepted significant if p values < 0.05. n = 3. Phosmet cause mortality in a dose- and time-dependent manner with a significant lethality starting with 25 and 1 µM at 24 and 48 h, respectively. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Figure 2. Phosmet increased reproduction.

Daphnia magna neonates (<24 h old) were exposed to phosmet (0.01 and 0.1 µM) and number of offspring was recorded daily until all Daphnids were dead. For each concentration, 10 organisms were used and experiments were performed in triplicates. The average of all replicates for the total number of individuals that one single Daphnia produced over the exposure period was taken. Statistical analyses were performed using one-way ANOVA followed by Dunnett post-test and the difference were accepted significant if p values < 0.05. n = 3.0.1 µM exposure group started to give offspring later than the control and 0.01 µM groups, however, the average number of offspring per adult was significantly higher. An asterisk (*) indicates p < 0.05.

Figure 3. Phosmet changed the expression of genes involved in stress response and xenobiotic metabolism.

Daphnia magna neonates (<24 h old) were exposed to 0.01 and 0.1 μM phosmet for 24 h, and the expression levels of mt-1 (A), NOS1 (B), gst (C), CYP4 (D), and CYP314 (E) were determined using qPCR. Statistical analyses were performed using one-way ANOVA followed by Dunnett post-test and the difference were accepted significant if p values < 0.05. n ≥ 4. All the genes were significantly downregulated by phosmet. *p < 0.05; **p < 0.01.

Figure 4. Phosmet decreased the expression of genes associated with lipid metabolism and respiration.

D. magna neonates (<24 h old) were exposed to 0.01 and 0.1 μM phosmet for 24 h, and the expression levels of hr96 (A), magro (B), NPC1b (C), SM3 (D), hif1a (E), and ftn3 (F) were determined using qPCR. Statistical analyses were performed using one-way ANOVA followed by Dunnett post-test and the difference were accepted significant if p values < 0.05. *p < 0.05; **p < 0.01. n ≥ 4.