Trans- and Multigenerational Effects of Isothiazolinone Biocide CMIT/MIT on Genotoxicity and Epigenotoxicity in Daphnia magna

Abstract

The mixture of 5-chloro-2-methylisothiazol-3(2H)-one and 2-methylisothiazol-3(2H)-one, CMIT/MIT, is an isothiazolinone biocide that is consistently detected in aquatic environments because of its broad-spectrum usage in industrial fields. Despite concerns about ecotoxicological risks and possible multigenerational exposure, toxicological information on CMIT/MIT is very limited to human health and within-generational toxicity. Furthermore, epigenetic markers altered by chemical exposure can be transmitted over generations, but the role of these changes in phenotypic responses and toxicity with respect to trans- and multigenerational effects is poorly understood. In this study, the toxicity of CMIT/MIT on Daphnia magna was evaluated by measuring various endpoints (mortality, reproduction, body size, swimming behavior, and proteomic expression), and its trans- and multigenerational effects were investigated over four consecutive generations. The genotoxicity and epigenotoxicity of CMIT/MIT were examined using a comet assay and global DNA methylation measurements. The results show deleterious effects on various endpoints and differences in response patterns according to different exposure histories. Parental effects were transgenerational or recovered after exposure termination, while multigenerational exposure led to acclimatory/defensive responses. Changes in DNA damage were closely associated with altered reproduction in daphnids, but their possible relationship with global DNA methylation was not found. Overall, this study provides ecotoxicological information on CMIT/MIT relative to multifaceted endpoints and aids in understanding multigenerational phenomena under CMIT/MIT exposure. It also emphasizes the consideration of exposure duration and multigenerational observations in evaluating ecotoxicity and the risk management of isothiazolinone biocides.

Keywords: aquatic toxicity; epigenotoxicity; genotoxicity; isothiazolinone; multigenerational effect; transgenerational effect.

Figure 1

Experimental workflow for multigenerational studies: (A) Illustration of three different exposure designs (control, parental exposure, and multigenerational exposure) across four generations (P0: parental generation; F1: first filial generation; F2: second filial generation; F3: third filial generation). The third clutch of female daphnids was used for the next generation. (B) Detailed experimental schedules for the assessment of various endpoints within a single generation. Exposure was initiated with neonates on day 0 and ended on day 21.

Figure 2

Effects of CMIT/MIT on mortality and reproductive capacity in D. magna: (A) Survival rate of daphnid neonates exposed for 48 h to 20, 40, 80, 160, and 320 µg/L CMIT/MIT. Data are presented as means ± SE (n = 4). (B) Total offspring numbers of adult daphnids exposed for 21 days to 5, 10, 20, 40, and 80 µg/L CMIT/MIT. Data are presented as means ± SE (n = 10). Asterisks indicate the significant differences between the exposure and control groups: * p < 0.05 and *** p < 0.001.

Figure 3

Reproductive capacity, growth, and swimming behavior of D. magna after exposure to EC20 CMIT/MIT (7 µg/L): (A) Number of offspring per clutch and total number of offspring produced by adult daphnids exposed to CMIT/MIT for 21 days. (B) Growth of daphnids was observed on days 0, 7, and 21 after exposure to CMIT/MIT. (C) The quantitative value of behavioral parameters (speed, locomotory rate, stop number, turning rate, and path length). (D) The representative pictures of two-dimensional pathway (four images were selected) in the control and exposure groups. Data are presented as means ± SE (n = 10), with the asterisks indicating significant differences between the exposure and control groups: ** p < 0.01 and *** p < 0.001.

Figure 4

Protein–protein interaction networks and functional enrichments of differentially expressed proteins (DEPs) in D. magna after exposure to EC₂₀ CMIT/MIT: KEGG pathways and GO molecular functions were enriched among (A) upregulated proteins and (B) downregulated proteins.

Figure 5

Phenotypic responses of daphnids to parental and multigenerational exposure to CMIT/MIT EC₂₀. Data are normalized to the means of each control group and presented as normalized values ± SE (n = 10). Asterisks indicate significant differences between the exposure and control groups: * p < 0.05, ** p < 0.01, and *** p < 0.001. (A) Total number of neonates produced by adult daphnids. (B) First reproduction time of daphnids. (C) Body length of 7-day-old daphnid. (D) Representative images of the morphology and egg-holding rate (%) of daphnids under the two exposure scenarios. % indicates the percentage of organisms that had eggs or progenies in the brood chamber.

Figure 6

Genotoxic and epigenetic responses of daphnids to parental and multigenerational exposure to EC₂₀ CMIT/MIT: (A) Comparison of the measured DNA damage with the representative images of tail moment. (B) Comparison of % global DNA methylation. Asterisks indicate significant differences between the exposure and control groups: * p < 0.05, ** p < 0.01, and *** p < 0.001. Letters denote the homogeneity between groups (Bonferroni’s test).