Both MPs and pharmaceuticals can be accumulated in aquatic organisms, and transferred to offspring (Junaid et al., 2023; Li et al., 2022; Minguez et al., 2015), thereby causing toxic effects on both generations. The MPs can act as carriers of pharmaceuticals and facilitate their exposure to aquatic organisms (Zhou et al., 2020). In the present study, we investigated the transgenerational reproductive toxicity of PS MPs alone, CBZ alone, and their mixtures on D. magna, and the mechanisms were revealed at the transcriptional level.
In the acute toxicity, the 48 h LC50 value of CBZ was 88.77 mg/L, which is within the range identified for D. magna in previous studies (Di Poi et al., 2018; Oropesa et al., 2016). In terms of the toxicity of PS MPs, the results indicated that 1 µm PS MPs showed the lowest 48 h LC50, illustrating that it posed the highest toxicity among all tested PS MPs. Previous studies have demonstrated that 1 µm PS MPs was more toxic to D. magna than 10 µm PS MPs, most probably because it is more readily ingested by zooplankton, and then cause more immobilization and physical damage (Zhang et al., 2019). Similarly, it has been demonstrated that the MPs of nanosize (e.g. 20 nm; 100 nm) are more bioavailable and could persist in the organism for a longer time than the MPs of microsize (e.g. 1 µm; 2 µm) (Rist et al., 2017;), resulting in more accumulation in D. magna, thereby, potentially causing a chronic sublethal toxicity rather than an acute toxicity (Besseling et al., 2014). Such findings might explain the fact that, in the present study, the 1 µm PS MPs caused the heaviest toxicity in the acute exposure.
In the presence of CBZ, the acute toxicity was enhanced for PS MPs of different particle sizes except for MPs of 1 µm (Table S2). A similar result was observed in a previous study. Lin et al. (2021) reported that, in the presence of pyrene, the immobilization of neonatal D. magna increased after 48 h exposure to PS MPs of different particle sizes. Interestingly, in the present study, the order of acute toxicity of PS MPs with different particle sizes in the presence of CBZ was almost opposite to those of PS MPs of different particle sizes alone. Among those, the acute toxicity of PS MPs of 700 nm nearly doubled in the presence of CBZ, possibly because the MPs of nanosize had a larger specific surface area than those of microsize, which facilitates more CBZ to enter into D. magna (Zhang and Goss, 2020). Compared to the MPs of microsize, the MPs of nanosize may remain longer time in D. magna and consequently had more time to discharge the carried CBZ, resulting in the enhanced bioaccumulation of CBZ in D. magna (Shi et al., 2020). Therefore, we speculated that the toxicity variation of PS MPs of different particle sizes in the presence of CBZ might be attributed to the toxicity amplification effect of CBZ.
In the presence of PS MPs, the acute toxicity of CBZ increased (Table S3). This can be probably attributed to the vector effect of PS MPs, which can adsorb CBZ and subsequently enter the organism together, thus elevating the bioaccumulation of CBZ in D. magna (Ma et al., 2016). Additionally, the variation of CBZ toxicity in the presence of PS MPs was correlated with the particle size of PS MPs in the present study. Among these, the toxicity of CBZ was significantly enhanced in the presence of 5 µm PS MPs, followed by 700 nm, and 1 µm. Moreover, the acute toxicity of CBZ in the presence of 5 µm PS MPs was enhanced to the largest extent in all combined acute toxicity exposure groups. The sublethal effects, however, still need further investigation.
The 21 d chronic reproduction tests of F0 generation showed that the combined exposure of 5 µg/L CBZ and 5 mg/L PS MPs exerted a serious reproductive threat to F0, notably in terms of the total neonate number (Fig. 3C). The total neonate number is a key and sensitive parameter to measure the reproductive capacity of D. magna (Dang et al., 2012). The impaired reproduction in organisms caused by environmental pollutants is also reflected in gene expression, especially for the genes related to the detoxification metabolism and reproduction (Bao et al., 2020). Regarding the toxic metabolism of D. magna, the expression of cyp4 was measured, which was a vital detoxification enzyme in Phase I detoxification systems of invertebrates (Snyder, 2000). And gst, which is involved in Phase II detoxification of various xenobiotics, plays an important role in the detoxification of oxidative stressors (Kim et al., 2021). Figure 5 observed that the expression of cyp4 from the exposure group of 5 µg/L PS MPs alone, 5 µg/L CBZ alone, and the co-exposure group of 5 mg/L PS MPs and 5 µg/L CBZ, and gst from the exposure group of 5 µg/L PS MPs were significantly upregulated, which indicates that the metabolism system was activated. Vitellogenin is considered to be the major precursor of vitellin, an egg-yolk protein, which provides an essential source of energy during the embryogenesis of oviparous organisms (Tokishita et al., 2006). In the D. magna, vitellogenin was encoded by at least two vtg genes, vtg1 and vtg2, which share > 99% identity and have been identified on a single cluster in neighboring but in opposite orientations ( Tokishita et al., 2006). Dmrt93b is associated with the determination of sex in D. magna and is involved in the process of changing reproductive strategies (Kato et al., 2008). In the present study, vtg2 from the exposure group of 5 µg/L PS MPs alone, 5 µg/L CBZ alone, and co-exposure group of 5 µg/L CBZ and 5 µg/L PS MPs all expressed a significant down-regulation (Fig. 5). However, there was no significant difference in the total neonate number for these groups (Fig. 3C). It suggests that the exposure to PS MPs alone, CBZ alone, or their mixture had affected the expression of reproduction related genes, but not further affected the reproduction physiological outcome. It has been demonstrated that the low or moderate doses of toxic compounds may induce compensatory responses in individuals, which facilitates the activation of repair mechanisms in response to damage (Bao et al., 2020; Rix et al., 2022). In the present study, the insignificant effects on reproductive physiological parameters of individuals exposed to PS MPs and CBZ at low doses, which may be attributed to the activation of toxic metabolism system in D. magna. The compensatory could also be found in exposure group of 5 mg/L PS MPs alone, where vtg 1 and vtg 2 were up-regulated and cyp4 and gst were down-regulated. This compensatory effect seemed to be broken in the high dose co-exposure group of 5 µg/L CBZ and 5 mg/L PS MPs. In this exposure group, vtg1 also showed upregulated expression, the reasons behind need further research.
Successful molting is essential for the growth and development of crustacea, which is predominantly regulated by ecdysteroids (Sumiya et al., 2014). Cyp314, a subfamily of cytochrome P450, plays an important role in the biosynthesis of ecdysone and is responsible for the conversion of ecdysone into its active form 20-hydroxyecdysone (20E), which regulates molting (Liu et al., 2022b). Ecr-b is an ecdysone receptor that is involved in crustacea growth and development by binding to 20E (LeBlanc, 2007). Both are upstream genes that regulate molting. Cut is regulated by 20E and responsible for the production of the neo-epidermal cuticle protein, which belongs to the downstream genes regulating molting (Song et al., 2017). In 21 d exposure, both the first molting time and the molting frequency of the F0 showed no significant difference compared to those from the control. However, from the transcriptional level, the expression of molting related genes increased in F0 from all the exposed groups, except ecr-b in the 5 µg/L PS MPs exposed group (Fig. 5). It is probably due to the fact that the molting process of D. magna involves multiple signal pathway besides the expression of cyp314, ecr-b and cut (Cho et al., 2022; Wei et al., 2022).
Compared with the control group, a significant reduction in the total neonate number was observed in the F1 exposure groups, except for the exposure group of 5 µg/L PS MPs and the co-exposure group of 5 µg/L CBZ with 5 µg/L PS MPs. The result was consistent with the gene transcription, i.e., vtg1 and vtg2 significantly downregulated in the F1 co-exposure group of 5 µg/L CBZ and 5 mg/L PS MPs), suggesting that the co-exposure of high-dose PS MPs and CBZ caused more adverse reproductive effects in F1 than F0. Previous studies have also demonstrated that continued exposure to MPs and PPCPs resulted in serious reproductive toxicity to offspring (Liu et al., 2022a).. Moreover, the molting frequency of the 5 µg/L PS MPs exposure group was significantly increased (Fig. 4E), which may be related to the irritant effect, caused by the low dose of PS MPs (Rix et al., 2022). It has been demonstrated that low doses of toxicants may promote the evolution of organisms (Costantini, 2019) via inducing transgenerational excitation and stimulating the biological processes (Rix et al., 2022).
Likewise, the exposure still showed a reproduction inhibition at both physiological and gene transcriptional level, even when the exposure was removed in the F1, except the offspring that their parents were exposed to 5 µg/L PS MPs (Fig. 4C and Fig. 6). It indicates a transgenerational reproductive toxicity under the long-term exposure to PS MPs alone, CBZ alone or their mixture. Previous studies have obtained similar results for other MPs and pharmaceuticals. For instance, De Liguoro et al. (2019) reported that, when F0 generations of D. magna were exposed to flumequine, the inhibition of reproduction could persist up to the three unexposed generations (F1, F2, F3).
Surprisingly, the gene expression of toxic metabolism (cyp4, gst) was significantly activated in both the F1-recovery and F1-exposure groups, meanwhile the vtg1 and vtg2 were found to be significantly down-regulated (Fig. 6). This was also translated into the physiological performance, according to the results shown in Fig. 4C that the total neonate number exhibited a significant decrease in the F1-recovery group whose parents were exposed to 5 mg/L PS MPs and 5 µg/L CBZ, as well as in the F1 group co-exposed to 5 µg/L CBZ and 5 mg/L PS MPs. It could be explained by the trade-off mechanism between toxic metabolism and reproduction (Metcalfe and Alonso-Alvarez, 2010), that is, F1 invested more energy in detoxification metabolism, resulting in the reduced investment of energy to reproduction. It has been reported that the reduction in reproduction of generations may be due to the increased energy requirements (e.g., synthesis of antioxidant enzymes) to restore the internal maintenance damaged by toxic-induced stress (Im et al., 2020; Kim et al., 2014).