Developmental and Reproductive Impacts of Four Bisphenols in Daphnia magna

Bisphenol A (BPA) is a typical endocrine-disrupting chemical (EDC) used worldwide. Considering its adverse effects, BPA has been banned or strictly restricted in some nations, and many analogs have been introduced to the market. In this study, we selected three representative substitutes, BPS, BPF, and BPAF, along with BPA, to assess the developmental and reproductive effects on Daphnia magna. The F0 generation was exposed to bisphenols (BPs) at an environmentally relevant concentration (100 μg/L) for 21 d; then the embryo spawn at day 21 was collected. Behavior traits, the activity of antioxidant enzymes, and gene transcription were evaluated at three developmental stages (days 7, 14, and 21). Notably, body length, heart rate, and thoracic limb beating were significantly decreased, and D. magna behaved more sluggishly in the exposed group. Moreover, exposure to BPs significantly increased the antioxidant enzymatic activities, which indicated that BPs activated the antioxidant defense system. Additionally, gene expression indicated intergenerational effects in larvae, particularly in the BPAF group. In conclusion, BPA analogs such as BPF and BPAF showed similar or stronger reproductive and developmental toxicity than BPA in D. magna. These findings collectively deepen our understanding of the toxicity of BPA analogs and provide empirical evidence for screening safe alternatives to BPA.


Introduction
Bisphenols (BPs) are a class of compounds used to synthesize materials such as polycarbonate and epoxy resin, which affect the daily lives of people worldwide [1]. Bisphenol A (BPA), an environmental endocrine disruptor, is one of the most widely used compounds [2,3]. Owing to its heavy estrogenic-like effects, its use has been banned or strictly restricted in some nations [4]. In recent years, with the ban or restriction of BPA, the production of BPA analogs with structures similar to those of BPA, including bisphenol S (BPS), bisphenol F (BPF), and bisphenol AF (BPAF), has continued to expand [5,6]. Owing to the rapidly expanding applications of bisphenol analogs, increasing attention has been paid to their toxicity and environmental side-effects.
Several studies have shown that BPs are commonly detected in various environmental media such as air [7], water [8], sediment [8], organisms [9], and human bodies [10,11]. Residual concentrations of BPA and its analogs have been reported to vary from nanograms per liter to milligrams per liter [5,8,12]. Previous studies reported that the highest levels of BPA in Africa were 251 ng/mL, 384.8 ng/mL, and 208.55 ng/mL for water, wastewater, and biological fluids, respectively [13]. Moreover, Wang, et al. [14] found that BPA, BPAF, and BPS were the most predominant analogs in Taihu Lake, China, with residual concentrations ranging from 49.7 to 3480 ng/L.

Development and Reproduction of the F0 Generation
No mortality of the F0 generation was observed during the experimental period. The growth, development, and reproduction of the F0 generation are shown in Figure 1. Compared with the control group, the body length of D. magna was significantly shorter after exposure to both BPF and BPAF for 7 d (Figure 1b,c). As time progressed, D. magna in the BPA group became significantly shorter on 14 d and 21 d. Following BPA, BPF, and BPAF treatment, the heart rates were all significantly reduced at each time point (Figure 1d). Thoracic limb jittering, which can represent movement ability at some levels, exhibited a similar trend to heart rate ( Figure 1e). With respect to reproduction, BPF and BPAF treatments may postpone the time to the first brood ( Figure 1f). There was also a significant reduction in the number of cumulative offspring in the BPA, BPF, and BPAF groups (Figure 1g). D. magna reproduction is usually accompanied by molting. Molting frequency was also reduced in the three groups ( Figure 1h). In addition, D. magna in the BPS treatment group did not show any significant differences in all of the six parameters. Figure 2 shows the growth, development, and reproduction of the F1 generation. The body length of the offspring was similar to that of the F0 generation, with D. magna exposed to BPF and BPAF for 14 or 21 d showing a significant decrease in body length (Figure 2a,b). Offspring of the BPA, BPF, and BPAF maternal treatment groups exhibited a reduced heart rate compared with that of the control group. Over time, this occurrence gradually became more evident (Figure 2c). The trend in thoracic limb tremors was similar to that of the heartbeat, both showing a reduction in the beating rate of the offspring after BPA, BPF, and BPAF treatment at 14 d and 21 d (Figure 2d). Additionally, the reproduction of the F1 generation was influenced by exposure to BPA and its analogs, and this manifested specifically 3 of 15 as the deferred first brood time, reduction in cumulative offspring, and decreasing molting frequency (Figure 2e-  , body length (c), heart rate (d), thoracic limb jittering (e), time to first brood (f), cumulative offspring (g), and molting frequency (h). Asterisks indicate significant differences between exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Figure 2 shows the growth, development, and reproduction of the F1 generation. The body length of the offspring was similar to that of the F0 generation, with D. magna exposed to BPF and BPAF for 14 or 21 d showing a significant decrease in body length (Figure 2a,b). Offspring of the BPA, BPF, and BPAF maternal treatment groups exhibited a reduced heart rate compared with that of the control group. Over time, this occurrence gradually became more evident (Figure 2c). The trend in thoracic limb tremors was similar to that of the heartbeat, both showing a reduction in the beating rate of the offspring after BPA, BPF, and BPAF treatment at 14 d and 21 d (Figure 2d). Additionally, the reproduction of the F1 generation was influenced by exposure to BPA and its analogs, and this manifested specifically as the deferred first brood time, reduction in cumulative offspring, and decreasing molting frequency (Figure 2e-g) Figure 1. A schematic of the experimental design used (a). Development and reproduction of the F0 gener-ation induced by BPs: D. magna with microscopic observation (b), body length (c), heart rate (d), thoracic limb jittering (e), time to first brood (f), cumulative offspring (g), and molting frequency (h). Asterisks indicate significant differences between exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Behavioral Response
The behavioral response of the F0 generation is shown in Figure 3. As shown in Figure 3a-c, D. magna tended to be static in the treatment group, especially in the BPF and BPAF treatment groups, after 21 d of exposure. After 7 d of exposure, there was no significant difference in swimming speed between the control and exposed groups. The swimming speeds of D. magna treated with BPS, BPA, BPF, and BPAF were 1%, 9%, 22% (p < 0.05), and 36% (p < 0.01) of the control group, respectively, after 14 d of exposure and 1%, 20% (p < 0.001), 31% (p < 0.001), and 33% (p < 0.001) after 21 d (Figure 3d).  . Asterisks indicate significant differences between exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Behavioral Response
The behavioral response of the F0 generation is shown in Figure 3. As shown in Figure 3a-c, D. magna tended to be static in the treatment group, especially in the BPF and BPAF treatment groups, after 21 d of exposure. After 7 d of exposure, there was no significant difference in swimming speed between the control and exposed groups. The swimming speeds of D. magna treated with BPS, BPA, BPF, and BPAF were 1%, 9%, 22% (p < 0.05), and 36% (p < 0.01) of the control group, respectively, after 14 d of exposure and 1%, 20% (p < 0.001), 31% (p < 0.001), and 33% (p < 0.001) after 21 d (Figure 3d).  . Asterisks indicate significant differences between the exposed and control groups (** p < 0.01, *** p < 0.001). Figure 4 shows the behavioral response of the F1 generation. The behavioral traits of the F1 generation had a similar tendency to those of the F0 generation. Compared with the control group, the F1 generation in the exposed group moved more sluggishly, especially in the BPF and BPAF groups (Figure 4a-c). The F1 generation differed from the F0 generation with regard to a significant decrease in swimming speed after 7 d of exposure, which increased after 14 d and 21 d (Figure 4d). These results indicated that these chemicals had intergenerational effects on the behavioral response of D. magna. . Asterisks indicate significant differences between the exposed and control groups (** p < 0.01, *** p < 0.001). Figure 4 shows the behavioral response of the F1 generation. The behavioral traits of the F1 generation had a similar tendency to those of the F0 generation. Compared with the control group, the F1 generation in the exposed group moved more sluggishly, especially in the BPF and BPAF groups (Figure 4a-c). The F1 generation differed from the F0 generation with regard to a significant decrease in swimming speed after 7 d of exposure, which increased after 14 d and 21 d (Figure 4d). These results indicated that these chemicals had intergenerational effects on the behavioral response of D. magna. Figure 4 shows the behavioral response of the F1 generation. The behavioral traits of the F1 generation had a similar tendency to those of the F0 generation. Compared with the control group, the F1 generation in the exposed group moved more sluggishly, especially in the BPF and BPAF groups (Figure 4a-c). The F1 generation differed from the F0 generation with regard to a significant decrease in swimming speed after 7 d of exposure, which increased after 14 d and 21 d (Figure 4d). These results indicated that these chemicals had intergenerational effects on the behavioral response of D. magna.  . Asterisks indicate significant differences between the exposed and control groups (*** p < 0.001). . Asterisks indicate significant differences between the exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001). Figure 6 shows that, in the F0 generation, the expression of genes related to development significantly increased after BPAF exposure, whereas cyp314, ecra, ecrb, and usp expression significantly increased after BPA exposure. BPF significantly increased the expression of cyp314 and ecrb, whereas BPS significantly increased the expression of ftz-f1. The reproduction of D. magna was visibly influenced after exposure to BPA and its ana-  Figure 6 shows that, in the F0 generation, the expression of genes related to development significantly increased after BPAF exposure, whereas cyp314, ecra, ecrb, and usp expression significantly increased after BPA exposure. BPF significantly increased the expression of cyp314 and ecrb, whereas BPS significantly increased the expression of ftz-f1. The reproduction of D. magna was visibly influenced after exposure to BPA and its analogs; vtg1 and vtg2 expression improved significantly with BPA, BPF, and BPAF treatment, and vmo1 expression was inhibited by BPA and BPF treatment. Additionally, in the F1 generation, BPAF significantly increased the expression of ftz-f1 and cht in the offspring and BPF significantly increased the expression of usp, whereas BPS inhibited ecrb expression.  (m) (n) (o) Figure 6. Expression of genes related to development (a-f,j-o) and reproduction (g-i) in the F0 generation (a-i) and F1 generation (j-o). Asterisks indicate significant differences between the exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
With the strict restriction of BPA in recent years, BPA analogs have been increasingly used worldwide [5,6]. It has been reported that these chemicals potentially have toxic effects on vertebrates. Previous studies elaborated on the neurotoxicity of BPA and its analogs in zebrafish [15]. Ji, et al. [20] compared and evaluated the vascular toxicity and oxidative stress potency of the BPs in zebrafish and ranked them as follows: BPAF > BPF > BPA > BPS. Moreman, et al. [32] illustrated that BPA and its analogs (BPAF, BPF, and BPS) hindered the development of larval zebrafish, leading to cardiac edema, spinal malformation, and craniofacial deformities. However, few studies have tested the toxicity of BPs in arthropods. Here, we conducted a comprehensive study of the developmental and reproductive toxicity of BPA, BPS, BPF, and BPAF in D. magna and assessed their toxic effects. Our results showed that after exposure to BPA and its analogs, both the F0 and F1 generations of D. magna exhibited abnormalities in development and reproduction.
Body length can reflect the developmental situation at some levels. Our study showed that, compared with the control group, the body length in the exposed group was reduced, especially in the BPF and BPAF groups, and this inhibition became clearer over time. Heart rate and thoracic limb jittering are linked to feeding behavior or respiratory metabolism, indicating the health and growth of organisms and the stress response to pollutants [33]. Both were significantly decreased in the F0 and F1 generations after BPA, BPF, and BPAF treatment, whereas there was no significant difference in the group exposed to BPS. The thoracic limb is the food-intake organ of D. magna, and alterations in the thoracic limb may result in feeding difficulties. It is possible that the depression of heart rate led to a decrease in thoracic limb frequency or that BPAF affected the nervous system [34,35].
Reproductive ability is an important index for detecting the health of the population of D. magna and is widely used in contaminant toxicity risk assessment [30]. Although molting frequency is not directly related to spawning ability, some studies have shown a positive correlation between molting and spawning times [36]. We found that the time to the first brood was postponed, whereas the molting frequency was declining in the F0 generation. The cumulative offspring decreased in the F0 generation, which meant that reproduction was influenced by exposure. Moreover, this occurrence did not return to normal levels in juveniles, which indicated that the damage to reproductive capacity in the exposed group may have an intergenerational effect.
In this study, when exposed to BPA and its analogs, D. magna appeared to inhibit locomotor behavior in the F0 generation as well as in the F1 generation. Similar phenomena have also been observed in other animals, exhibiting inhibition of behavior. Gu illustrated that after BPS, BPF, BPA, and BPAF exposure, zebrafish appeared to suffer oxidative stress, larval hypoactivity, and dysregulated neuronal development [15]. Kim, et al. Figure 6. Expression of genes related to development (a-f,j-o) and reproduction (g-i) in the F0 generation (a-i) and F1 generation (j-o). Asterisks indicate significant differences between the exposed and control groups (* p < 0.05, ** p < 0.01, *** p < 0.001).

Discussion
With the strict restriction of BPA in recent years, BPA analogs have been increasingly used worldwide [5,6]. It has been reported that these chemicals potentially have toxic effects on vertebrates. Previous studies elaborated on the neurotoxicity of BPA and its analogs in zebrafish [15]. Ji, et al. [20] compared and evaluated the vascular toxicity and oxidative stress potency of the BPs in zebrafish and ranked them as follows: BPAF > BPF > BPA > BPS. Moreman, et al. [32] illustrated that BPA and its analogs (BPAF, BPF, and BPS) hindered the development of larval zebrafish, leading to cardiac edema, spinal malformation, and craniofacial deformities. However, few studies have tested the toxicity of BPs in arthropods. Here, we conducted a comprehensive study of the developmental and reproductive toxicity of BPA, BPS, BPF, and BPAF in D. magna and assessed their toxic effects. Our results showed that after exposure to BPA and its analogs, both the F0 and F1 generations of D. magna exhibited abnormalities in development and reproduction.
Body length can reflect the developmental situation at some levels. Our study showed that, compared with the control group, the body length in the exposed group was reduced, especially in the BPF and BPAF groups, and this inhibition became clearer over time. Heart rate and thoracic limb jittering are linked to feeding behavior or respiratory metabolism, indicating the health and growth of organisms and the stress response to pollutants [33]. Both were significantly decreased in the F0 and F1 generations after BPA, BPF, and BPAF treatment, whereas there was no significant difference in the group exposed to BPS. The thoracic limb is the food-intake organ of D. magna, and alterations in the thoracic limb may result in feeding difficulties. It is possible that the depression of heart rate led to a decrease in thoracic limb frequency or that BPAF affected the nervous system [34,35].
Reproductive ability is an important index for detecting the health of the population of D. magna and is widely used in contaminant toxicity risk assessment [30]. Although molting frequency is not directly related to spawning ability, some studies have shown a positive correlation between molting and spawning times [36]. We found that the time to the first brood was postponed, whereas the molting frequency was declining in the F0 generation. The cumulative offspring decreased in the F0 generation, which meant that reproduction was influenced by exposure. Moreover, this occurrence did not return to normal levels in juveniles, which indicated that the damage to reproductive capacity in the exposed group may have an intergenerational effect.
In this study, when exposed to BPA and its analogs, D. magna appeared to inhibit locomotor behavior in the F0 generation as well as in the F1 generation. Similar phenomena have also been observed in other animals, exhibiting inhibition of behavior. Gu illustrated that after BPS, BPF, BPA, and BPAF exposure, zebrafish appeared to suffer oxidative stress, larval hypoactivity, and dysregulated neuronal development [15]. Kim, et al. [37] demonstrated that exposure to BPS or other EDs can cause anxiety and reduced social behavior in juvenile mice. In terms of effects during generations, Wolstenholme, et al. [38] found that BPA exposure may influence the social recognition of both parent and descendant rats and has a long-lasting and transgenerational effect even after three generations of BPA exposure, which was consistent with our results.
It has been reported that BPs enhance reactive oxygen species (ROS) formation and damage the structures of lipids and proteins, which can lead to oxidative damage of cells [39,40]. Previous studies have shown that BPA may induce oxidization system disorders in the liver and nervous system [41,42]. Our results showed that exposure to all four BPs significantly increased the activity of CAT and SOD, and three of the BPs (except BPS) reciprocally increased the levels of MDA, indicating that BPs may induce oxidative stress in D. magna.
As mentioned previously, developmental effects and reproductive impairment may have intergenerational effects, which can often be demonstrated using RT-qPCR analysis. The development and reproduction of D. magna may be linked to molting, which can be regulated by ecdysteroids (20-hydroxyecdysone, 20-E) and juvenile hormones (JH) [36]. Cytochrome P450s (CYPs) are a superfamily of heme proteins that are vital for drug metabolism, bioactivation, and breakdown of xenobiotics [43,44]. Cyp314 belongs to this family and is responsible for synthesizing the molting hormone 20-E and converting it to its active form, which can regulate the molting and reproduction cycles [45,46]. In contrast to previous studies [47], our study showed that there is an up-regulation of cyp314 expression in D. magna of F0 generation after BPA, BPF, and BPAF exposure, which may account for the different exposure conditions. When ecdysone 20-E is combined with the protein ecdysone receptor (EcR) and ultraspiracle (USP) to form a nuclear heterodimer complex (EcR/USP-20E), the expression of ecra, ecrb, and usp is altered [45,48]. Moreover, ftz-f1 may also be expressed after the presentation of 20-E and incentivize the expression of downstream cht and other genes [49]. The cht gene is involved in cuticle metabolism [47]. The dysregulation of ecra, ecrb, usp, ftz-f1, and cht in both the F0 and F1 generations may indicate that BPA and its analogs indeed had endocrine effects and deteriorated the molting and metabolism of D. magna. Owing to the abnormal fluctuation still occurring in the F1 generation, it can be deduced that the damage may have intergenerational effects in terms of development.
Exposure to BPA and its analogs led to abnormalities in the gene expression of D. magna, which were related to both development and reproduction. As the precursor of vitellin, vitellogenin (vtg) is considered an energy source for the development of offspring in oviparous organisms [50,51]. Gene vom1 prevents the yolk from mixing with the albumen and protecting the egg from bacterial infection [52]. In our study, the expression of vtg1 and vtg2 improved significantly with BPA, BPF, and BPAF treatment, whereas the expression of vmo1 was inhibited by BPA and BPF treatment, which indicated that BP exposure impaired the reproductive system of D. magna. Several studies have demonstrated that endocrine disruptors can alter the expression of reproductive genes in organisms, and dysregulation of genes can be transferred during generations [50,53,54].

Experimental Animals
D. magna was provided by the Institute of Hydrobiology (Wuhan, China). The culturing method was performed as previously described [55]. Before the experiment, the organisms were incubated for ten generations in deionized water after aeration for 48 h

Chronic Toxicity Test
The concentrations were determined to be 100 µg/L according to the environmentally relevant concentration [13], and the stock solution was diluted to the final test solution with deionized water. For the exposure treatment, each group had ten replicates and each replicate had one D. magna (F0 generation), which were randomly separated into 100 mL sterile beakers with 80 mL test solution. All the control groups of the other chemicals received 0.01% (v/v) DMSO. D. magna fasted during the test, and other culture conditions were consistent with those described in Section 4.1. The number of new offspring was counted and recorded daily. The offspring (F1 generation) born on days 7, 14, and 21 after maternal exposure (7 d, 14 d, and 21 d) were collected and transferred into clean water [56,57]. Furthermore, molting, survival, and reproduction (both F0 and F1 generations) were recorded at 7 d, 14 d, and 21 d. In addition, behavior tests were performed at these three time points. After 21 d of culture, samples (both F0 and F1 generations) were collected for the following tests.

Heartbeat, Body Length, and Thoracic Limb Beat Frequency Measurement
A D. magna, which was able to move (including its antennae) at least every 15 s was considered to be alive [58]. Ten F1 generations were randomly selected from each replicate. F0 and F1 generations were observed by a Research Stereo Microscope (SMZ25, Nikon, Japan). The length from the top of the helmet to the base of the tail (excluding the tail spur) was measured as the body length of D. magna. The number of heart beats and the number of thoracic limb beats that survived for 1 min were recorded by manual counting as heart beats (one per minute) and thoracic limb shakes (one per minute).

Behavioral Tests
Behavioral tests were conducted using a DanioVision observation chamber (Noldus, Wageningen, Netherlands). According to the method of a previous study [23], 10 D. magna F0 or F1 generations were selected from each exposure treatment. A 48-well plate was used for detection, with one D. magna in each well filled with 1 mL of exposure solution. The test program was set for 35 min, including 5 min of dark acclimation and three 10-min light-and-dark cycles, which consisted of 5 min of light and 5 min of darkness. Movement traits, heat maps, and swimming speed were analyzed using EthoVision XT 15 video tracking software (Noldus, Wageningen, The Netherlands).

Determination of Antioxidant Enzymatic Activity
The lysate was homogenized using an ultrasonic crushing instrument and the protein concentration was determined using a protein detection kit (Biyuntian, Shanghai, China). Superoxide dismutase (SOD; Biyuntian) and catalase (CAT; Biyuntian) (MDA; Biyuntian) detection kits were used to determine the enzymatic activities of the different treatment groups.

Statistical Analysis
The data were analysed with one-way analysis of variance (ANOVA) using GraphPad Prism 8.0.1 software. The results are expressed as the mean value ± standard error of mean (SEM). Asterisks indicate significant differences between exposure and control, * indicates a significant difference (* p < 0.05), ** indicates a very significant difference (** p < 0.01), and *** indicates a highly significant difference (*** p < 0.001).

Conclusions
BPA has been strictly restricted because of its strong endocrine effects, and analogs have been invented for broad applications. However, the toxicity of these substitutes has not been fully evaluated. In the present study, we found that BPA alternatives induce similar toxic and estrogenic effects to BPA, and the toxicity of its three BPA substitutes (BPS, BPF, and BPAF) on the growth and reproduction of D. magna may be not weaker than those of BPA, which manifest as growth inhibition, oxidative stress, and altered gene expression. Considering all aspects, the toxicity of these four chemicals in Daphnia magna should be in the order BPAF > BPF ≈ BPA > BPS, which is consistent with the findings of previous studies conducted on zebrafish. Given that BPAF and BPF have toxic effects comparable to those of BPA, BPAF and BPF should be carefully considered as alternatives to BPA. In addition, we should be more concerned about the long-term low-dose effects of such alternatives at environmental concentrations. We can step up publicity to establish the perceptions of BPs' impacts, adjust lifestyle habits to reduce daily exposure, and establish corresponding laws and regulations to cut off the emission of BPs [4].

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijms232314561/s1, Table S1: Abbreviation and description of the parameters in this study; Table S2: Primers used for qPCR validation.