*3.4. Joint Effects of BPA and Heavy Metals*

According to the toxicities of mixtures of BPA and heavy metals, the EC50 values of mixtures (EC50m) were calculated using Equations (2) and (3). Based on the EC50 values of the mixtures, the TUs of mixtures were obtained using Equation (4). In the case of the acute toxicity of the mixtures, the joint effects of BPA and heavy metals exhibited simple additive and antagonisms, and the TU values of mixtures ranged from 1.06 to 1.33 (Figure 4A). BPA and Cd showed an antagonistic effect at the equitoxic ratio (the TU of the mixture was 1.33), whereas simple additive effects were observed among BPA and other heavy metals at the equitoxic ratio. In addition, according to the EC50 values of BPA + Cd and BPA + As at a non–equitoxic ratio, the joint effects of BPA + Cd and BPA + As exhibited antagonisms and simple additives (Figure 4B,C). The TU values of BPA + Cd at a non–equitoxic ratio ranged from 0.97 to 1.38, and the values decreased with the increase in the ratio of BPA in mixtures.

The same conclusion could be drawn for BPA + As, and the range of EC50 values was from 0.94 to 1.59.

**Figure 4.** Joint effect of BPA and heavy metals. (**A**–**C**) BPA and heavy metals at equitoxic or non–equitoxic ratios in the acute toxicity test; (**D**–**F**) BPA and heavy metals at equitoxic or non– equitoxic ratios in a chronic toxicity test. The error bars indicate the standard deviations from three independent experiments.

In the case of the chronic toxicities of mixtures, the joint effects of BPA and heavy metals indicated a synergistic and additive effect (Figure 4D). The TU values of BPA + Cr, BPA + Cd, BPA + As, and BPA + Hg were 1.07, 0.80, 0.77, and 0.67, respectively, at an equitoxic ratio. The joint effect of BPA and Cr suggested an additive effect, while the others revealed a synergism. Meanwhile, the joint effects of BPA + Cd and BPA + As suggested synergistic and additive effects at non–equitoxic ratios. The TU values of BPA + Cd ranged from 0.62 to 0.94, while the TU values of BPA + As were shown to range from 0.63 to 0.79. These results suggest that the joint effects of BPA and heavy metals between acute and chronic exposure were different. The joint effects of BPA and heavy metals on acute exposure were shown to be antagonisms and additives, whereas additive effects and synergism were obtained with chronic exposure.

#### *3.5. Mechanism of the Synergistic Effects of BPA and Heavy Metals on V. qinghaiensis Q67*

In terms of the joint effects, BPA and heavy metals exhibited different effects between the acute and the chronic test. For example, the joint effects of BPA and Cd showed antagonistic and additive effects for the acute test at non–equitoxic ratios but synergistic and additive effects for the chronic test. To investigate whether the synergistic effect was due to the inhibition of growth and luminescence, the impact of the contaminants on cell growth and luminescence was tested. The concentrations of mixtures corresponded to the EC50 values from the test of the mixtures' toxicities, and the concentrations of BPA and heavy metals corresponded to their individual concentrations in mixtures. The final concentrations of BPA + Cr, BPA + Cd, BPA + As, and BPA + Hg in mixtures were 6.57, 4.25, 4.48, and 2.31 mg/L, respectively. The results showed that BPA barely inhibited cell growth, but heavy metals significantly inhibited that. All the binary mixtures inhibited the growth

of Q67 (Figure 5). Meanwhile, the luminescence inhibition ratio of mixtures was close to 50% after 6 h exposure but below 50% at earlier cultivation times (0.25, 2, and 4 h) except for BPA + Cr. The luminescence inhibitions of BPA and Cr were constantly close to 50% throughout the exposure time. In other words, the toxicities of mixtures of BPA and Cd, As, and Hg were shown to be time–dependent but BPA + Cr was not. It is worth mentioning that the joint effect of BPA and Cr displayed simple additive effects not only for 15 min exposure in the acute test but also for 15 min, and 2, 4, and 6 h exposure in the chronic test.

**Figure 5.** The impact of the mixtures of BPA and heavy metals (equitoxic ratio) on the growth and luminescence of *V. qinghaiensis* Q67. The concentrations of mixtures used in this work are the EC50 values of the mixtures in the test of the chronic toxicity of the mixtures, and the concentrations of BPA and heavy metals correspond to their individual concentrations in mixtures. The error bars indicate the standard deviations from three independent experiments. Impact of the individual contaminant on growth (**A**). The impact of mixtures on the growth and luminescence (**B**).

#### **4. Discussion**

BPA and heavy metals are widely spread in the environment. BPA is well known as an endocrine disrupter that appears in daily human life, even in human milk [11]. This study indicates that BPA exhibits a stronger acute toxicity than heavy metals. Studies regarding the toxicity of mixtures of BPA and other contaminants have mainly focused on BPA and its analogues, EDs. However, works on the toxicities of mixtures of BPA and heavy metals are lacking. The co–exposure of BPA and heavy metals in the environment should be further studied. In this study, the toxicities of binary mixtures of BPA and heavy metals were investigated using bioluminescent bacteria *V. qinghaiensis* Q67. The results indicated that BPA and heavy metals displayed an antagonistic and an additive effect in the context of acute exposure but synergistic and additive joint effects in the contexts of chronic exposure. Compared with other bioluminescent bacteria, such as *V. fischeri* and *P. phosphoreum* T3, Q67 is the only strain isolated from freshwater so far. Thus, the low concentration of NaCl added to samples decreased the impact of NaCl on the toxicities of heavy metals [31–33]. Q67 is therefore a suitable bioluminescent bacterial strain to detect the mixture toxicities of BPA and heavy metals. The results from bioluminescent bacteria might not be repeated in animal tests, but they could be considered a rapid screening approach used in the environmental field, especially in aqueous samples.

Studies regarding the mixture toxicities of BPA and heavy metals have been conducted in the past. The prenatal co–exposure of BPA and Pb has been associated with neurotoxicity, but the interaction of BPA and Pb should be further studied [16]. In addition, the toxicity of mixtures of BPA and Cd was investigated in HepG 2 cells, and the results showed that the co–exposure of BPA and Cd enhanced the cytotoxicity, oxidative stress, and genotoxicity, indicating an additive and synergistic effect [40]. The additive and synergistic effects of

BPA and Cd could be also obtained from a chronic test in this study. However, the acute joint effects of BPA and Cd were shown to be the antagonism and additive in this work. The chronic joint effect of BPA and Cd toward Q67 in this study was similar to a previous study. In fact, the detection of the toxicity of mixture contaminants to higher organisms and cells was often exhibited in the long term, which was similar to the chronic toxicity test of contaminants toward Q67. Otherwise, there are some similar studies that should be compared with this work. In one study, toxicities of mixtures of phthalate esters and Cd to *V. qinghaiensis* Q67 were investigated, and the results showed that mixtures of phthalate esters and Cd showed additive effects [57]. The EC50 value of Cd in that study (18.8 mg/L) was higher than in our study (13.274 mg/L). Further, the synergistic and antagonistic effects of the ternary mixtures of BPA, diethyl phthalate (DEP), and diglycidyl ether (BADGE) to *V. fischeri* were confirmed; however, BPA and other chemicals were dissolved in methanol in that study [37]. Indeed, methanol can affect the luminescence of bioluminescent bacteria. Therefore, the aqueous BPA solution was applicable for acute toxicity detection using bioluminescent bacteria.

The quantitative structure–activity relationship (QSAR) model, CA and IA models, and integrated CA and IA models such as TSP (two–stage prediction) have been used to predict the toxicity of mixtures of contaminants [56]. The CA model is usually used to predict the mixtures with the same MOA but IA with different MOA. The toxicities of mixtures of Cd, Pb, and Mn were predicted well by CA models [58]. However, the observed toxicities of these mixtures were often lower or higher than the predicted value. This was illustrated by the toxicities of mixtures of zinc oxide nanoparticles and chemicals with different MOAs, and neither CA nor IA models were suitable to predict the toxicities of mixtures of these contaminants [52]. Therefore, the prediction of the toxicity of mixtures in silicon was significant but should combined with the actual observed toxicity in experiments.

The difference in the joint effect of acute and chronic exposure was investigated (Figure 5), and the difference can be explained by mixtures inhibiting the growth of bioluminescent bacteria in 6 h long–term exposure. The luminescence in samples containing mixtures of BPA and heavy metals decreased by 50% relative to the control after 6 h of incubation; however, the biomass of these samples was lower than the control. In other words, the stronger inhibition of luminescence (over 50% ratio) should appear when the biomass of the samples equals the control. Consequently, mixtures of BPA and heavy metals inhibiting both cell growth and luminescence resulted in an antagonistic effect in acute exposure, which converted to a synergistic effect in the context of chronic exposure. The ecotoxicity of BPA to different classes of creatures, such as annelids, mollusks, crustaceans, insects, fish, and amphibians was investigated in past decades [20]. Additionally, such toxicity was also illustrated by BPA inhibiting algal and cyanobacterial growth [59,60]. Thus, it is reasonable that BPA inhibits the growth of bioluminescent bacteria. The effect of heavy metals on different creatures has been studied. Hg, Cd, Cu, Zn, Pb, and Cr affect the survival of ciliates [61]. Meanwhile, the influence of metals on the populations of microorganisms has been illustrated in the context of long–term exposure [62]. In this study, similar results were obtained in the chronic toxicity test (6 h exposure) (Figure 5A). Indeed, the mixture also inhibited the growth of Q67 (Figure 5B). Therefore, the joint effect of BPA and heavy metals revealed a synergism in the context of chronic detection. However, the joint effect of BPA and Cr exhibited an additive effect in both an acute and chronic test. In addition, the toxicities of mixtures of BPA and Cd, As, and Hg were shown to be time–dependent, but BPA and Cr were not, in the context of chronic exposure. BPA and Cr might present the same MOA, but this needs to be validated by further studies.

#### **5. Conclusions**

The acute and chronic toxicities of BPA and heavy metals, individually and in mixtures, were determined using bioluminescent bacteria *V. qinghaiensis* Q67 isolated from freshwater. The results showed that single chronic toxicities of BPA and heavy metals were time– dependent, and the toxicities of these contaminants were shown to be stronger in acute

exposure than in chronic exposure, except for Cd. Furthermore, neither CA nor IA models were suitable for the prediction of the mixture toxicities of BPA and heavy metals. Moreover, the acute joint effects of BPA and heavy metals mainly displayed antagonistic and additive effects, whereas the chronic joint effects of these mixtures mainly exhibited synergistic and additive effects. Indeed, the mixtures inhibiting both cell growth and luminescence resulted in antagonistic effects in the context of acute exposure, shifting to a synergistic effect in the context of chronic exposure.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/toxics10050255/s1, Figure S1: Arrangement of control and test samples in 96–well microplate. Gray solid circles are the control, and the blue solid circles are test samples. Each concentration of sample locates in four wells in same column; Figure S2: Comparison of aqueous BPA solution and methanol dissolved BPA using HPLC. Black line, methanol dissolved BPA solution. Red line, aqueous BPA solution; Figure S3: Observed acute mixture toxicity of BPA and Cd at non–equitoxic ratio; Figure S4: Observed acute mixture toxicity of BPA and As at non–equitoxic ratio; Figure S5: Observed chronic mixture toxicity of BPA and heavy metals at equitoxic ratio; Figure S6: Observed chronic mixture toxicity of BPA and Cd at non–equitoxic ratio; Figure S7: Observed chronic mixture toxicity of BPA and As at non–equitoxic ratio; Table S1: Concentration of individual contaminant used in single toxicity test; Table S2: Concentrations of individual contaminants in stocked mixtures.

**Author Contributions:** Conceptualization, J.Y. and Y.L.; methodology, J.Y., S.L., and Y.L.; software, J.Y.; validation, J.Y. and S.H. (Shulin Hu).; formal analysis, J.Y.; investigation, J.Y., A.L., S.H. (Shulin Hu), and Y.Z.; resources, J.Y. and Y.L.; data curation, J.Y., S.L., and Y.L.; writing—original draft preparation, J.Y.; writing—review and editing, J.Y., Y.L., and S.L.; visualization, J.Y.; supervision, Y.L. and S.L.; project administration, Y.L. and S.H. (Shuangyan Han); funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Key Areas Research Development Projects of Guangdong Province (Department of Science and Technology of Guangdong Provience: 2019B020210001).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The dataset is available upon the request of the corresponding authors.

**Acknowledgments:** We thank senior scientist Lingyun Mo from the Guilin University of Technology for providing the bioluminescent bacteria *V. qinghaiensis* Q67. The first author, Jun Yang, thanks Song Fan for his financial assistance for this work.

**Conflicts of Interest:** The authors declare that they have no competing interests.

#### **References**

