**1. Introduction**

BPA is an important industrial chemical that has been widely used as the monomer of polycarbonate plastics and epoxy resins. Materials containing BPA appear in our daily life, especially in food contact materials, such as plastic bottles, cups, plates, goblets, and storage containers [1,2]. Heavy metals, which are natural substances in the Earth's crust, are widely spread in environment and foods [3]. The increasing demand for BPA has resulted in its accumulation in the environment [4–6]. BPA has been detected in packaged foods, drinking water, dust, sewage sludge, urine, serum, etc. [2,7]. A mean value of 8.99 ng/g BPA was tested in 45 vegetable samples [8]. Previous studies have shown that the concentration of BPA reached 174.6 ug/mL in a river sample [9] and 0.98 mg/L BPA was measured in drinking water [10]. Significantly, the concentration of BPA ranged from 0.1 to 3.9 ng/mL in 3021 human milk samples in 50 studies [11]. Therefore, the pollution of BPA has become serious. Furthermore, BPA and heavy metals widely co–exist in the environment and in foods. The toxicities of their mixtures must therefore be investigated.

**Citation:** Yang, J.; Liao, A.; Hu, S.; Zheng, Y.; Liang, S.; Han, S.; Lin, Y. Acute and Chronic Toxicity of Binary Mixtures of Bisphenol A and Heavy Metals. *Toxics* **2022**, *10*, 255. https:// doi.org/10.3390/toxics10050255

Academic Editors: Stefano Magni, Valerio Matozzo and François Gagné

Received: 12 April 2022 Accepted: 15 May 2022 Published: 17 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The single toxicities of BPA and heavy metals have been well studied. BPA is not only well–known as an endocrine disrupter, but it also exhibits genotoxic activity [12] and reproductive [13,14], nervous [15,16], developmental [17,18], and immune toxicity [19,20]. Heavy metals can accumulate in the human body because of their long half–life, which results in diseases in target organs, such as the brain, kidney, and liver. [3]. Considering the long experimental time and poor repeatability for animal tests, especially the 3R principles regarding the ethical use of animals [21–23], a rapid and low–cost bioassay based on bioluminescent bacteria has been developed. Bioluminescent bacteria can rapidly respond to contaminants within a short time, and the light emission decreases with the increase in the concentration of contaminants [24,25]. To date, the acute toxicity of BPA evaluated by bioluminescent bacteria is lacking; however, the toxicities of heavy metals to bioluminescent bacteria are well studied in the environmental field. Most natural bioluminescent bacteria are isolated from the ocean, including four genera, namely *Photobacterium*, *Photorhabdus*, *Shewanella*, and *Vibrio* [25,26]. *Photobacterium phosphoreum* T3, *Vibrio fischeri,* and *V*. *qinghaiensis* Q67 are widely used to detect the acute toxicity of contaminants [25,27]. The acute toxicities of heavy metals, such as chromium, cadmium, copper, mercury, lead, and zinc were tested using *V. fischeri*, *V. qinghaiensis* Q67, and *P. phosphoreum* [28–30]. Indeed, it is known that high concentrations of NaCl (2–3%, *w*/*v*) impact the toxicities of contaminants, especially heavy metals [31–33]. Therefore, Q67 is suitable to measure the toxicities of heavy metals because it is the only strain isolated from freshwater so far. Thus, a lower concentration of NaCl (0.85%, *w*/*v*) needs to be added to samples [28].

In recent decades, binary- and ternary-mixture toxicities regarding BPA and other chemicals have been studied. However, the toxicities of mixtures of BPA and other chemicals have mainly focused on BPA and other endocrine disrupters (Eds) [16,34,35]. Studies regarding the combined toxicities of BPA and its analogues are increasing because BPA was replaced by its analogues due to its adverse effect [2,36–38]. In addition, the toxicities of mixtures of BPA and TiO2 [15,17,39], Pb [16], and Cd [40] have been investigated using different types of wildlife; however, no clear synergism, additive, or antagonism effects were obtained in these works. The mixture toxicities of BPA and phthalates were studied using different organisms [41–44]. The joint effect of BPA and dibutyl phthalate was investigated, and the results showed that the co–exposure of these contaminants resulted in increased cytotoxicity, oxidative stress, and genotoxicity [40]. The mixtures of BPA and diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) when exposed to human amniotic fluid reduced INSL3/RXFP2 signaling, which revealed an antagonist effect [45]. The antagonism effect of BPA and DEHP was observed in juvenile rats [46]. In addition, the joint effects of BPA and its analogues BPS, BPAF, BPB, and BPF were assessed by a highly sensitive micro–biosensor, and additive and synergistic effects were observed [47]. It is worth noting that *V. fischeri* was used to assess the joint effects of bisphenols and plasticizers and pharmaceuticals, and synergistic and antagonistic effects were observed [37]. Nonetheless, studies regarding the toxicities of mixtures of BPA and heavy metals are lacking, and their joint effects are still unknown. Bioluminescent bacteria have rarely been used to measure toxicities of mixtures of BPA and other contaminants until now. The rapid and low–cost approach based on bioluminescent bacteria should be used to estimate the joint effects of BPA and heavy metals in the environmental field.

In this study, the toxicities of binary mixtures of BPA and heavy metals were evaluated using bioluminescent bacteria *V. qinghaiensis* Q67. Further, the conventional models, CA and IA, were used to predict the toxicities of the mixtures. Moreover, the joint effects of BPA and heavy metals were assessed using the toxicity–unite method. This work proposes the application of Q67 for the detection of the toxicities of mixtures of BPA and other contaminants. Q67, which was isolated from Qinghai Lake in China, was the only strain isolated from the freshwater, and it might be the appropriate model from the aquatic system. The toxicity evaluated by Q67 might indicate the real toxicity of samples, which would contribute to ecotoxicology and risk assessment.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Contaminants*

The bioluminescent bacteria used in this work was *V*. *qinghaiensis* Q67. The freeze–dried Q67, used for the detection of the acute toxicity alone and in the mixture, was prepared according to a previous study [48]. The contaminants used in this study were bisphenol A (BPA), K2Cr2O7, Cd(NO3)2, ZnSO4·7H2O, Pb(NO3)2, arsenic (As), mercury (Hg), and nickel (Ni). BPA (GC, purity > 99%) was purchased from Macklin (Shanghai, China). All the other chemicals were purchased from Aladdin (Shanghai, China). All the concentrations of contaminants used in this work are listed in Table S1. It is worth mentioning that an aqueous BPA solution was prepared and compared with BPA dissolved in methanol. BPA was dissolved in deionized water at 75 ◦C for 2 h; then, the quantities of BPA dissolved in water and methanol at the same concentrations were compared using the e2695–series high–performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) [49]. Briefly, the column was ZORBAX Eclipse XDB–C18 (Agilent Technology, Santa Clara, CA, USA), the liquid phase was 60% (*v*/*v*) acetonitrile, the injection volume of the sample was 20 μL, the temperature was kept at 30 ◦C, and the detection wavelength was 276 nm.
