*2.3. Single Toxicity*

The detection of acute and chronic single toxicity was performed according to the method used in a previous study [51,52]. For the test of the acute toxicity, the freeze–dried Q67 was resuscitated in a 0.85% NaCl solution for 15 min; then, 10 μL of resuscitated Q67 was transferred into a 150 μL contaminant solution. The contaminant solution was diluted using 0.85% NaCl over a range of concentrations. In terms of the chronic toxicity, 90 μL contaminant solution and 90 μL of 2–fold culture medium were first mixed; then, 20 μL of the fresh culture of Q67 was transferred into the last mixture. Additionally, the mixture was oscillated uniformly and incubated at 23 ◦C for 6 or 12 h. Finally, the bioluminescence of the mixtures was recorded by a SYNERGY H1 microplate reader (BioTek, Winooski, VT, USA). The arrangement of control and samples in a 96–well plate are shown in Figure S1. The inhibition ratio of the luminescence was calculated according to Equation (1). The concentration–inhibition data were fitted by the logistic (Equation (2)) and the dose–response model (Equation (3)) [52,53].

$$I = \frac{L\_{\text{C}} - L\_{\text{S}}}{L\_{\text{C}}} \times 100\% \tag{1}$$

where *I* is the inhibition ratio of luminescence. *LC* and *LS* indicate the relative luminescence unit of control and contaminants solution, respectively.

$$I = A\_2 + \frac{A\_1 - A\_2}{1 + \left(\mathbb{C}/\mathbb{C}\_0\right)^P} \tag{2}$$

$$I = A\_2 + \frac{A\_2 - A\_1}{1 + 10^{(\log \mathbb{C}\_0 - \mathbb{C})P}} \tag{3}$$

where *A*<sup>1</sup> and *A*<sup>2</sup> are the bottom and top values, respectively, of the inhibition ratio. *C* is the concentration of tested samples, and *C*<sup>0</sup> indicates the value of *C* at 50% of the inhibition ratio of luminescence. *P* is the parameter of slope for the concentration–inhibition curve.

#### *2.4. Mixture Toxicity*

The toxicities of mixtures of BPA and heavy metals were analyzed. The test of the toxicity of the binary mixture was carried out using the same method as that for single toxicity. Based on the *EC*<sup>50</sup> values of individual contaminants, the mixture toxicities of BPA and Cr, Pb, Hg, and Ni at equitoxic ratios were detected, while BPA and Cd and As at non–equitoxic ratios were prepared at 1:10−1.5, 1:10−1, 1:10−0.5, 1:1, 100.5:1, 101:1, and 101.5:1; then, the mixture toxicities were tested. The concentrations of individual contaminants in stocked mixtures are listed in Table S2. Stocked mixtures were diluted for further tests. The toxic unit of the mixture (*TUmix*) was used to evaluate the joint effects of mixtures, which were calculated using Equation (4). According to a previous study, 0.8 < *TU* < 1.2 was considered simple additivity, while *TU* < 0.8 revealed synergism; additionally, *TU* > 1.2 indicated antagonism [54,55].

$$Tll\_{mix} = \frac{\mathcal{C}\_A}{EC\_{50A}} + \frac{\mathcal{C}\_B}{EC\_{50B}} \tag{4}$$

where *CA* and *CB* are the individual concentrations of *A* and *B*, respectively, in a mixture that inhibits 50% of luminescence. *EC*50*<sup>A</sup>* and *EC*50*<sup>B</sup>* are effective concentrations of individual contaminants at 50% inhibition according to single toxicity.
