**2. Materials and Methods**

#### *2.1. Cell Cultures and Chemical Treatments*

Axenic algal cultures of the diatom *P. tricornutum* CCMP2561 were obtained from the National Center of Marine Algae and Microbiota (USA) and cultured with an f/2 medium at 20 ◦C (12:12 h light-dark cycles with 65 μmol photons/m2/s). The chemical toxicity studies used exponential growth phase cultures with an initial cell density of 5.2 × <sup>10</sup><sup>5</sup> cells. To evaluate the influence of chemical pollutants on cultures, a gradient of individual BDE-47 (Dr. Ehrenstorfer GmbH, Germany), individual nickel (NiCl2·6H2O, Sigma-Aldrich, Louis, MO, USA), and mixtures of the compounds were added to *P. tricornutum* cultures (concentrations shown in Table S1), followed by harvesting cells at 24, 48, 72, and 96 h to assess cell abundance and photosynthetic efficiency. Each 200 mL of *P. tricornutum* cultures was maintained in glass flasks, and all the flasks were maintained in the same incubator.

The actual concentrations of nickel and BDE-47 were verified with ICP-MS (inductively coupled plasma-mass spectrometry) and GC-MS (gas chromatograph-mass spectrometry), respectively. Moreover, relationships among the actual nickel concentrations and BDE-47 in mixtures were assessed with regression analysis (Table S1). BDE-47 was dissolved in dimethyl sulfoxide (DMSO, CNW, China), and untreated DMSO-amended cultures were used as controls. All treatments were performed in triplicate.

#### *2.2. Physiological Parameter Analysis*

Cell density, photosynthetic efficiency, and reactive oxidative species (ROS) levels were measured to evaluate the physiological responses of *P. tricornutum* to chemical exposure. *P. tricornutum* cells were fixed with glutaraldehyde (Sigma, Louis, MO, USA) and counted using a Countstar automated algae counter (Countstar algae, Shanghai, China). Photosynthetic efficiency (*F*v/*F*m) was directly determined using a pulse amplitude-modulated fluorometer (Water-PAM fluorometer, Walz, Germany) after adapting cultures to the dark. ROS production was determined by measuring fluorescence density. *P. tricornutum* cells were stained with 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). At the same time, ROS fluorescence density was measured using a fluorospectrophotometer (LS45 Fluorescence Spectrometer, PerkinElmer, Llantrisant, UK) with an excitation and emission wavelength of 515 nm and 550 nm, respectively. A sampling of the microalgae was performed in triplicate throughout the experiments.
