*3.5. P. tricornutum Genes Differentially Expressed after Exposure to Mixtures of Nickel and BDE-47*

A total of 565 and 325 genes were upregulated and downregulated after 48 h of exposure to nickel and BDE-47, respectively (i.e., those exhibiting |fold changes| > 2 and *p* < 0.05). After 72 h of exposure, these DEG numbers increased to 841 and 435 upregulated and downregulated genes, respectively (Figures S1 and S2).

**Figure 3.** Relative ROS production of *P. tricornutum* after individual BDE-47 treatment and treatment with mixtures of BDE-47/nickel. \*\*\* *p* < 0.001; \*\* *p* < 0.01, and \* *p* < 0.05. Error bars indicate ± SD. *p*-values are shown in Table S5.

Among the DEGs, 532 genes defined as core genes were upregulated or downregulated (|fold changes|>2, *p* < 0.05) after exposure to the mixtures for 48 h and 72 h (Figure S3). KEGG pathway analysis (Figure 4) of the core genes revealed that many of the DEGs were involved in the biosynthesis of secondary metabolites, carbon metabolism, biosynthesis of amino acids, glyoxylate and dicarboxylate metabolism, ribosome biogenesis in eukaryotes, fatty acid metabolism, fatty acid biosynthesis, and porphyrin and chlorophyll metabolism. Further, enrichment GO analysis of the core genes revealed many were involved in oxoacid metabolism, organic acid metabolism, carboxylic acid metabolism, oxidation-reduction reactions, cellular amino acid metabolism, and small molecule metabolism. In addition, oxidoreductase activity and ligase activity were significantly enriched GO terms involved in molecular functions.

#### *3.6. DEGs Related to Photosynthesis and Nitrogen Metabolism*

At least 31 genes related to photosynthetic metabolism were responsive to exposure to mixtures of BDE-47 and nickel (Figure 5A, Table S6). Among these, *HemA*, *UbiA*, *Aminotran\_3*, *PHATRDRAFT\_50740* (magnesium chelates), *hemE*, *GluRS\_2*, and *CHLH*, which are involved in porphyrin and chlorophyll metabolism, were downregulated due to exposure to a mixture of BDE-47 and nickel. The photosystem II gene *PsbP* did not exhibit significant changes in expression after 48 h of exposure but decreased expression after 72 h. Most differentially expressed photosynthesis-related genes involved in carbon fixation were upregulated, including *Fba4*, *Fba3*, *FbaC5*, *TIM\_1*, *PEPCase\_2*, *TPI/GapC3*, *GapC4*, and *GapC1*. The antenna protein-encoding genes *Lhcf15* and *Lhcf11* exhibited increased expression due to exposure to the mixtures, while *Lhcx1* was downregulated after exposure to mixtures between 48 and 72 h. These results indicate that photosynthetic processes display different responses due to exposures to mixtures of BDE-47 and nickel, although the overall photosynthetic balance was disturbed.
