**4. Discussion**

The toxic effects of nickel by itself on the diatom *P. tricornutum* have been evaluated in our previous studies. Specifically, nickel exposure exhibits an EC50-72 h value of 2.48 ± 0.33 mg/L and leads to ROS production and decreased photosynthesis [16]. In addition, the toxic effects of BDE-47 on *P. tricornutum* have also been reported [24]. Previous studies have indicated that nickel and BDE-47 can cause toxic effects on the diatom *P. tricornutum* by inhibiting cell growth and affecting photosynthesis [24]. However, the present study is the first to investigate the combined toxicity of BDE-47 and nickel on microalgae.

BDE-47 and nickel exhibited synergistic interactions, with enhanced toxicity when mixed. Photosynthetic efficiency was strongly inhibited in *P. tricornutum* due to exposure to mixtures of BDE-47 and nickel, while inhibitory effects increased with increasing exposure time. However, BDE-47 by itself only slightly inhibited the photosynthetic efficiency of *P. tricornutum*, while nickel has been shown to strongly inhibit the photosynthetic efficiency of *P. tricornutum* [16]. Other studies of microalgae have shown that BDE-47 inhibits the photosynthetic efficiency of *Skeletonema costatum* and *Alexandrium minutum*. However, BDE-47 also reduced photosynthetic efficiency within 24 h of exposure in *Dunaliella salina* and *Thalassiosira pseudonana*, followed by gradual recovery to normal levels within 120 h [26–28]. Several differentially expressed photosynthetic genes at the transcriptional level, including those involved in chlorophyll synthesis, antenna proteins, oxygen evolution, electron transport, and downstream carbon fixation, were identified as DEGs of *P. tricornutum* after individual BDE-47 exposure, with most being downregulated [24]. Further, BDE-47 also suppressed the expression of some genes involved in photosystem II and antenna proteins in the diatom *T. pseudonana* [25]. In the present study, several genes involved in photosynthesis components such as carbon fixation, chlorophyll synthesis, photosystem II, and antenna proteins exhibited differential expression after exposure to mixtures of BDE-47 and nickel. In addition, nickel might impair the photosystem's oxygen-evolving complex (OEC) in *P. tricornutum*, leading to decreased photosynthetic efficiency [16]. These data indicate that photosynthetic complexes may be one of the toxic effect targets of BDE-47 in microalgae and that its toxic effects on *P. tricornutum* photosynthesis could be enhanced with additional nickel supplementation.

Increased ROS is an indicator that cells were exposed to oxidative stress. Exposure to mixtures of BDE-47 and nickel significantly induced oxidative stress in *P. tricornutum*. Transcriptomic analysis identified DEGs involved in oxidation-reduction processes, with some involved in nitrogen metabolism, photosynthesis, and amino acid metabolism. Among these genes, antioxidant-associated genes, including those encoding thioredoxin (*Thx*) and glutathione *S*-transferase (*GST*), exhibited increased expression, implying that they might be important for oxidative stress resistance after exposure to mixtures of BDE-47 and nickel. Specifically, Thx could be involved in oxidative damage mitigation [36]. Thx possesses a conserved redox site that supports intracellular redox homeostasis, reduces protein thiols, and is one of the most important components of the thioredoxin system [37,38]. Increased expression of Thx has also been detected in individual nickel treatments of *P. tricornutum* [16]. In addition, *GST* plays an important role in the detoxification and reduction of ROS in multiple stress processes [39]. BDE-47-induced *GST* expression has been detected in *T. pseudonana* [25], while *GST* has been identified as a target of Thx in *Arabidopsis* plants [40]. Increased expression of *Thx* and *GST* indicates that detoxification activities were activated to protect cells from oxidative stress due to exposures to mixtures of BDE-47 and nickel. Thus, *Thx* and *GST* might simultaneously protect *P. tricornutum* cells from oxidative stress.

Transcriptomic analysis revealed that exposure to mixtures of BDE-47 and nickel could affect genes involved in oxoacid metabolism, organic acid metabolism, carboxylic acid metabolism, and organic acid biosynthesis (Figure 4B). Organic acids, such as citric acid and malic acid, play important roles in detoxifying chemical stresses, including those from BDE-47 and nickel [41–43]. The regulated organic acid BDE-47 has been detected in *Oryza sativa* [42,43], while nickel is also known to modulate organic acid compositions in *Alyssum murale* [41]. Isocitrate lyase (encoded by *PHATRDRAFT\_51088*) is an enzyme that produces succinic acid and exhibited increased expression with 1.95 and 1.65 log2-fold change values after 48 and 72 h exposure to nickel and BDE-47 mixtures in this study. Succinic acid is involved in abiotic stress resistance in some plants [41,44,45]. For example, additional succinic acid concentrations can alleviate aluminum toxicity in alfalfa [44], while exogenous succinic acid concentrations lead to the enhanced tolerance of lead by *Larix olgensis* [45]. These data suggest that some organic/oxo-/carboxylic acids might be involved in the stress responses of *P. tricornutum* to mixtures of BDE-47 and nickel. In addition, organic/oxo- /carboxylic acids could serve as electron donors via their involvement in the dehalogenation process. For example, exogenous organic/oxo-/carboxylic acid-related compounds can be used as carbon sources (e.g., formate, acetate, and lactate) that could enhance the degradation of BDE-47 by microorganisms under anaerobic conditions [46,47]. Some microalgae, such as *Chlorella*, have been shown to transform BDE-47 into the debrominated

product of BDE-47 [48]. We consequently hypothesized that some organic/oxo-/carboxylic acids produced by microalgae could be electron donors and are involved in transforming BDE-47 within algal cells.

The biosynthesis of secondary metabolites (Table S9), amino acids, and fatty acids were also extensively affected by exposure to mixtures of BDE-47 and nickel. Many organic acids are precursors for fatty acids, amino acids, and secondary metabolites [49]. Similarly, some secondary metabolites, amino acids, and fatty acids exhibited increased concentrations after BDE-47 treatment of rice [42,43]. Moreover, the biosynthesis of amino acids and fatty acid synthesis pathways were also affected by the BDE-47 treatment of the diatom *T. pseudonana*. Likewise, individual nickel treatment of *P. tricornutum* led to differentially expressed biosynthesis of amino acid and fatty acid synthesis pathways [16]. These results indicate that exposure to mixtures of BDE-47 and nickel extensively affected the primary and secondary metabolite profiles of *P. tricornutum*.

Nitrogen metabolism-associated genes were also detected among the DEGs of this study following exposure to the mixture of pollutants. The most expressed nitrogen metabolismassociated gene was *PHATRDRAFT\_8155* (nitrite reductase), which is involved in nitrate assimilation, although other nitrogen metabolism-associated genes were also highly upexpressed. The enhanced expression of nitrogen metabolism-related genes was most prevalent in the individual BDE-47-treated *T. pseudonana* and nickel-treated *P. tricornutum*. These results suggest that the nitrogen metabolism of microalgae is highly sensitive to BDE-47 and/or nickel pollution. In addition, mixtures of BDE-47 and nickel can also enhance toxicity effects on nitrogen metabolism relative to the effects from individual chemicals.
