*2.1. Quality Measurements*

Heavy metals can lead to chronic heart disease, cancer, and death [19]. Heavy metals have been banned by law from foods, cosmetics, and drugs [20]. The metal concentrations in pufferfish skin were found to be Pb (0.562 ± 0.052 mg kg−1), Cd (0.042 ± 0.004 mg kg−1), As (0.075 ± 0.002 mg kg−1), and Cr (1.152 ± 0.067 mg kg<sup>−</sup>1), which are below the permissible limit recommended by the Chinese national standards (Table 1) [20]. Some previous works have suggested that the accumulation of heavy metals in fish depends on the dietary ingestion rate and the concentration of heavy metals in the ingested food [21]. Thus, the low level of heavy metal concentration in pufferfish skin might be due to the artificial feed of lower heavy metals in aquaculture pufferfish. Moreover, the metal concentration in the collagen sample in this study was Pb (0.421 ± 0.015 mg kg−1), Cd (0.023 ± 0.002 mg kg−1), As (0.052 ± 0.001 mg kg−1), and Cr (0.863 ± 0.072 mg kg−1), which are also below the permissible limit recommended by the Chinese national standards (Table 1) [20].

The pufferfish (*T. flavidus*) skin contains 1.7 ± 0.5 MU/g of tetrodotoxin. According to Tani [22], the oral lethal dose of tetrodotoxin for a human being is 10,000 MU, and pufferfish with a tetrodotoxin content of less than 10 MU/g is considered to be nontoxic. Thus, pufferfish (*T. flavidus*) skin is safe for use in food, cosmetics, and pharmaceutical raw materials. Additionally, the content of

tetrodotoxin in the collagen sample in this study was below the detection limit, indicating the safety of pufferfish collagen.


**Table 1.** Heavy metal concentrations (mg kg−<sup>1</sup> dry weight).

<sup>a</sup> Chinese national standards were obtained from the literature described by Chen et al. (2016).
