**5. Conclusions**

We report an efficient and sensitive LC and GC-MS/MS-based method for detecting sulfonamide and organophosphorus insecticide residues in fish. The most abundant and the only sulfonamide and organophosphorus insecticide residues detected in all fish samples were sulfamethazine and chlorpyrifos, respectively. Nevertheless, all the detected chemicals were present in trace amounts, much below the TFDA-recommended MRLs, in all 52 fish samples. This indicates that the presence of sulfamethazine and chlorpyrifos use during large-scale breeding of fish in Taiwan does not lead to severe contamination. Furthermore, EDIs for these chemicals in Taiwanese adults were considerably lower than the JECFA-defined ADIs—confirming that no immediate health risk is posed by consuming the aquacultured fish. Therefore, the low sulfamethazine and chlorpyrifos intake through the consumption of contaminated fish in Taiwan seems to present a negligible threat to the health of Taiwanese people. Taiwanese regulatory authorities and aquafarmers may use our findings as a reference for improving aquaculture-related food safety regulation.

We, however, are unaware of contaminants other than sulfonamides and organophosphorus insecticides used in fish production that may be consumed during daily meat intake and may exceed their ADIs to a hazardous extent within the general population. Regardless of our findings, global concern regarding veterinary antibiotic and insecticide contamination and adverse effects on the environment and human health is increasing. Thus, a background information system on veterinary antibiotic and insecticide consumption through fish must be established and improved so as to provide an appropriate monitoring and management framework. Moreover, aquatic samples should be continually surveyed for detecting residues of chemicals and ensuring food safety.

**Supplementary Materials:** The following are available online, Table S1: MS/MS fragmentation conditions for 12 sulfonamides and LC-amenable 6 organophosphorus insecticides. Table S2: MS/MS fragmentation conditions for GC-amenable 12 organophosphorus insecticides. Table S3: Recovery, repeatability, and limit of quantification of veterinary drugs spiked into tilapia samples. Table S4: Recovery, repeatability, and limit of quantification of organophosphorus insecticides spiked into tilapia samples. Figure S1: LC-MS/MS chromatogram of the detected 12 sulfonamides residues at the quantification ion for sulfamethazine in the positive samples. Figure S2: GC–MS/MS chromatograms of the detected 18 organophosphorus insecticide residues at the quantification ion for chlorpyrifos in the positive samples. Figure S3: Location of 52 sampling areas in Taiwan.

**Author Contributions:** C.-P.C. and P.-H.H. conceived the idea and executed experiments. W.-C.Y., C.-F.W., C.-C.C., M.-Y.T., and H.-P.T. provided assistance in recombinant construction. C.-T.L., Y.-J.X., and J.-H.W. executed data analysis. G.-R.C. wrote, reviewed, and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by theMinistry of Science and Technology (Taiwan) (MOST 107-2313-B-415-012) and, in part, by Grant 108AS-8.2.4-BQ-B2 and 108AS-8.2.4-BQ-B3 from the Council of Agriculture, Executive Yuan

(Taiwan) and the Taichung Veterans General Hospital (Taiwan) and National Chung-Hsing University (Taiwan) (TCVGH-NCHU-1087608). This manuscript was edited by Wallace Academic Editing.

**Conflicts of Interest:** No potential conflict of interest was reported by the authors.
