**3. Discussion**

We analyzed 52 fish samples from aquaculture regions in Taiwan for the residues of 12 sulfonamides (i.e., sulfamerazine, sulfaethoxypyridazine, sulfathiazole, sulfadiazine, sulfamethoxypyridazine, sulfapyridine, sulfadoxine, sulfamethazine, sulfadimethoxine, sulfamethoxazole, sulfamonomethoxine, and sulfameter,) and 18 organophosphorus insecticides (i.e., chlorfenvinphos, chlorpyrifos, diazinon, fenamiphos, fenitrothion, fenthion, formothion, iprobenfos, malathion, methacrifos, methamidophos, methidathion, phoxim, profenophos, prothiofos, pyrazophos, triazophos, and trichlorfon). The TFDA allows the presence of only sulfadimethoxine and sulfamonomethoxine in aquacultured products at total residual levels of <0.1 mg/kg and prohibits the use of any other sulfa drugs and organophosphorus insecticides in fish aquaculture. These compounds' residues existing in fish were thus identified; moreover, the identified levels of the residues were noted to adequately demonstrate the level of legal compliance concerning the application of these products.

The limit of quantification (LOQ) of all sulfonamides in fish samples was 10 ng/g—identical to the LOQ recommended by the TFDA for sulfonamide contamination in edible livestock, chicken, milk, and aquacultured foods and half of that for sulfonamide contamination in animal viscera [16]. However, the TFDA has not recommended a strict LOQ for organophosphorus insecticide contamination in aquacultured foods. Nevertheless, the TFDA-recommended LOQ is 10 ng/g for organophosphorus insecticide contamination in livestock (e.g., pork) and chicken muscles [17]. Previous methods developed for analyzing organophosphorus insecticide contamination in bivalves, crustaceans, fish, and cephalopods have involved the use of various apparatuses. For instance, gas chromatography flame ionization revealed the LOQs of chlopyriphos, ethion, ethoprophos, fensulfothion, isoxathion, and parathion to be 2–50 ng/g [18]; gas chromatography–mass spectrometry revealed the LOQs of propetamphos, diazinon, disulfoton, malathion, fenthion, and triazophos to be 7–15.2 ng/g [19]; and high-performance liquid chromatography (HPLC)–tandem mass spectrometry (LC-MS/MS) coupled with gel permeation chromatography revealed the LOQs of profenofos, chlorpyrifos, malathion, phosmet, triazophos, trichlorphon, and dimethoate to be 0.05–0.2 ng/g [20]. Compared with the aforementioned LOQs, our present analytical method detected a lower LOQ (i.e., 5 ng/g) for organophosphorus insecticide multiresidues, indicating the usefulness of our method in detecting trace organophosphorus insecticide residues.

Here, we adopted the analytical approach for sulfonamide residues suggested by the TFDA [16] and that for organophosphorus insecticide residues developed by the European Committee for Standardization, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) method [4,21]—both of which are suitable for detecting trace chemical residues. For the validation of its analytic method, the TFDA [2,22] recommends an acceptable recovery rate of 70–120% [relative standard deviation (RSD) < 15%], 70–120% (RSD < 20%), and 50–125% (RSD < 35%) for chemical residues in food matrices detected in the 0.1–1, 0.001–0.01, and <0.001 mg/kg ranges, respectively. In this study, residues of sulfonamides and organophosphorus insecticides that were identified within the 0.01–0.1 mg/kg ranges were noted to exhibit a recovery rate of 90–120% (RSD < 15%) and 80–120% (RSD < 15%), respectively. Moreover, the quantities of spiked analytes used at the lower and higher levels were respectively 5 and 25 ng/g for the 12 sulfonamides (Table S3) and 10 and 50 ng/g for the 18 organophosphorus insecticides (Table S4). Thus, all analyzed sulfonamide and organophosphorus insecticide concentrations were within the acceptable range [22]. Accordingly, our method of extraction proposed herein was robust for the analysis of the investigated sulfonamides and organophosphorus insecticides in the fish samples.

LC-MS/MS demonstrated a positive result for sulfonamide in only 3.85% of the 52 fish samples, and in all positive samples, only sulfamethazine, a TFDA-banned sulfonamide, was observed. Sulfonamide residues were identified in 77.59% of a total of 116 samples of fish in 2016 in China [23], 4.27% of 101 samples of fish over 1994–1998 in Slovenia [24], 17.39% of 138 samples of fish in 2012 in Iran [25], and 1.75% of 171 samples of fish over May September 2008 in South Korea (specifically sulfadiazine and sulfamethoxazole residues) [26]. The TFDA conducted surveys for sulfonamide residues in aquacultured products over 2013–2018 in Taiwan and detected increased illegal use of sulfonamides as antibiotic agents in marine products in order to increase production [27–32]: the detection rate of banned sulfonamide residues in fish samples was 0.50% in 2013 (1/199) [27], 0% in 2014 (0/194) [28], 4.29% in 2015 (3/70) [29], 1.49% in 2016 (1/67) [30], 2.94% in 2017 (2/68) [31], and 2.67% in 2018 (2/75) [32]. However, our current findings are inconsistent with the aforementioned findings from the TFDA surveys. These differences may possibly be due to varied sample sizes. In addition, our study samples

were derived from areas of fish production in Taiwan; by contrast, those derived by the TFDA could have been imported fish. Moreover, we included a larger fish sample size and analyzed for more sulfonamides. Therefore, a wider spectrum of banned sulfonamide residues was analyzed in fish in Taiwan.

The predominant sulfonamide residue was sulfamethazine, with its maximum concentration being 0.03 mg/kg (in one tilapia sample). This is consistent with the results observed by Sampaio et al. [1] and Nunes et al. [14]: sulfamethazine is a frequently applied sulfonamide in fish aquaculture. Moreover, sulfamethazine has previously been found to be present at high concentrations (>100 μg/kg) frequently [23]. The aforementioned studies have positively identified sulfathiazole [27], sulfamethoxazole [29,30,32], and sulfadiazine [31] in fish samples, and a TFDA survey positively identified sulfaquinoxaline and sulfathiazole in soft-shell turtle samples [28]. These findings confirm the increased use of sulfonamides as feed additives in aquaculture [11]. In addition, the water sulfamethazine level of 100 ng/L could affect greenhouse gas release, atmospheric ozone depletion, and eutrophication control through denitrification inhibition and N2O release stimulation [33]. Moreover, high sulfonamide usage appears to be strongly correlated with bacterial resistance to sulfonamides [34], achieved through the development of antibiotic resistance genes, which can pose a potential human health risk [23]. Our results, in combination the findings derived from other surveys described here, demonstrate the exposure of Taiwan's population to trace sulfonamide levels through fish consumption.

Of all 18 analyzed organophosphorus insecticides, only chlorpyrifos was detected in the fish samples. In all fish samples, 5.77% was the detection rate for all studied organophosphorus insecticide residues, with the highest rate being for chlorpyrifos (6.25%) in milk fish and perch samples, followed by tilapia samples (5.0%). During their aquaculture activities, some farmers in Taiwan apply organophosphorus insecticides for ectoparasite treatment [4]. Sun et al. [35] reported an organophosphorus insecticide detection rate of 11.37% in 607 fish samples from traditional markets, regional supermarkets, fish markets, and fish farms in Taiwan over 2001–2003; however, over 2002–2004, the authors reported an organophosphorus insecticide detection rate of 16.83% (only chlorpyrifos) in 814 fish samples from Taiwan markets [36]. Our results concerning organophosphorus insecticide detection rates are much lower than those of Sun et al. [35,36]. These discrepancies are likely due to differences in the sample sizes and collection sources as well as the differences in the organophosphorus insecticide categories analyzed. These could also have occurred because of the Taiwanese government's implementation of the national action plan for reducing veterinary drug use in 2006 [37].

The predominant organophosphorus insecticide in our fish samples was chlorpyrifos—similar to the observations of a similar survey in Egypt [13]. Even though chlorpyrifos rapidly degrades in the environment, the Egyptian study reported that extensive chlorpyrifos use polluted aquatic habitats and caused increased general toxicity to all vertebrates compared with other classes of insecticides. Chlorpyrifos remains a widely used organophosphorus insecticide for controlling pests in agriculture as well as in sanitation industries around the globe [38]. Because of neurotoxicity concerns, Taiwan's government has banned the use of organophosphorus insecticides (except for trichlorfon) in aquaculture and specified strict MRLs for animal husbandry [4]. In the current study, the insecticides detected may have been illegally applied for the direct treatment of ectoparasite infections occurring in fish or for the treatment of infectious diseases caused by the presence of other aquacultured organisms during mixed breeding in aquaculture ponds, as we revealed previously [4,6,7]. Moreover, we detected chlorpyrifos levels of 0.002 mg/kg, which are much lower than the level revealed by Sun et al. (0.463 mg/kg) [36]. The aquacultured fish also exhibited more variation in chlorpyrifos residues. The chlorpyrifos contamination sources could be fish feed and aquaculture environments, including water and sediments. A study reported that chlorpyrifos rapidly degrades in the marine ecosystem [39], but data on chlorpyrifos contamination in fish feed are limited. Thus, we recommend additional studies examining phthalate content in fish feeds. Information on organophosphorus insecticide use for ectoparasite infection treatment in other special fish species, however, remains limited. The detected levels of organophosphorus insecticide varied among all three fish species; this variation

is attributable to differences in the sample sizes used. We thus recommend the execution of further research employing large samples of cultured fish.

Several guidelines provide parameters that can aid in assessing human and other organisms' risks of various conditions after exposure to chemical residues through food; these parameters include ADI, target hazard quotient (THQ), and tolerable daily intake (TDI) [2,4,40]. ADI is recommended by JECFA, but apparent discrepancies in consumption rates and eating habits are not considered in ADI [7]. Through the use of TDI, one could evaluate the risks that are related to consuming specific food products tainted with plasticizers and could identify whether exposure to plasticizers such as phthalates at various degrees is harmful to human health [41]. THQ indicates the assessment of health risk of noncarcinogenic harmful effects [40]. The JECFA [42] and US Environmental Protection Agency [43] have proposed EDI, which can be used to estimate chronic dietary intake at a relatively high accuracy level. In the current study, EDIs were not exceeded by the corresponding ADIs. Because few sulfonamide residues were detected, the EDIs revealed that fish consumption led to a substantially lower dietary intake of sulfamethazine, relative to that specified by the corresponding ADIs, in Taiwan's population. In addition, when evaluated against the ADIs, the EDI corresponded to <1% of the ADIs, signifying that the corresponding risk is negligible [2,7,44]. Farmers engaged in aquaculture in Taiwan may also apply organophosphorus insecticides for ectoparasite treatment [4,45]. In the present study, the consumption of aquacultured fish led to exposure to very low levels of the organophosphorus insecticide chlorpyrifos: in the Taiwanese population, its EDI was much lower than its JECFA-recommended ADI (<0.1% of the ADI for organophosphorus insecticide residues). Therefore, these results were similar to those for sulfamethazine for fish consumption. The risk assessment is negligible because the EDI is <1% of the corresponding ADI [2,7,44]. Taken together, sulfonamide and organophosphorus insecticide residue levels in aquacultured fish in Taiwan may not affect human health adversely.

Our results suggest that to ensure commercial food safety, regulatory authorities as well as producers in Taiwan must endeavor to continually monitor aquacultured products and potential contamination sources. Moreover, considering that antibiotics may exert adverse effects on health and aquatic environments, additional studies on the effects exerted by such pollutants are imperative.

#### **4. Materials and Methods**
