Spatial distribution, source identification, and risk assessment of organochlorines in wild tilapia from Guangxi, South China

Seventy-five wild tilapia samples from six rivers (ten sites) in Guangxi province were collected and analyzed for 53 organochlorine compounds. DDTs, endosulfan, and PCBs were the most dominant compounds found in this study. Tiandong County (TD) and Guigang City (GG) sites were found to be heavily contaminated with high levels of endosulfan (385–925 ng/g lw) and/or DDTs (20.1–422 ng/g lw). The diagnostic ratios indicated that the residues of DDTs and endosulfan in wild tilapia are associated with historical applications as well as the recent introduction of technical DDTs and endosulfan at some sampling sites. The correlation between total length, body mass, and organochlorines (OCs) was higher than the correlation between age and lipid content. There was no significant correlation between organochlorine pesticides (OCPs) and lipid content. Therefore, for organisms, the feeding intensity (related to length and mass) of fish could better reflect degree of pollution than exposure time (age) of fish. The hazardous ratios for the 50th and 95th percentile data of OCPs and PCBs in fish were both below 1, suggesting that daily exposure to OCPs and PCBs yields a lifetime cancer risk lower than 1 in 10,000.


Contents
Text S1. Study area Five ecologically important main rivers were selected from the southern of Guangxi province, including Youjiang River (YO), Zuojiang River (ZU), Yujiang River (YU), Qianjiang River (QI) and Xunjiang River (XU) (Fig. 1). With a total drainage area of approximately 175,412 square kilometers, it is the most important water system in Guangxi. The southern river system of Guangxi has become an important resource support and pollution bearer for regional economic development. The basin is home to more than 73.4% of the province's industrial enterprises, and is a veritable industrial area, yet until recently their ecological condition has not been well quantified. This basin is also China's largest sugarcane producing area (about 52.1% of the country's total planting area), and is also a high incidence area for pests and diseases (Guangxi Statistics Bureau, 2019). In order to control pests and diseases, a large number of chemical pesticides have been used for a long time. In 2015-2018, the average annual use of pesticides in Guangxi was 13,800 tons (Guangxi Statistics Bureau, 2019). The escalating anthropogenic activities (agricultural run-off, industrial waste and municipal discharge) along the basin may seriously pollute the region with OCs and may cause high accumulations of OCs in the riverine biota. Many studies have reported the concentration of POPs in their downstream water bodies (Xijiang River Basin and Pearl River Delta) (Mai, et al., 2005;Guo, et al., 2008;Qiao, et al., 2010;Sun, et al., 2015), but studies on the concentration of POPs in the southwestern Guangxi have not been reported.

Text S2 Chemicals
Fifty-three OCs congeners were selected as target compounds. The OCPs were dicided into seven groups including DDTs, HCHs, Drins, CHLs, Endos, HCB and MXC, The 28 PCBs from the groups of tri-, tetra-, penta-, hexa-, hepta-CBs were selected as target compounds in this study. Physicochemical properties, purchasing channels and handling procedures of all the target compounds and chemicals are listed in Table S2.
Dichloromethane (DCM) and n-hexnane (HEX) were purchased from CNW (ANPEL Laboratory Technologies (Shanghai) co. Ltd). Aceton were purchased from KNOWLES (Chengdu chren chemical co. Ltd). Deactivated neutral alumina: 70-230 mesh (Merck), extracted using DCM for 48 h, roasted in an oven at 250 °C for 12 h and burned in a muffle furnace at 450 °C for 6 h, added with 3% distilled water after cooling to activate; Acid silical gel: 80-200 mesh (Merck), extracted using DCM for 48 h, roasted in an oven at 180 °C for 12 h and burned in a muffle furnace at 450 °C for 6 h; added with sulfuric acid accounting for half of the total weight after cooling; Anhydrous anhydrous sodium sulfate was burned in a muffle furnace at 450 °C for 6 h.
All of the three reagents was placed in a desiccator before use. Ultra-pure water was prepared with a Milli-Q water-purification system (Millipore, Bedford, Massachusetts, USA). ENVI™-Florisil cartridges (500 mg, 3 mL) were purchased from Supleco (Bellefonte, PA, USA).

Text S3 QA/QC
The standard solutions with nine concentration gradients (concentration range was between 1-500 ng/mL) were detected and analyzed, and the regression coefficient of > 0.97. Each sample was analyzed three times and the average was used for data analysis. A standard solution (2 ng/mL for each OC compounds) with a fixed concentration was injected every day to monitor the sensitivity of the instrument.
Procedural blanks (n = 8), spiked blanks (n = 8), and spiked matrices (n = 8) were set to ensure method quality control. The laboratory procedural blanks were analyzed using extracted organism samples (fish muscle). No targeted OC congeners were detected in the procedural blank. The spiked compounds included 24 OCP congeners and 28 PCB congeners. The mean recoveries of the surrogates in all samples were 72.3 ± 8.4%, 87.6 ± 20.0%, and 92.4 ± 17.8% for TCMX, PCB30, and PCB204, respectively. The concentrations reported in this paper were after subtracting the mean concentration in the blanks analyzed in the same batch, but not adjusted by surrogate recoveries. Recovery of OC standards was 68.6-96.2% in the spiked blanks and 73.8-105.6% in the matrix-spiked samples, with a relative standard deviation of < 17%.

Instrumental detection limits (IDLs) and instrumental quantification limits (IQLs)
were defined as three and ten times the signal-to-noise (S/N) ratio, respectively. The LODs and LOQs for the tilapia samples were 0.006-0.087 ng/g lw (lipid weight) and 0.020-0.289 ng/g lw (Table S3).

Text S4 Calculation of Estimated Daily Intakes (EDIs).
EDI values for the OC compounds (Table GG) were calculated using the following equation:

EDI = PR × C BW
Where EDI is the estimated daily intake of the OC compounds in ng/kg body weight (bw)/day (d); PR is the annual per capita fish consumption of 59.3 g/person/day in Southern China ; C is the average concentration of PCBs and OCPs in fish muscle (ng/g wet weight (ww)); BW is the average body weight of 56.6 kg (Ding, et al., 2019); C is selected the maximum concentration of each OC compounds (ng/g

/ 25
ww) detected in wild tilapia from the main river in the southern China.
Text S5 Risk assessment.
Hazard ratios (HRs) were computed to assess risk associated with fish consumption in humans (Dougherty, et al., 2000;Jiang, et al., 2005). Hazard ratios (HRs) were calculated by dividing the estimated daily intakes (EDIs) by the benchmark concentrations (BMC). There would be no obvious hazard if HRs is less than 1 (Jiang, et al., 2005). The hazard ratios were assessed by the equation:

HRs =
The benchmark concentration (BMC) for carcinogenic effects consists of two Where RL is the maximum acceptable risk level (1×10 −6 ); CSF is the cancer slope factor (mg/kg / d); CR is the consumption rate (g/d).
Two hazard ratios (HRs), i.e., from the 50th and the 95th percentile measured concentrations (50th and 95th MEC), were used for assessing the potential health risk to humans. The two HRs provided a simple way for screening chemicals that might require a more detailed analysis. When 95th centile HR was greater than unity, a refined 7 / 25 risk assessment would subsequently be conducted to further ascertain the real risk.