Analysis of bacterial biodiversity and ecological risk evaluation of organophosphorus in Lianhuan Lake

PLOS One | https://doi.org/10.1371/journal.pone.0332712 September 26, 2025 6 / 20 2.3.4 Analysis of integrated water quality index. The IWQI provides a comprehensive assessment of a water body’s pollution degree and overall quality status. The IWQI model was first proposed by Mukate et al. [13] to integrate multiple water‐quality indicators into a single value for rapid evaluation of pollution levels and potability. In this study, three key parameters—NH₃–N, TN, and TP—were selected for IWQI calculation based on their dominant influence on eutrophication and overall water‐quality variability, and the assignment criteria were refined accordingly (Table 3). The formula used to calculate the IWQI is as follows: IWQI = ∑n i=1 CiPi ∑n i=1 Pi (8) The Water quality is based on the IWQI scores. The range of Urgent rectification is IWQI < 10. The range of Red line is 10 ≤ IWQI ≤ 20. The range of Serious pollution is 20 < IWQI ≤ 40; Moderate pollution is 40 < IWQI ≤ 60. The range of Minor pollution is 60 < IWQI ≤ 80, The range of Good is IWQI > 80. 2.4 Method for analyzing the organization of microbial communities The microbial community structure was assessed using the identical methodology as described in the work conducted by Zhao et al. [31]. For microbial community analysis, two of the three 5 L duplicate water samples collected at each sampling site (total of 10 L) were pooled for DNA extraction, while the remaining 5 L duplicate sample was retained for other sup-

plementary tests. Water DNA was isolated from 10L freshwater samples using the E.Z.N.A. Soil DNA Kit (Omega, USA), following the instructions provided by the manufacturer. DNA quality and quantity were measured with a Qubit 2.0 (Life, USA) fluorometer. The V3-V4 region of the 16S rRNA gene is amplified using a forward primer and a reverse primer. The forward primer has a sequence of CCTACGGGNGGCWGCAG, whereas the reverse primer has a sequence of GAC- TACHVGGGTATCTAATCC. The PCR results were analyzed by electrophoresis in 1% (w/v) agarose gels in TBE buffer (Tris, boric acid, EDTA) that are stained with ethidium bromide (EB) and seen under UV light. 2.5 Evaluation of the ecotoxicity 2.5.1 Analysis of organic phosphorus in water and sediment samples. Organic phosphorus compounds were quantified by ultrasonic-assisted extraction followed by gas chromatography (GC). Briefly, 10 mL of water or 10 g of wet sediment was extracted with 20 mL of acetonitrile under ultrasonication (500 W, 30 min). After centrifugation (5 000 rpm, 15 min), the supernatant was filtered through a 0.22 μm membrane and stored at 4 °C until analysis. Samples (1 μL) were injected without split into an RT-1701 capillary column (30 m × 0.25 mm × 0.25 μm). The oven program was 40 °C (hold 0 min), ramp at 30 °C/min to 130 °C, then 5 °C/min to 250 °C (hold 10 min). Carrier gas was N₂ (1.0 mL/min), with H₂ as auxiliary gas; inlet and detector temperatures were set at 270 °C and 280 °C, respectively.

2.5.2 ECOSAR. An expert tool for precisely predicting the effects of chemicals on the environment and ecotoxicity is the ECOSAR program. It is especially advantageous in assessing the potential harm that chemicals might do to aquatic plants, fish, and invertebrates. The ECOSAR model is employed in this study to evaluate the short- and long-term Table 3. Normalized values and weights for IWQI. Water quality indicators Unit Weight (pi) Normalization factor (Ci) 100 90 80 70 60 50 40 30 20 10 0 TN mg/L 2 <0.2 <0.5 <1 <1.5 <2 <3 <5 <7 <10 ≤15 >15 NH3 – N mg/L 1 <0.15 <0.5 <1 <1.1 <1.2 <1.4 <1.6 <1.8 <2 ≤5 >5 TP mg/L 1 <0.02 <0.1 <0.2 <0.22 5 <0.25 <0.27 5 <0.3 <0.35 <0.4 ≤0.5 >0.5 https://doi.org/10.1371/journal.pone.0332712.t003