Due to the advantages such as good inactivation effect on pathogenic microorganisms and low usage cost, chlorinated disinfectants have been widely employed for disinfection in various aspects. However, the large amount of chlorinated disinfection by-products produced by chlorinated disinfectants have biological toxicity, and they will have toxic effects on aquatic organisms and pose potential threats to the aquatic ecosystem once entering aquatic environment. Therefore, the development of on-site and rapid detection methods for the toxicity of chlorinated disinfection by-products in water is of significance for ensuring the aquatic environment safety. As the main primary producers and energy converters in aquatic ecosystems, microalgae play an important role in maintaining the balance and stability of aquatic ecosystems and indicating the water environment quality. Additionally, the microalgae photosynthesis has a rapid response characteristic to the toxicity of pollutants in water. Based on this, fluorescence kinetics technique which could rapidly and non-destructively detect the photosynthetic status of living plants has good development prospects in rapid toxicity detection of chlorinated disinfection by-products in water. However, at present, it is still unclear which photosynthetic fluorescence parameter obtained based on fluorescence kinetics technique can serve as the optimal response indicator for rapidly and sensitively detecting the toxicity of chlorinated disinfection by-products. Therefore, we study the response characteristics of different photosynthetic fluorescence parameters to the toxicity of chlorinated disinfection by-products for determining the optimal response indicator with sensitive response characteristics to chlorinated disinfection by-products. This is of practical significance for the development of on-site and rapid detection methods for the toxicity of chlorinated disinfection by-products based on fluorescence kinetics technology.

We employ a common freshwater microalgae Chlorella pyrenoidosa as the test organism, and two typical toxic chlorinated disinfection by-products chloroacetic acid and trichloroacetonitrile as the research objects. Meanwhile, we first investigate the change rule of photosynthetic activity of Chlorella pyrenoidosa with incubation time and the influences of algal cell density and environmental temperature on the response sensitivity of Chlorella pyrenoidosa to the toxicity of two chlorinated disinfection by-products to determinate the optimal experimental conditions for the response of Chlorella pyrenoidosa to chlorinated disinfection by-products toxicity. The fluorescence kinetics method is adapted to study the response of four photosynthetic fluorescence parameters F_v/F_m, F_v/F_o, F_m/F_o, and PI_ABS to different mass concentrations of chloroacetic acid and trichloroacetonitrile, and then the dose-response relationships between each photosynthetic fluorescence parameter and each chlorinated disinfection by-product are constructed. On this basis, the response sensitivities of four photosynthetic fluorescence parameters to chlorinated disinfection by-products are compared in four aspects. The aspects include response performance to low mass concentration of chlorinated disinfection by-products, inhibition degree by equal mass concentration of chlorinated disinfection by-products, 10% effective mass concentration (EC₁₀), and 50% effective mass concentration (EC₅₀) values.

By studying the change of photosynthetic activity of Chlorella pyrenoidosa with incubation time, the results show that when Chlorella pyrenoidosa is cultured to the third to fourth days, all the four photosynthetic fluorescence parameters F_v/F_m, F_m/F_o, F_v/F_o, and PI_ABS are at a high level (Fig. 1), which indicates that Chlorella pyrenoidosa has the best photosynthetic activity at this time. Therefore, the best growth period of Chlorella pyrenoidosa for studying the response characteristics of different photosynthetic fluorescence parameters to the toxicity of chlorinated disinfection by-products is the third day to the fourth day during culture. The results of influences of algal cell density and environmental temperature on PI_ABS inhibition ratio of Chlorella pyrenoidosa exposed to chloroacetic acid and trichloroacetonitrile demonstrate that when the algal cell density is in the range of 0.5×10⁵-100×10⁵ cells·mL^-1, PI_ABS inhibition ratio is at a high level and tends to stabilize [Fig. 2 (a)]. Under the environmental temperature of 25 ℃, the inhibition ratio of PI_ABS is the maximum [Fig. 2 (b)]. Therefore, 0.5×10⁵-100×10⁵ cells·mL^-1 and 25 ℃ are the optimal algal cell density and the optimal environmental temperature for the response of Chlorella pyrenoidosa to chlorinated disinfection by-products toxicity respectively. In the optimal toxicity response conditions, when the exposure time is 2 h, all the four photosynthetic fluorescence parameters F_v/F_o, F_m/F_o, F_v/F_m, and PI_ABS values gradually decrease with the mass concentration increase in chloroacetic acid and trichloroacetonitrile (Figs. 3 and 4). When the mass concentrations of chloroacetic acid and trichloroacetonitrile are in the ranges from 5.06 to 36.80 mg·L^-1 and from 1.55 to 19.32 mg·L^-1 respectively, all the inhibition ratios of F_v/F_o, F_m/F_o, F_v/F_m, and PI_ABS show good Logistic curve relationships with the mass concentrations of chloroacetic acid and trichloroacetonitrile, and the adjustive correlation coefficients Radj2 are all greater than 0.993 (Figs. 5 and 6). These results indicate that all four photosynthetic fluorescence parameters could be adopted as response indicators for quantitative toxicity detection of chlorinated disinfection by-products. For low mass concentrations of chloroacetic acid and trichloroacetonitrile, PI_ABS has the most sensitive response performance compared to F_v/F_o, F_m/F_o, and F_v/F_m (Figs. 3 and 4). Meanwhile, by comparing the inhibition ratios of F_v/F_o, F_m/F_o, F_v/F_m, and PI_ABS by equal mass concentration chloroacetic acid and trichloroacetonitrile at the exposure time of 2 h, the inhibition degree order of the four photosynthetic fluorescence parameters is PI_ABS>F_v/F_o>F_m/F_o>F_v/F_m. This indicates that compared with the other three photosynthetic fluorescence parameters, PI_ABS is the most sensitive to the toxicity of chlorinated disinfection by-products at the same mass concentration. Additionally, the EC₁₀ and EC₅₀ values of chloroacetic acid and trichloroacetonitrile calculated according to F_v/F_o, F_m/F_o, F_v/F_m, and PI_ABS are further compared. The results show that the photosynthetic fluorescence parameters corresponding to the EC₁₀ and EC₅₀ values of chloroacetic acid and trichloroacetonitrile in descending order are F_v/F_m>F_m/F_o>F_v/F_o>PI_ABS (Table 2). This indicates that the sensitivity of the four photosynthetic fluorescence parameters to the toxicity of chloroacetic acid and trichloroacetonitrile is PI_ABS>F_v/F_o>F_m/F_o>F_v/F_m.

All the photosynthetic fluorescence parameters F_v/F_m, F_v/F_o, F_m/F_o, and PI_ABS of Chlorella pyrenoidosa have mass concentration-dependent response characteristics to two chlorinated disinfection by-products chloroacetic acid and trichloroacetonitrile. The four photosynthetic fluorescence parameters have good logistic curve dose-response relationships with the mass concentration of chloroacetic acid and trichloroacetonitrile, which could be utilized for quantitative toxicity detection of chlorinated disinfection by-products in water. In addition, among the four photosynthetic fluorescence parameters, PI_ABS exhibits the most sensitive response characteristics to chloroacetic acid and trichloroacetonitrile. Therefore, it is a suitable response indicator for rapidly and sensitively detecting the toxicity of chlorinated disinfection by-products. We provide a more suitable toxicity response parameter for the development of on-site and rapid detection methods and technologies for disinfection by-products toxicity based on fluorescence kinetics technique.