Potential health risks to disinfection workers from exposure to active substances in COVID-19 biocidal products.

The importance of disinfection has recently been emphasized due to the increasing risk of the spread of infections such as coronavirus disease-2019 (COVID-19). In addition, disinfection for preventing the spread of COVID-19 is highly recommended. The increased use of biocidal products raises concerns regarding the potential health risks from exposure among disinfection workers. This study aimed to assess these exposure and health risks using questionnaires targeting disinfection workers who were exposed to the active substances in biocidal products used for disinfection during the COVID-19 pandemic. A follow-up survey was conducted among 271 disinfection workers for 10 working days within two weeks, and exposure factors with reference to disinfection were evaluated through interview-administered questionnaires. An exposure algorithm was used to evaluate the exposure of disinfection workers during disinfection. The hazard index (HI) was calculated by dividing the inhalation concentration obtained using the exposure algorithm and the dermal dose according to occupational exposure limits (OEL). A sensitivity analysis was conducted to identify the exposure factors with the greatest impact on the inhalation and dermal exposure algorithms. A logistic regression analysis was performed to verify the relationship with health effects and sociodemographic and exposure characteristics. The average number of disinfections performed during 10 working days was 17.5 ± 12.3 times. The type of disinfection work was divided into 2806 cases of COVID-19 prevention and disinfection and 1956 cases of regular pesticide application to prevent and remove any pests. The HI was ≥1, indicating a potential health risk, with the use of ethanol (6.50E+00), quaternary ammonium compounds (QACs; 1.49E+01), and benzalkonium chloride (BKC; 1.73E+00). Dermal exposure was more hazardous than inhalation exposure for 6 of the 11 active substances in biocidal products. The weight fraction and exposure time were the factors that most significantly influenced the inhalation and dermal exposure algorithms in the sensitivity analysis. Higher exposure concentrations were more likely to affect health (AOR: 3.239, 95% CI: 1.155-9.082). This study provides valuable information regarding the exposure and risk of disinfection workers to 11 biocidal active substances included in common disinfectants. Our results suggest that the use of ethanol, BKC, and QACs has potential health risks to disinfection workers, with a higher possibility of negative health impacts with increasing exposure concentration.

Developing a Novel Read-Across Concept for Ecotoxicological Risk Assessment of Phosphate Chemicals: A Case Study.

This study introduces a novel concept approach for a read-across assessment, considering species sensitivity differences among phosphate chemicals within structurally similar compound groups. Twenty-five organic chemicals, with a log Kow of 5 or less, were categorized into three functional groups based on acetylcholinesterase (AChE) inhibition as a specific mode of action (MOA). The short-term aquatic toxicity data (LC50) for fish, crustaceans, and insects were collected from the U.S. EPA Ecotoxicology (ECOTOX) Knowledgebase. A geometric mean calculation method was applied for multiple toxic endpoints. Performance metrics for the new read-across concept, including correlation coefficient, bias, precision, and accuracy, were calculated. Overall, a slightly higher overestimation (49.2%) than underestimation (48.4%) in toxicity predictions was observed in two case studies. In Case study I, a strong positive correlation (r = 0.93) between the predicted and known toxicity values of target chemicals was observed, while in Case study II, with limited information on species and their ecotoxicity, showed a moderate correlation (r = 0.75). Overall, the bias and precision for Case study I were 0.32 ± 0.01, while Case study II showed 0.65 ± 0.06; however, the relative bias (%) increased from 37.65% (Case study I) to 91.94% (Case study II). Bland-Altman plots highlight the mean differences of 1.33 (Case study I) and 1.24 (Case study II), respectively. The new read-across concept, focusing on AChE inhibition and structural similarity, demonstrated good reliability, applicability, and accuracy with minimal bias. Future studies are needed to evaluate various types of chemical substances, diverse modes of action, functional groups, toxic endpoints, and test species to ensure overall comprehensiveness and robustness in toxicity predictions.

Transcriptome analysis reveals differences in developmental neurotoxicity mechanism of methyl-, ethyl-, and propyl- parabens in zebrafish embryos.

Studies on the comparison of developmental (neuro) toxicity of parabens are currently limited, and unharmonized concentrations between phenotypic observations and transcriptome analysis hamper the understanding of their differential molecular mechanisms. Thus, developmental toxicity testing was conducted herein using the commonly used methyl- (MtP), ethyl- (EtP), and propyl-parabens (PrP) in zebrafish embryos. With a benchmark dose of 5%, embryonic-mortality-based point-of-departure (M-POD) values of the three parabens were determined, and changes in locomotor behavior were evaluated at concentrations of 0, M-POD/50, M-POD/10, and M-POD, where transcriptome analysis was conducted to explore the underlying neurotoxicity mechanism. Higher long-chained parabens were more toxic than short-chained parabens, as determined by the M-POD values of 154.1, 72.6, and 24.2 µM for MtP, EtP, and PrP, respectively. Meanwhile, exposure to EtP resulted in hyperactivity, whereas no behavioral effect was observed with MtP and PrP. Transcriptome analysis revealed that abnormal behaviors in the EtP-exposed group were associated with distinctly enriched pathways in signaling, transport, calcium ion binding, and metal binding. In contrast, exposure to MtP and PrP mainly disrupted membranes and transmembranes, which are closely linked to abnormal embryonic development rather than neurobehavioral changes. According to the changes in the expressions of signature mRNAs, tentative transcriptome-based POD values for each paraben were determined as MtP (2.68 µM), EtP (3.85 µM), and PrP (1.4 µM). This suggests that different molecular perturbations initiated at similar concentrations determined the extent and toxicity outcome differently. Our findings provide insight into better understanding the differential developmental neurotoxicity mechanisms of parabens.

Effects of the chorion on the developmental toxicity of organophosphate esters in zebrafish embryos.

Many toxicological studies have utilized zebrafish embryos to investigate the developmental toxicity of organophosphate esters (OPEs). However, in respect of the presence or absence of the chorion, a consistent experimental methodology has yet to be developed. In this study, we used a fixed exposure scheme to compare the developmental toxicities of six major OPEs in chorionated and dechorionated zebrafish embryos. Removal of the chorion increased sensitivity to OPEs: we found higher incidence of mortality and malformation in dechorionated embryos. In a behavioral assay, the locomotive activity of zebrafish larvae was consistently inhibited by OPEs except tris (1-chloropropyl) phosphate regardless of chorion presence. However, at the molecular level, the expression of ZHE1 and mmp9 was affected by the presence of the chorion in zebrafish embryos exposed to tributyl phosphate and triphenyl phosphate (TPHP), respectively. Furthermore, in zebrafish embryos exposed to TPHP, the increased expression of miR-137 and miR-141 was abolished by the presence of the chorion. Our results demonstrate for the first time that the presence of the chorion influences phenotypic morbidity, organismal behavior, and gene expression in zebrafish embryos exposed to chemicals; thus, we suggest that dechorionation is desirable for exploring the toxicity mechanisms that underlie effects of chemical exposure.

Predicting PBT and CMR properties of substances of very high concern (SVHCs) using QSAR models, and application for K-REACH.

Quantitative structure-activity relationship (QSAR) models have been applied to predict a variety of toxicity endpoints. Their performance needs to be validated, in a variety of cases, to increase their applicability to chemical regulation. Using the data set of substances of very high concern (SVHCs), the performance of QSAR models were evaluated to predict the persistence and bioaccumulation of PBT, and the carcinogenicity and mutagenicity of CMR. BIOWIN and Toxtree showed higher sensitivity than other QSAR models - the former for persistence and bioaccumulation, the latter for carcinogenicity. In terms of mutagenicity, the sensitivities of QSAR models were underestimated, Toxtree was more accurate and specific than lazy structure-activity relationships (LAZARs) and Computer Assisted Evaluation of industrial chemical Substances According to Regulations (CAESAR). Using the weight of evidence (WoE) approach, which integrates results of individual QSAR models, enhanced the sensitivity of each toxicity endpoint. On the basis of obtained results, in particular the prediction of persistence and bioaccumulation by KOWWIN, a conservative criterion is recommended of log Kow greater than 4.5 in K-REACH, without an upper limit. This study suggests that reliable production of toxicity data by QSAR models is facilitated by a better understanding of the performance of these models.

Validation of a biotic ligand model on site-specific copper toxicity to Daphnia magna in the Yeongsan River, Korea.

The objective of this study was to determine whether the water effect ratio (WER) or biotic ligand model (BLM) could be applied to efficiently develop water quality criteria (WQC) in Korea. Samples were collected from 12 specific sites along the Yeongsan River (YSR), Korea, including two sewage treatment plants and one estuary lake. A copper toxicity test using Daphnia magna was performed to determine the WER and to compare to the BLM prediction. The results of the WER from YSR samples also indicated significantly different copper toxicities in all sites. The model-based predictions showed that effluent and estuary waters had significantly different properties in regard to their ability to be used to investigate water characteristics and copper toxicity. It was supposed that the slight water characteristics changes, such as pH, DOC, hardness, conductivity, among others, influence copper toxicity, and these variable effects on copper toxicity interacted with the water composition. The 38% prediction was outside of the validation range by a factor of two in all sites, showing a poor predictive ability, especially in STPs and streams adjacent to the estuary, while the measured toxicity was more stable. The samples that ranged from pH 7.3-7.7 generated stable predictions, while other samples, including those with lower and the higher pH values, led to more unstable predictions. The results also showed that the toxicity of Cu in sample waters to D. magna was closely proportional to the amounts of acidity, including the carboxylic and phenolic groups, as well as the DOC concentrations. Consequently, the acceptable prediction of metal toxicity in various water samples needs the site-specific results considering the water characteristics such as pH and DOC properties particularly in STPs and estuary regions.

Application of toxicity identification evaluation procedure to toxic industrial effluent in South Korea.

Toxicity identification evaluation (TIE) was applied to the effluent from a pharmaceutical industrial complex, following the US EPA TIE guidelines. The whole effluent toxicity (WET) test found toxicity greater than 16toxic units (TU) in the effluent. Dissolved non-polar organic compounds were identified as the major contributor to the observed toxicity in the TIE manipulations in phases I and II. Among the 48 organic compounds identified, three compounds (i.e., acetophenone, benzoimide, and benzothiazole) were related to the pharmaceutical production procedure; however, no contribution to toxicity was predicted in the compounds. The results of the ECOSAR model, which predicts toxicity, indicated that the alkane compounds caused significant toxicity in the effluent. The toxicity test and heavy metal analysis, which used IC and ICP/MS, identified that particulate and heavy metals, such as Cu and Zn, contributed to the remaining toxicity, except dissolved organics. The results showed the applicability of the TIE method for predicting regional effluents produced by the industrial pharmaceutical complex in this study. Although the location was assumed to be affected by discharge of pharmaceutical related compounds in the river, no correlations were observed in the study. Based on the results, advanced treatment processes, such as activated carbon adsorption, are recommended for the wastewater treatment process in this location.

Human health risks associated with dietary exposure to persistent organic pollutants (POPs) in river water in Korea.

Because of their persistence, bioaccumulation tendency, and toxicity, organochlorine pesticides (OCPs) were banned several decades ago. However, they are still detected in natural water and pose a serious risk to human health and the environment. In the present study, seven OCPs were measured in river water collected from 28 sampling sites between 2010 and 2011 in the Yeongsan (YS) and Seomjin (SJ) Rivers, Korea. Gas chromatography/high-resolution mass spectrometry (HRMS) was used for the chemical analysis. The potential health risks of consuming OCPs in fish were assessed in the monitoring region (YS and SJ River basins) by measuring the residual level of OCPs. The human health risks were characterized by considering both the exposure level and the corresponding cancer slope factors; we incorporated both deterministic and probabilistic approaches. The risks were calculated using the probabilistic Maximum Likelihood Estimation (MLE) method. Risk uncertainty and sensitivity were calculated by performing a Monte Carlo analysis. Of the six OCPs, dieldrin, heptachlor epoxide, and hexachlorobenzene exceeded the acceptable risk level in the heavy and light fish consumer groups. The cancer risks associated with these OCPs were 2.99×10(-6), 5.81×10(-6) and 3.72×10(-6), respectively, using the deterministic approach and 1.64×10(-5), 3.36×10(-2) and 5.52×10(-5), respectively, at the 95th percentile using the probabilistic approach. Because there is a high cancer risk associated with these three OCPs, individuals who consume large amounts of freshwater fish may be subject to a high risk of cancer. However, the calculations may have over- or underestimated the risk because of the uncertainty of the environmental concentration and fish intake rate or the use of left-censored monitoring data.

Occurrence of estrogenic chemicals in South Korean surface waters and municipal wastewaters.

Broad scale monitoring of estrogenic compounds was performed at 19 sampling points throughout the Yeongsan and Seomjin river basins and 5 wastewater treatment plants (WWTPs) adjacent to the Gwangju area, Korea, from December 2005 to August 2007. The concentrations of estrogenic compounds, including estrone (E1), 17ß-estradiol (E2), 17α-ethynylestradiol (EE2), bisphenol-A, nonylphenol (NP) and 4-octylphenol (OP), in the samples was measured with gas chromatography/mass spectrometry (GC-MS). In addition, the estrogenic activities throughout the river were investigated using the E-screen assay. Of the six estrogenic chemicals, NP (114.6-336.1 ng L(-1)) and EE2 (0.23-1.90 ng L(-1)) were detected at the highest and lowest levels, respectively in both the river waters and the WWTP effluents. Bisphenol-A showed the largest concentration range, from 7.5 to 335 ng L(-1). The concentrations of E1, E2 and octylphenol ranges were 3.6-69.1, 1.2-10.7, and 2.2-16.9 ng L(-1), respectively. According to the calculated estradiol equivalent concentration (EEQ); however, no estrogenic contribution was observed due to the phenolic compounds in the river waters and effluents. E1 and E2 dominated in both the river water and effluent samples, with contributions to the calculated EEQ of over 79 and 77%, respectively. Conversely, EE2 was rarely detected in the river waters (21%) and effluents (0%). The largest contribution of EE2 to the calculated EEQ was 21% in the river water at S-7. The levels of E1, E2, and EE2 were remarkably decreased in the effluents, indicating that the 5 WWTPs did not contribute to the estrogenic effect of the receiving streams. Overall, the WWTPs did not contributed to the estrogenic activity of the receiving waters, but the livestock industry or wildlife may play an important role in the estrogenic contribution to river water.

Bioaccumulation of perfluorochemicals in Pacific oyster under different salinity gradients.

Despite the reports of widespread occurrence of perfluorinated compounds (PFCs) in estuarine and coastal waters and open seas, little is known on the effect of salinity on bioaccumulation. In this study, effects of salinity on bioaccumulation of PFCs in Pacific oysters (Crassostrea gigas) were investigated. Furthermore, partitioning of PFCs between water and particles (oysters' food) was examined at different salinities. The distribution coefficients (K(d); partitioning between water and particles) for selected PFCs, that is, PFOS, PFOA, PFDA, and PFUnDA, increased by 2.1- to 2.7-fold with the increase in water salinity from 10 to 34 psu, suggesting "salting-out" effect, and the salting constant (delta) was estimated to range from 0.80 to 1.11. The nonlinear regression analysis of bioaccumulation suggested increase in aqueous and dietary uptake rates (K(w) and K(f)), with the increase in salinity, which resulted in elevated bioaccumulation, although the depuration rates (K(e)) also increased. The relative abundance of long carbon chain length PFCs (i.e., PFDA and PFUnDA) increased as salinity increased, while the proportion of PFOS and PFOA decreased, which is explained by the positive relationship between delta and carbon chain length. The contribution of diet to bioaccumulation in oysters ranged from 18 to 92%. Overall, salinity not only affected the chemistry of PFCs, but also the physiology of oysters, contributing to sorption and bioaccumulation of perfluorochemicals in oysters.

Estrogenic chemicals and estrogenicity in river waters of South Korea and seven Asian countries.

The effects of treatment processes on estrogenicity were evaluated by examining estradiol equivalent (EEQ) concentrations in influents and effluents of sewage treatment plants (STPs) located along Yeongsan and Seomjin rivers in Korea. The occurrence and distribution of estrogenic chemicals were also estimated for surface water in Korea and compared with seven other Asian countries including Laos, Cambodia, Vietnam, China, Indonesia, Thailand and Malaysia. Target compounds were nonylphenol (NP), octylphenol (OP), bisphenol A (BPA), estrone (E1), 17beta-estradiol (E2), 17alpha-ethynylestradiol (EE2) and genistein (Gen). Water samples were pretreated and analyzed by liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). The results showed that the treatment processes of Korean STPs were sufficient to reduce the estrogenic activity of municipal wastewater. The concentrations of phenolic xenoestrogens (i.e., NP, OP and BPA) in samples of Yeongsan and Seomjin rivers were smaller than those reported by previous studies in Korea. In most samples taken from the seven Asian countries, the presence of E2 and EE2 was a major contributor toward estrogenic activity. The EEQ concentrations in surface water samples of the seven Asian countries were at a higher level in comparison to that reported in European countries, America and Japan. However, further studies with more sampling frequencies and sampling areas should be carried out for better evaluation of the occurrence and distribution of estrogenic compounds in these Asian countries.

Sorption of estrogens onto different fractions of sediment and its effect on vitellogenin expression in male Japanese medaka.

This study investigated the sorption capacity of estrogenic compounds-such as estrone (E1), 17beta-estradiol (E2), and 17alpha-ethynylestradiol (EE2)-of different sediment particle fractions. Two-sized fractions of sediment were used in the experiments, with a particle size <1 microm (mostly from 450 to 800 nm) and >1 microm up to 50 microm. Sorption kinetics were followed using a two-step reaction in which the major amount of chemicals was sorbed rapidly within minutes and then gradually increased until equilibrium was reached after 48 h. The sorption capacity of the fine particle fraction (particle size <1 microm) was shown to be significantly higher than that of the large fraction (1 microm < particle size < 50 microm). The sorption kinetics and isotherm were adequately predicted by using a pseudo second-order model and the Freundlich equation, respectively. Total organic carbon (TOC) content and surface area of particle fractions were also measured. Although the effects of TOC on the sorption of estrogens could not be verified, a higher surface area of fine particle fractions may significantly increase sorption capacity to target compounds. Sorption of estrogens onto sediment particles could be used to explain the differences of estrogenic activity of E2 spiked into different size fractions of particle suspensions.

Comparative whole effluent toxicity assessment of wastewater treatment plant effluents using Daphnia magna.

An approach to compare the toxicities employing the whole effluent toxicity (WET) test, using Daphnia magna and chemical analysis with GC/MS and ICP/MS, was conducted to the nine South Korean wastewater treatment plants (WWTPs). From the chemical analysis and bioassay experiments, heavy metals (i.e., Cu and Zn) were found to be the major compounds causing toxic effects toward D. magna. In the whole effluent toxicity (WET) tests using D. magna, toxicities were observed in 34% of the effluent samples. However, the biological toxic unit (TU) value showed a non-toxic response (i.e., 0 TU) in many samples despite the response indicated by the chemical TU values. This may be due to the species sensitivity, environmental parameters, mixture effects, and limitation of the chemical analyses.

The effect of suspended particles coated by humic acid on the toxicity of pharmaceuticals, estrogens, and phenolic compounds.

The sorption characteristics of 10 organic chemicals, categorized as pharmaceuticals, estrogens and phenols, onto synthetic suspended particle (i.e., alumina) coated with humic acid were investigated according to their octanol-water partition coefficient (K(ow)). Chemical analyses were performed with gas chromatography and mass spectrometry (GC/MS) and high performance liquid chromatography (HPLC). The effects of particles on the toxicity reduction were evaluated using bioassay tests, using Daphnia magna and Vibrio fisheri for phenols and pharmaceuticals, and the human breast cancer cell MCF-7 for estrogens. Sorption studies revealed that 22 and 38% of octylphenol and pentachlorophenol, respectively, were removed by suspended particle, whereas 2,4-dichlorophenol was not removed, which was directly proportional to the logK(ow) value. Similar to the sorption tests, suspended particles significantly reduced the acute toxicities of octylphenol and pentachlorophenol to D. magna and V. fisheri (p<0.01), but there was no significant difference in the toxicity of 2,4-dichlorophenol to D. magna (p=0.8374). Pharmaceuticals, such as ibuprofen, gemfibrozil and tolfenamic acid, showed no discernible sorption to the suspended particle, with the exception of diclofenac, which revealed 11% sorption. For estrogens, such as estrone, 17beta-estradiol and 17alpha-ethynylestradiol, the results indicated no reduction in the sorption test. This may be attributed to the polar interaction by functional groups in sorption between pharmaceuticals and estrogens and suspended particles. In the bioassays, presence of suspended particles did not significantly modify the toxicity of pharmaceuticals (regardless of their K(ow) values) to D. magna, V. fisheri or E-screen.

Effect of copper complexation on the estrogenic activities of endocrine-disrupting compounds using E-screen bioassay.

The complexation reaction between Cu and four targeted endocrine-disrupting compounds (EDCs)-17beta-estradiol (E2), 17alpha-ethynylestradiol (EE2), 4-octylphenol (OP), and bisphenol A (BPA)-was studied using the fluorescence quenching method, with the changed EDC forms evaluated for their estrogenic activities using an E-screen assay. The results of the E-screen showed no estrogenic activity of Cu, whereas results with the EDCs indicated high cell proliferation (decreasing order of induction: E2, EE2, OP, and BPA). The results of the fluorescence quenching response and FTIR (Fourier transformation infrared) strongly support the formation of Cu complexes on the -OH functional group of the targeted EDCs when Cu was spiked at concentrations between 10(-7) and 10(-4) M. The degree of complexation was in the following order: E2, EE2, OP, and BPA. The E-screen result for three of the EDCs, with the exception of BPA, was similar when estimating the effects of Cu on the estrogenic activities of the EDCs, which were significantly reduced at alpha = 0.05 with increasing Cu concentration. Consequently, the results of the complexation reaction and E-screen assay with the Cu-EDC mixtures indicated that Cu plays a significant role in the assessment of the estrogenic activities of EDCs in natural waters.

Ecological health assessments based on whole effluent toxicity tests and the index of biological integrity in temperate streams influenced by wastewater treatment plant effluents.

Whole effluent toxicity (WET) tests and ecosystem health assessments, based on test guidelines of U.S. Environmental Protection Agency (U.S. EPA) and index of biological integrity (IBI), were conducted on various streams located in Youngsan River watershed, Korea. The WET tests showed that about 33 and 82% of wastewater treatment plants (WWTPs) exhibited significant toxicity to Daphnia magna and Selenastrum capricornutum, respectively. Small WWTPs with low discharge volumes contributed less than 1% to the total stream toxicity. Fish community compositions and trophic guild analysis showed that the diversity index was greater in the control than in impacted streams, and the proportion of omnivore species was less in the control. Also, ecosystem health assessments, based on the IBI, showed distinct differences between the control and impacted sites of WWTPs. Model values of the IBI, based on 12 stream data sets, averaged 28, which is judged as a fair to poor condition according to the U.S. EPA criteria. The mean IBI in the control sites was 42, indicating good stream condition, whereas the impacted sites was scored 21, indicating poor condition. Overall, WET tests and ecosystem health assessments suggested the WWTP effluents had evident toxic effects on the biota, and impacted the species compositions and trophic guilds, resulting in degradation of the stream ecosystem health.

Whole effluent toxicity (WET) tests on wastewater treatment plants with Daphnia magna and Selenastrum capricornutum.

Whole effluent toxicity (WET) tests, with Daphnia magna and Selenastrum capricornutum, were introduced to evaluate the biological toxicities of effluents from the wastewater treatment plants (WWTPs) in Korea. In WET tests of WWTPs effluents, 33.3% (33/99) for D. magna and 92.6% (75/81) for S. capricornutum revealed greater than 1 toxic unit (TU), even though all the treatment plants investigated were operating in compliance with the regulations, as assessed using conventional monitoring methods (i.e., BOD and total concentration of N or P, etc). There were only minor differences in toxicities according to the types of influents (municipal and agro-industrial) in all treatment plants. However, the effluents treated by an activated sludge treatment process were found to exhibit significantly lower toxicity than those treated by rotating biological contactor (RBC) and extended aeration processes. The seasonal variations in the toxicity were lower in the summer compared to winter, which may have been due to the rainfall received to the sewage intake system during the former period. The impact of WET on river water was also investigated based on the discharge volume. At sites A and B, the total impact of toxicity to stream and river waters was observed to be 70.9% and 90.4% for D. magna and S. capricornutum, respectively. The other four small treatment plants (sites F, G, H and I), with relative discharging volumes between 0.001 and 0.002, contribute less than 1% to the total toxicity.

Estimating the combined toxicity by two-step prediction model on the complicated chemical mixtures from wastewater treatment plant effluents.

The toxicities of chemical mixtures containing 10 compounds, detected in wastewater treatment plant (WWTP) effluents, were investigated using Daphnia magna in a two-step prediction (TSP) model. The 10 chemicals determined by gas chromatography/ mass spectrometry in WWTP effluents included three groups: Three acetylcholinesterase inhibitors, six narcosis inhibitors, and one seedling root inhibitor. In the first step, a concentration addition (CA) model was used to predict the mixture toxicities for the three component groups with similar modes of action; in the second step, an independent action (IA) model was used for the newly developed concentration-response curves from the three CA predictions. The CA predictions did not show a statistically significant difference from the observed results with respect to the three groups of chemicals, whereas the IA model did not conform to the experimental results. Therefore, the concentration-response curves obtained from the mixture toxicity tests in each group was considered as a single curve and applied in the next step of the mixture toxicity prediction. However, the observed toxicity of the 10-chemical mixture showed large differences from the results of the IA and CA model predictions, whereas the TSP model predicted the toxicity well and with statistical significance (p = 0.0501, n = 17). This suggests that the TSP model would provide a valid prediction for a randomly selected chemical mixture having various modes of action if the concentration-response function for an individual component is obtained.