key: cord-0871728-ra9y4jnb authors: Won, Eun-Ji; Byeon, Eunjin; Lee, Young Hwan; Jeong, Haksoo; Lee, Yoseop; Kim, Min-Sub; Jo, Hyeong-Wook; Moon, Joon-Kwan; Wang, Minghua; Lee, Jae-Seong; Shin, Kyung-Hoon title: Molecular evidence for suppression of swimming behavior and reproduction in the estuarine rotifer Brachionus koreanus in response to COVID-19 disinfectants date: 2022-02-08 journal: Mar Pollut Bull DOI: 10.1016/j.marpolbul.2022.113396 sha: f48be9ffa6ff519af4f19a1bc6f3873085df6f60 doc_id: 871728 cord_uid: ra9y4jnb The increased use of disinfectants due to the spread of the novel coronavirus infection (e.g. COVID-19) has caused burden in the environment but knowledge on its ecotoxicological impact on the estuary environment is limited. Here we report in vivo and molecular endpoints that we used to assess the effects of chloroxylenol (PCMX) and benzalkonium chloride (BAC), which are ingredients in liquid handwash, dish soap products, and sanitizers used by consumers and healthcare workers on the estuarine rotifer Brachionus koreanus. PCMX and BAC significantly affected the life table parameters of B. koreanus. These chemicals modulated the activities of antioxidant enzymes such as superoxide dismutase and catalase and increased reactive oxygen species even at low concentrations. Also, PCMX and BAC caused alterations in the swimming speed and rotation rate of B. koreanus. Furthermore, an RNA-seq-based ingenuity pathway analysis showed that PCMX affected several signaling pathways, allowing us to predict that a low concentration of PCMX will have deleterious effects on B. koreanus. The neurotoxic and mitochondrial dysfunction event scenario induced by PCMX reflects the underlying molecular mechanisms by which PCMX produces outcomes deleterious to aquatic organisms. When a novel coronavirus (SARS-CoV-2) caused the COVID-19 outbreak, disinfection of surfaces was recommended as one of the most effective ways to prevent infection (Ijad et al., 2021; Nowak et al., 2021) . However, the chemicals recommended for hard-surface disinfectants and hand sanitizers for COVID-19 are classified as biocide and could have adverse effects, especially when high demand for them causes wide spread release (Merchel Piovesan Pereira and Tagkopoulos, 2019; Dev Kumar et al., 2020; Tan et al., 2021) . In fact, the increase in disinfectant usage caused by the COVID-19 pandemic has created a large effluent of these chemicals into aquatic environments, and inappropriate use leads to severe environmental problems caused by the chemicals themselves or their byproducts (Yadav et al., 2020) . Chloroxylenol (para-chloro-meta-xylenol, PCMX), commonly known as Dettol, is a chemical currently recommended to inactivate viruses, including COVID-19. It is also used to remove bacteria, algae, and fungi (Dev Kumar et al., 2020) and commonly used in households, hospitals, and other institutions to sanitize bathrooms, laundry equipment, bedding, and pet living quarters (EPA, 1994; Dev Kumar et al., 2020) . In particular, it has long been used because of its low toxicity to target vertebrates such as humans, fish, and birds (EPA, 1994) . Benzalkonium chloride (BAC) is another widely used chemical with broad-spectrum antimicrobial characteristics against bacteria, fungi, and viruses (Elersek et al., 2018; Merchel Piovesan Pereira and Tagkopoulos, 2019) . Aquatic ecosystems are a major target of increased disinfectant impacts caused by COVID-19, and non-target organisms are likely to be affected (Zhang et al., 2020) . In fact, these disinfectants were measured in sediment (Li and Brownawell, 2010; Khan et al., 2017) and found to persist in soil and estuarine environments. However, no studies have been conducted on the adverse effects that these disinfectants have on aquatic organisms. Furthermore, information is lacking about the genotoxicity and carcinogenicity of these chemicals, even though they show minimal systemic toxicity in vertebrates (EPA, 1994) . To date, the global expression of defensive genes by aquatic organisms in response to disinfectants has been reported in only a few species. For example, in the rainbow trout Oncorhynchus mykiss, toxic results were found in response to three general disinfectants (triclosan, PCMX, and methylisothiazolinone) (Capkin et al., 2017) , demonstrating that these disinfectants cause DNA damage to the transcriptional regulation of superoxide dismutase (SOD), glutathione peroxidase, glutathione Stransferase, heat shock protein, catalase (CAT), and heat shock 70 cognate genes. Furthermore, in the Japanese medaka Oryzias latipes, significant upregulation of the vitellogenin gene was shown in juvenile fish following exposure to BAC . However, transcriptional profiling studies in response to disinfectants (to evaluate ecophysiological effects and uncover toxic mechanisms) have not been reported, although molecular studies have been deemed a promising approach to molecular ecotoxicological studies (Anderson et al., 1994; Connon et al., 2012) . Brachionus spp. are rotifers, fundamental microzooplankton in the ecological structure of aquatic ecosystems that make energy available for other trophic levels (Dahms et al., 2011) . They link the food web from primary producers to large consumers and also participate in the microbial loop by consuming bacteria (Turner and Tester, 1992; Yúfera, 2002) . These organisms are thus suitable species for conducting life history research in a short time frame (Snell and Janssen, 1995; Dahms et al., 2011) . In ecotoxicological studies, the advantages of using these organisms to assess adverse effects have been fully addressed for metals, pharmaceuticals, and persistent organic pollutants due to their short life cycle (~1 day) and ease of maintenance in the laboratory (Dahms et al., 2011; Lee et al., 2020; Li et al., 2020) . In this paper, several in vivo endpoints of the estuarine rotifer Brachionus koreanus were measured to evaluate the ecotoxicological and ecophysiological effects of two disinfectants, PCMX and BAC. We focused on the defensive role of antioxidant enzymes in response to PCMX and BAC. In addition, to evaluate the relationship between molecular mechanisms and COVID19 disinfectant-induced adverse effects on behavior and reproduction, an ingenuity pathway analysis (IPA), a powerful method using web-based software for data analysis in genomics, was conducted to examine the signaling pathways under PCMX exposure. A population of the estuarine monogonont rotifer B. koreanus has been stably maintained at the Department of Biological Sciences in Sungkyunkwan University in South Korea for several years. This species was originally collected at Uljin (36 • 58 ′ 43.01 ′′ N, 129 • 24 ′ 28.40 ′′ E) and kindly provided by Prof. Heum Gi Park, Gangneung-Wonju National University, South Korea. The species was identified using both morphological information and the CO1 and ITS1 genes (Hwang et al., 2013) . All chemicals, including 4-chloro-3,5-dimethylphenol (PCMX), alkylbenzyldimethyl ammonium chloride (BAC), and dimethyl sulfoxide (DMSO), were purchased from Sigma-Aldrich (St. Louis, MO, USA) at HPLC grade (>99%). The analytical kits for the SOD (EC 1.15.1.1) and CAT (E.C.1.11.1.6) assays were purchased from Abcam (ab65354, Cambridge, UK) and Sigma-Aldrich (CAS 9001-05-2), respectively. All reagents for molecular studies and chemical exposure were of ultrapure grade for molecular biology and analytical grade. : lethal concentrations and life table parameters B. koreanus were exposed to PCMX and BAC to measure the median lethal concentrations (LC 50 -24 h) with waterborne exposure. Stock solutions of PCMX and BAC (conc. 100 mg PCMX mL − 1 and 20 mg BAC mL − 1 ) were prepared using DMSO and distilled water, respectively, because of their molecular weight and water solubility (Table S2 ). The final concentration of DMSO was 0.001% in each PCMX exposure group, the lowest concentration that showed no adverse effects in the preliminary study. For the test organisms, 2 h post-hatching individuals (n = 10) were used in biological triplicates, and mortality was measured after 24 h of exposure following the reported procedures for rotifers (Snell and Persoone, 1989) . The effects of PCMX (0, 1, 2, 5, and 10 mg L − 1 ) and BAC (0, 0.03, 0.06, 0.15, and 0.3 mg L − 1 ) on the population growth, reproduction, and lifespan of B. koreanus were measured as shown in Lee et al. (2018) and Kang et al. (2021) . Briefly, population growth of B. koreanus following exposure to PCMX and BAC was measured by counting the number of individuals produced by ten individuals for 7 days. Reproduction was measured by counting the number of neonates produced by one adult for 6 days. Lifespan indicates the average number of days from hatching to death. The individuals used for the lifespan study were selected from stable individuals hatched simultaneously. The details for measuring the life table parameters are presented in Text S1. The dissolved concentrations of PCMX and BAC were analyzed to validate the exposure concentrations. The chemical analyses of PCMX and BAC were conducted using liquid chromatography (LC) and LCmass spectrometry (MS)/MS, respectively and the detailed conditions are described in Text S2. The PCMX and BAC that accumulated in B. koreanus were also analyzed. For this step, a large-volume exposure test was conducted using approximately 50,000 B. koreanus individuals. In brief, 600 mL of test solution was prepared for exposure, and the B. koreanus were dampdried at 24 h post-exposure. For analysis, biological samples were sonicated in 1 mL of methanol (40 • C, 30 min). Then, the samples were filtered with an appropriate filter and injected into the LC system (Text S2). All tests were conducted in triplicate. The intracellular reactive oxygen species (ROS) levels were measured using 2 ′ ,7 ′ -dichlorodihydrofluorescein diacetate (H 2 DCFDA) as described in Jeong et al. (2017) . The experimental groups were sampled 24 h after exposure to PCMX and BAC at the NOEC level. Additionally, N-acetyl-L-cysteine (NAC), a ROS scavenger, was administered to confirm scavenging of the ROS generated in response to PCMX and BAC. Approximately 5000 individuals were used to analyze antioxidant enzymatic activity, and ten individuals were used for microscopic observation. Fluorescent images of the organisms were observed using a fluorescence microscope (Olympus IX71, Olympus Corporation, Tokyo, Japan). The enzymatic activities of SOD and CAT were measured using cellular activity assay kits according to the manufacturers' protocols. The enzymatic activities were normalized with proteins as explained in the manufacturers' protocols. Protein quantitation followed the Bradford assay as described in Park et al. (2020) . Swimming behavior (swimming speed and rotation rate) were measured with a minor modification of the procedures of Garaventa et al. (2010) . The swimming pattern was recorded under a stereomicroscope (M205A, Leica Microsystem Ltd., Wetzlar, Germany), and alterations in swimming speed and rotation rate were compared with the control. Briefly, the movement was recorded using LAS software ver. 4.3 (https://www.leica-microsystems.com; M205A, Leica Microsystems Ltd.) after placing rotifers onto slide glass and covering them with a cover glass; movies were acquired in AVI file format (.avi). Then, the recorded videos were processed using ImageJ software (https://imagej. nih.gov/ij/). A binary 8-bit image converted from the colored video was used to determine the boundary between the rotifers and the background based on a user-defined threshold value. To measure the movement of 2D tracks of the rotifer B. koreanus in response to NOEC levels of PCMX and BAC, the particle tracking plugin MTrack2 was used as described in Lee et al. (2020) . In brief, MTrack2 (https://imagej.nih. gov/ij/plugins/multitracker.html) identifies objects based on the defined particle size and determines which objects in successive frames are closest together. The traveled distance was calculated using the x and y coordinates of successive frames and then divided by the total play time of the video to calculate movement (μm/s). To examine molecular signaling pathway changes in B. koreanus in response to PCMX, we chose the NOEC level and a lower concentration that produced significant adverse effects on the lifespan parameters of B. koreanus. First, RNA-seq was conducted using samples treated with PCMX at concentrations of 1, 5, and 10 mg L − 1 , which was lower than the NOEC-24 h. For this analysis, RNA was extracted in triplicate from PCMX-exposed B. koreanus and the control and converted to complementary DNA (Text S3). Then, sequencing libraries were constructed. Sequencing was conducted using an Illumina Novaseq 6000 at DNA LINK (Seoul, South Korea). The paired-end raw data were manufactured, normalized, and trimmed by Trimomatic v0.38. To compare gene expression, the RNA-seq data were analyzed by the new tuxedo protocol (Pertea et al., 2016) . The cleaned RNA-seq data were mapped onto the reference genome by HISAT v2.2.1. From the mapped files, fragments were counted on the gene-level with annotated genome files using Stringtie v2.1.4, and estimated expressions were calculated and normalized as Fragments Per Kilobase Million and Transcripts Per Kilobase Million (TPM) values. To check the normalized TPM value calculation and variation of samples within replicates and within groups, a principal component analysis (PCA) was used and visualized as a plot. Using the counted data, the differential analysis proceeded with the edgeR method using the TCC package in R (Sun et al., 2013) . Significant fold change differences (absolute log[fold change] > 1, FDR < 0.05) between the control group and the PCMX treatment groups (1, 5, and 10 mg L − 1 ) were counted as differentially expressed genes (DEGs), and those results were visualized as volcano plots with logFC and logFDR axes. To acquire detailed information about the functions and pathways changed by the DEGs, we conducted gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. We used functional annotation information about the B. koreanus genome and did a BLAST search in the Swiss-Prot database with high correctness (e-value <1e − 10 ). The enriched GO term and KEGG pathway information was analyzed using the DAVID v6.8 functional annotation analysis tool (Huang et al., 2009) . Moreover, from the enriched biological processes identified using GO terms and KEGG pathways, we filtered significant pathways at Fisher's P < 0.05. The IPA web-based software (Ingenuity Systems, Redwood City, CA, USA) was used to visualize the biological functions of genes and molecules and find the canonical pathways and disease and function responses of B. koreanus to PCMX. Using the fold change value of the DEGs from each experiment, the enriched canonical pathways and networks between genes were analyzed using an IPA core analysis of the Ingenuity database. To observe the biological states of networks across all experiments, we used an IPA comparison analysis to compare canonical pathways and functions. In all processes, the networks and canonical pathways were explored as significant at P < 0.05. Data are expressed as the mean value (± S.D). All statistical analyses were performed using SPSS ver. 17.0 (SPSS Inc., Chicago, IL, USA). First, the homogeneity of variance and normal distribution of all data were analyzed by Levene's test. Significant differences between exposure groups were analyzed by one-way analysis of variance (ANOVA) with a post hoc analysis following Tukey's method (P < 0.05). The LC 50 -24 h of PCMX and BAC in B. koreanus was approximately 24.264 mg L − 1 and 0.483 mg L − 1 , respectively ( Fig. 1A and B). A reverse S-curves analysis of each test on the survival rate produced 15 mg L − 1 of PCMX and 0.3 mg L − 1 of BAC as the NOEC level. Those concentrations were used directly in this study as marginal levels of exposure for measuring biological responses in B. koreanus. In the exposure test, the initial concentrations of dissolved PCMX and BAC were 8.7 ± 0.2 mg L − 1 and 0.39 ± 0.05 mg L − 1 , respectively, which differed slightly from the expected concentrations (10 mgL − 1 of PCMX and 0.3 mgL − 1 of BAC), but were within the range of concentration used for exposure when considering the recovery rate. The concentrations of these two disinfectants did not show any significant changes in the experimental condition ( Fig. 1C and Fig. S1 ), although the half-life of PCMX in the environment was reported in several studies (NCBI, 2021) . The bioaccumulated concentrations of these two disinfectants increased significantly (P < 0.05) during 24 h of exposure, whereas no levels were detected in the controls. In particular, PCMX, a disinfectant that was observed to be less toxic at the lethal concentration, had a lower concentration than BAC in B. koreanus ( Fig. 1C and Fig. S1 ). In the control, the population of B. koreanus increased by up to approximately 300 individuals during 7 days of incubation, and that population growth was significantly retarded (P < 0.05) in response to all concentrations of PCMX, even though all the concentrations of PCMX were less than the NOEC. In the groups treated with 2 and 5 mg L − 1 of PCMX, the final number of rotifers was less than 50. Interestingly, BAC did not cause any conspicuous retardation in population growth at any exposure, although statistical differences were observed at the highest concentrations of BAC ( Fig. 2A , Table S3 ). In the fecundity experiments, PCMX produced a significant decrease (P < 0.05) in reproduction ability. The number of newly hatched neonates decreased in a dosedependent manner (Fig. 2B) , resulting in zero offspring at 10 mg L − 1 PCMX. However, the number of hatched individuals did not change significantly (ANOVA, Tukey's post hoc, P > 0.05) in response to BAC exposure. The lifespan, a representative parameter to examine failure beyond compensation in stress conditions, also decreased significantly (P < 0.05) in response to PCMX, by up to 33% compared with the control. However, the lifespan did not change significantly (P > 0.05) upon BAC exposure (Fig. 2C ). H 2 DCFDA staining showed that only PCMX without NAC produced fluorescence under the green stimulus (Fig. 3A) . The ROS levels in B. koreanus were increased by approximately 10% in response to PCMX, whereas lower ROS levels compared with the control were measured following BAC exposure (Fig. 3B) . Experiments with NAC, a ROSscavenging chemical, support our finding that PCMX caused ROS generation in B. koreanus (Fig. 3B ). The increase in the antioxidant enzymatic activities of SOD and CAT were in accordance with the results observed in the ROS experiments ( Fig. 4A and B) , showing a series of these enzymes as antioxidant mechanisms to oxidative stress (Fig. 4C) . Both swimming speed and movement patterns were altered in response to PCMX and BAC exposure (Fig. 5) . Swimming speed was measured as 840.4 μm/s in normal conditions and decreased to 422.4 and 672.0 μm/s upon exposure to PCMX (10 mg L − 1 ) and BAC (0.3 mg L − 1 ), respectively. The alterations in swimming speed are expressed as percentages relative to the control and were reduced by both disinfectants (Fig. 5A ). However, BAC reduced the swimming speed by only 15% compared with the control (Fig. 5B ), whereas PCMX exposure caused a difference of about 40%, which explains the abnormal swimming in the figure showing the movement path. The changes in sinuosity observed in the swimming track were also significant (P < 0.05), and the rotation rate was significantly enhanced (ANOVA, Tukey's post hoc, P < 0.05) in response to PCMX (Fig. 5C ). These results are well matched with the swimming path measured by MTrack2, which showed abnormality in the rotifers' swimming around in place (Fig. 5D ). In transcriptomic analysis, we identified 12,250 expressed genes, of which 399, 1329, and 1069 were differentially expressed upon exposure to three different concentrations of PCMX (1 mg L − 1 , 5 mg L − 1 , and 10 mg L − 1 , respectively) ( Table S4 ). The PCA showed clear differences across PCMX concentrations, suggesting that PCMX induced global gene expression differences between treatment groups in a concentrationdependent manner (Fig. 6A ). Apparent differences in upregulated and downregulated genes were also exhibited upon exposure to 1 mg L − 1 PCMX (upregulated: 92, downregulated: 148), 5 mg L − 1 PCMX (upregulated: 145, downregulated: 379), and 10 mg L − 1 PCMX (upregulated: 145, downregulated: 379) (P < 0.05) (Fig. 6B, Fig. S2 , and Table S5 ). It is particularly noteworthy that the highest concentration of PCMX (10 mg L − 1 ) caused a significant increase (P < 0.05) in the number of downregulated DEGs, compared with the control group. The DEGs shared among the three PCMX treatment groups accounted for only 14.9% and 12.6% of the total upregulated and downregulated DEGs, respectively ( Fig. 6C and Table S4 ). The DEGs among the three different PCMX concentrations were further examined using GO term and KEGG (Fig. 6C) analyses. Clearly, 5 mg L − 1 and 10 mg L − 1 PCMX had significant effects (P < 0.05) on metabolic process-related biological processes such as oxidationreduction, hydrogen sulfide metabolism, lipid catabolism, phospholipid biosynthesis, glucose metabolism, and long-chain fatty-acyl-CoA biosynthesis. Similar characteristics were observed in the KEGG path enrichment analysis. At all three PCMX concentrations, DEGs were significantly enriched (P < 0.05) in the metabolic pathway. Notably, B. koreanus was exposed to PCMX (left) and BAC (right) for 7 days. Differences between exposed groups were analyzed for significance using Tukey's post hoc analysis. Different letters above columns indicate significant differences (P < 0.05). compared with the lower concentrations (1 mg L − 1 and 5 mg L − 1 PCMX), the DEGs in the lysosome pathway of B. koreanus exposed to the highest concentration of PCMX were significantly enriched (P < 0.05). In combination with autophagy, lysosomes play a crucial role in resistance to exogenous substances by recognizing foreign materials and attempting to degrade them (Medina et al., 2011) . The excessive oxidative stress induced by a high PCMX concentration can negatively affect lysosomal membrane stability (Pascua-Maestro et al., 2017) . As a result, several DEGs were related to the expression of the lysosome pathway. To further explore the functions and interactions among DEGs unique to the PCMX-exposed rotifers, we performed an IPA at all three PCMX concentrations (1, 5, and 10 mg L − 1 ). When we compared those three concentrations of PCMX, we found that 10 mg L − 1 PCMX inactivated more canonical pathways than the two lower concentrations (Fig. 7A) . The canonical pathway analysis revealed that calcium signaling, estrogen receptor signaling, notch signaling, and NRF-2mediated oxidative stress response were significantly enriched (P < 0.05) upon exposure to 10 mg L − 1 PCMX. The disease and function analysis predicted disorders of energy metabolism, movement disorders, and organismal death (Fig. 7B) . Notably, the top enriched functional network in the 10 mg L − 1 PCMX-treated groups consisted of cellular assembly organization, lipid metabolism, and reproductive system disease (score above 30) (Fig. S3) . The other intriguing enriched network involved embryonic development, nervous system development and function, and neurological disease (score 37) (Fig. 8A ). HSPA5 and SOX2 were the main hub genes connected to several genes in this network. IPA also provided a biological picture of the calcium signal pathway associated with behavioral impairments in the 10 mg L − 1 PCMX-treated groups (Fig. 8B) . DEGs (CREB3L1, MYH7, SLC2A1, and SPR) associated with movement disorder in the neurological disease category in the 10 mg L − 1 PCMX-treated groups are presented in a heat map (Fig. 8C and Table S6 ). In previous studies, the environmental relevant levels of disinfectants have wide ranges according to the environment from ng L − 1 up to several hundreds of μg L − 1 (Table 1) . In recent years, the use of disinfectants has increased dramatically due to frequent epidemic outbreaks. In fact, the use of those antimicrobials, which are permitted by United States Food and Drug Administration, has increased significantly since the outbreak of the COVID-19 pandemic. Of them, PCMX in particular is widely used in antibacterial hand sanitizers and household disinfectants in several countries (DeLeo et al., 2020; Kimura et al., 2014; Tan et al., 2021) , whereas BAC is widely used for medical purposes. Near wastewater sites, including hospitals, laundries, dairies, swimming pools, and paper production facilities, the concentrations of these disinfectants were near or higher than the lethal concentrations measured in our study. Furthermore, as the degradation rates of these chemicals in municipal sewage treatment plants is less than 100% (Yu et al., 2006; Choi and Oh, 2019; Ebele et al., 2017; Tan et al., 2021) , the fate of these disinfectants raises environmental issues in aquatic ecosystems (DeLeo et al., 2020) . Therefore, it is timely to examine the adverse effects of PCMX and BAC in aquatic environments. In our study, the mortality data in B. koreanus indicate that these two disinfectants cause toxic effects. The different lethal concentration and NOEC levels (more than one order of magnitude) in response to the two chemicals indicate that PCMX is less toxic to B. koreanus than BAC (BAC LC 50 = 0.483 mg L − 1 and PCMX LC 50 = 24.264 mg L − 1 at 24 h). This difference is similar to previously reported data that screened several species, including both land and aquatic organisms (Table S7 ). BAC is more toxic than PCMX, with acute lethality occurring at environmentally relevant concentrations or less (Sreevidya et al., 2018) . BAC at lower concentrations induced similar adverse effects on the survival, reproduction, lifespan, and germline toxicity of two model organisms (worm Caenorhabditis elegans and zebrafish Danio rerio) compared with PCMX and benzethonium chloride (Sreevidya et al., 2018) . Those differences are well reflected in the maximal permissible concentrations of these chemicals in ready-to-use PCPs. Typically, BAC is an acceptable preservative in cosmetics with a maximum allowable concentration of 0.1% in ready-for-use formulations (EU Cosing, ANNEX V), whereas PCMX can be in the range of 0.3% to 3.75% and 0.5% according to EU Cosing ANNEX V and WHO, respectively (EU, 2021; WHO, 2009) . Those results reflect the potentially different modes of action of these two chemicals. However, the rotifer B. koreanus had different susceptibility to PCMX than the other tested organisms, particularly more resistance than the freshwater cladoceran species D. magna (Table S8) . Although toxicity results are likely chemical-and species-specific, D. magna were more resistant to lindane, endosulfan, pentachlorophenol, 3,4- dichloroaniline, and copper sulphate than the freshwater rotifer Brachionus calyciflorus (Ferrando et al., 1992) . Contrary to the lethal concentrations, the results observed upon long-term exposure (>4 days) were significant. Thus, the growth, reproduction, and lifespan were prominently reduced in response to PCMX in a dose-dependent manner, whereas the NOEC levels of BAC did not cause any significant alterations to B. koreanus. Particularly, the population-growth rate was significantly reduced at 5 mg L − 1 PCMX, which is one-third of the NOEC level (NOEC, 15 mg L − 1 PCMX), whereas no mortality was observed at even the highest concentration of BAC (NOEC, 0.3 mg L − 1 BAC). In terms of accumulated PCMX, an average individual exposure of 2.3 pg of PCMX was calculated. Actually, in a study conducted in vertebrates (rat and dogs), rapid excretion of PCMX was found, with almost complete elimination within 24 h (Havler and Rance, 1977; EPA, 1994) . However, the lipophilic and longer chain BACs used in this study (C12, C14, and C16) showed metabolic stability, with high accumulated concentrations. Nonetheless, the inhibitory effect on growth in response to BAC did not differ significantly from that of the control group after 7 days of exposure. The two disinfectants tested here are classified differently: BAC is a quaternary ammonium compound, and PCMX is a phenol antiseptic compound. Therefore, the mode of toxic action might be different, although the general responses in organisms can be similar due to integrated stresses. For example, a previous study of PCMX and other disinfectants, including BAC, showed that these chemicals caused hatching delays (hatching inhibition), embryo mortality, morphological malformations, and neurotoxicity in zebrafish (Sreevidya et al., 2018) . The toxic results measured in the zebrafish D. rerio showed that, among five different disinfectants, only PCMX induced a 'body curvature' phenotype, suggesting that it has a unique mode of toxic action in zebrafish. The increased ROS patterns measured in B. koreanus in response to these two disinfectants also differed. PCMX is widely used because it has low toxicity to humans and is biodegradable (Nowak et al., 2021) . However, in aquatic environments, PCMX is classified as a moderately toxic chemical based on its low LC values (7.7 mg L − 1 for D. magna LC 50 -96 h) for invertebrates, unlike in vertebrates such as birds and fish (Table S7 ) (EPA, 1994) . Various in vivo and in vitro studies of its toxicity have demonstrated a lack of toxic effects in several organisms, including humans (Yost et al., 2016) . Non-target invertebrates were not expected to be exposed to PCMX due to its indoor usage patterns. However, our results show that PCMX induced oxidative stress in the estuarine rotifer B. koreanus. The general reactions to PCMX in bacteria show that hydroxyl (-OH) groups of the chloroxylenol (Fig. 1A) bind directly to and disrupt bacterial cell membranes. In addition, the protein and nucleic acid contents of targeted bacterial cells also become coagulated and cease to function, leading to rapid cell death (NCBI, 2021) . Similarly, after four days of exposure to PCMX, the number and fertility (reproduction) of B. koreanus were noticeably affected in a dose-dependent manner. Furthermore, the antioxidant enzymatic activity of SOD and CAT in B. koreanus increased upon exposure to 10 mg L − 1 of PCMX for 24 h. The increase in antioxidant enzyme levels in the PCMX-exposed groups supports the finding of physiological alterations with significant repercussions on various life cycle parameters (e.g., population growth and fecundity) in chronically exposed populations. However, in the BAC-exposed groups, ROS and consequent antioxidant enzymatic responses were not induced, implying that BAC exposure within the NOEC range did not induce ROS or ROS-related physiological changes in B. koreanus, as supported by the H 2 DCFDA fluorescence observed in the presence of NAC, a ROS scavenger. Oxidative stress, its antioxidant responses, and adverse outcomes such as delayed growth, productivity, and lifespan are a common series of responses to environmental stressors in aquatic invertebrates (Buttemer et al., 2010; Lister et al., 2016; Jeong et al., 2017) . They can also be interpreted as changes in energy allocation: the stress generated by PCMX causes the reallocation of the cellular energy budget from reproduction and growth to the expression of defensome components (e. g., antioxidative stress-related genes and enzymes), leading to delayed growth, productivity, and lifespan. For example, in the copepods Tigriopus japonicus and Paracyclopina nana, crude oil extraction and microplastics caused oxidative stress, which reduced fertility by reducing energy allocation for reproduction (Han et al., 2014; Jeong et al., 2017) . In our findings, the lifespan of B. koreanus was significantly reduced in response to PCMX, indicating that cellular defense mechanisms activated by ROS are closely related to changes in life parameters in vivo through a modulated energy budget. In B. koreanus, the antimicrobial agents triclosan and triclocarban produced similar effects on the endpoints of lifespan and oxidative stress-mediated expression of the defensome (Han et al., 2016) . Conversely, in the freshwater rotifer Asplanchna brightwelli, the antioxidant agent thiazolidine-4-carboxylic acid extended the lifespan by 7.8% (Bozovic and Enesco, 1986) , demonstrating that ROS is both directly and indirectly associated with longevity. Of the variables measured in B. koreanus, the reproduction rate (the number of neonates produced by one adult during 6 days) is highly likely to be related to the population increase (total number of rotifers after 7 days of incubation) because B. koreanus have an absolute parthenogenetic reproductive system that shows no resting eggs. Moreover, a shortened lifespan can predict the possible consequences of long-term exposure to PCMX in B. koreanus. Taken together, the reduced fecundity and shortened lifespan observed in PCMX-exposed rotifers Swimming is important for aquatic organisms (Charoy and Janssen, 1999; Obertegger et al., 2018) . For rotifers, swimming abnormalities can affect several aspects of fitness in terms of encountering food and predators (Charoy and Janssen, 1999; Chen et al., 2014; Obertegger et al., 2018) . In this study, the locomotory behavior of B. koreanus was affected after relatively short exposures to PCMX and BAC. Particularly, the abnormal circular motion measured in response to PCMX and BAC indicates that these two disinfectants disrupt the balance in rotifer swimming patterns. PCMX had stronger effects on rotifer swimming behavior than BAC. In PCMX-exposed rotifers, the ROS level increased significantly in a concentration-dependent manner, which is closely related to intracellular metabolic disorders (Liang et al., 2021) . As mentioned in previous studies, metabolic capacity is closely associated with swimming performance (Yan et al., 2013) . Rotifers use ciliary locomotion, which requires them to spend energy. A theoretical and calculational study conducted by Epp and Lewis (1984) reported that rotifers use about 62% of their energy for ciliary movement. The demand for metabolic energy in response to PCMX might exceed the energy supply available from a rotifer's accumulated energy resources, which could make the animal unable to sustain its routine metabolism (Sokolova et al., 2012) . The detoxification and metabolic pathways of xenobiotics (e.g., toxicants) also require energy and could interfere with the energy-making process in mitochondria. Thus, stress and the reallocation of energy budgets in response to PCMX might also affect swimming behavior. Although PCMX is less toxic than BAC, it reduced reproduction and lifespan and led to abnormal swimming behavior in B. koreanus. Furthermore, fundamental changes in the transcriptional landscape of the rotifers were observed with all PCMX treatments, as demonstrated by the numerous DEGs identified in this study. Nevertheless, the specific functional response was concentration-dependent: the 10 mg L − 1 PCMX treatment produced the largest treatment-specific response. Several macromolecule metabolic process-related genes, including TCEA2 and RVSE2, were overexpressed in the group that received the highest PCMX concentration. The upregulation of those metabolic process pathways is likely associated with energy homeostasis for energy metabolic regulation in PCMX-exposed rotifers. The available energy that organisms use for growth and reproduction can be affected by external factors such as toxicants (De Coen and Janssen, 2003) and microbial agents (Maynou et al., 2021) . Those previous studies are consistent with our findings, indicating that PCMX causes changes in the metabolic processes of rotifers. That process can change both the amount and ratio of energy stored in macromolecules (carbohydrates, lipids, and proteins) (Sancho et al., 2009) . Lipids in particular constitute an important source of energy that is typically allocated to offspring (Goulden and Place, 1993) . In cladocerans, for example, the offspring consume approximately 50% of the lipid reserves allocated in the brood pouch of parthenogenetic females, and the remaining reserves are maintained as a major energy source during the postembryonic-period (Goulden and Place, 1993) . Therefore, our result suggests that PCMX adversely affects metabolic processes in rotifers, leading to in vivo toxicity. In our RNA-seq analyses, oxidative stress-induced DNA damage was predicted, with consequent adverse effects on individuals. In addition, a series of toxic effects was observed when we used various approaches to uncover adverse outcomes such as acute and chronic toxicity. To further confirm the PCMX-induced adverse effects on cellular and molecular responses, we performed IPA. The results indicate that PCMX disrupts swimming behavior by affecting the calcium signaling pathway and nervous system (Fig. 9) . The contraction of muscles associated with movement depends on an increase in the concentration of cytoplasmic Patrauchan and Oriel, 2003 calcium. First, calcium is stored in the endoplasmic reticulum through the activity of the sarcoendoplasmic reticulum Ca 2+ ATPase (Brennan et al., 2005) . The cascade of neurotransmitter released from nerve terminals induces action potentials, and that process produces postsynaptic calcium signals for muscle contraction through mechanical binding of ryanodine receptors (Altringham and Ellerby, 1999) . The increasing cytosolic calcium promotes the contraction of cardiac and skeletal muscle, along with troponin and myosin complexes (Schiaffino and Reggiani, 1996) . Those reactions are also important mediators of swimming behavior in marine organisms. For example, in the fathead minnow Pimephales promelas, alterations in swimming behavior can involve changes in calcium signaling that can be measured by disruption of ryanodine and dihydropyridine receptors, which are directly related to muscle function in response to the antimicrobial chemical triclosan (TCS) (Fritsch et al., 2013) . TCS, which is in wide use, causes sporadic wriggling movement in the larvae of rainbow trout (Oncorhynchus mykiss) (Capkin et al., 2017) . Ajao et al. (2015) suggested that the interference in calcium-dependent K + leakage and excitationcontraction of skeletal muscles induced by TCS are the main causes of abnormal swimming behavior in rainbow trout. Our canonical pathway results indicate that calcium signaling and muscle-related genes (actin, myosin, and troponins) were downregulated, indicating that PCMX hindered muscle contraction and calcium signaling processes, resulting in decreased rotifer movement. Furthermore, changes in intracellular calcium concentration can lead to reductions in the function and viability of neurons (Jones and Smith, 2016) . As expected, the IPA predicted a significant effect on movement disorders in the neurological category in rotifers exposed to 10 mg L − 1 PCMX. Moreover, embryonic and nervous system development and neurological disease were identified as the main canonical pathways associated with PCMX exposure. Indeed, our swimming behavior analyses indicate that PCMX not only reduced swimming speed but also caused abnormal swimming patterns in rotifers. These results indicate that neurotoxic environmental pollutants can affect the behavior of marine organisms. In zebrafish, the neurotoxicants copper chloride and chlorpyrifos produced freeze responses (an anti-predator behavior) and a decrease in swimming speed, respectively (Tilton et al., 2011) . Moreover, the freshwater rotifer B. calyciflorus, which possesses a well-developed neuromuscular system despite its small size, exhibited uncoordinated movements upon exposure to neurotoxic cyclodiene-type toxicants (Charoy and Janssen, 1999) . Therefore, PCMX-induced abnormal swimming behavior might be entirely due to changes in muscle function and neurotoxicity mediated by the calcium signaling pathway (Fig. 9) . Taken together, the adverse effects of PCMX on rotifers at the behavioral, physiological, and molecular levels reveal that this compound might pose a much more serious threat to aquatic animals than currently perceived. Fig. 9 . Schematic summary of the suggested chloroxylenol-mediated molecular mechanism for suppressed swimming behavior and reproduction in Brachionus koreanus. PCMX and BAC are widely used as topical antiseptic products in over-the-counter medications and hard-surface disinfectants. However, our data indicate that these compounds can have serious repercussions in aquatic environments. In previous studies, the modes of toxic action of PCMX and BAC were reported in terms of their genotoxic, cytotoxic, and neurotoxic effects in other species. However, although ROS plays a central role in cell signaling for oxidative stress and regulating the main pathways of apoptosis mediated by mitochondria, previous studies did not directly demonstrate ROS generation in response to PCMX. In this paper, we are first to report that PCMX induces ROS generation in B. koreanus and produces various adverse effects in in vivo life parameters, producing deleterious effects on the molecular responses required for swimming behavior and reproduction. Furthermore, this study can cover the relevant concentrations of disinfectants, while many works on this chemical, focusing on its degradation or elimination by bacteria, and its biological toxicity, have been conducted at much higher levels of concentration. The expected mode of action described by IPA after ROS generation supports all the results observed at the in vivo and in vitro levels, including behavioral and life table parameters and alterations in enzymatic activity. It is also consistent with the chemical structural properties of PCMX, which contains hydroxyl groups that affect cell membranes. In conclusion, in vivo life parameter observations, antioxidant responses, and RNA-seq-based IPA all shed light on how PCMX induces deleterious effects at various levels that produce the observed in vivo and in vitro endpoints. These findings can be used to examine the underlying molecular mechanisms of rotifers using to cope with PCMXinduced oxidative stress and its resulting effects. The authors declare that they have no known competing financial interests that could have appeared to influence the work reported in this paper. Mitochondrial toxicity of triclosan on mammalian cells Fish swimming: patterns in muscle function Genetic and molecular ecotoxicology: a research framework Effect of antioxidants on rotifer lifespan and activity Acetylcholine and calcium signaling regulates muscle fibre formation in the zebrafish embryo The ecology of antioxidants & oxidative stress in animals: From bivalves to birds: oxidative stress and longevity Antimicrobial agents, triclosan, chloroxylenol, methylisothiazolinone and borax, used in cleaning had genotoxic and histopathologic effects on rainbow trout The swimming behaviour of Brachionus calyciflorus (rotifer) under toxic stress. II. Comparative sensitivity of various behavioural criteria The swimming speed alteration of two freshwater rotifers Brachionus calyciflorus and Asplanchna brightwelli under dimethoate stress Removal of chloroxylenol disinfectant by an activated sludge microbial community PubChem Compound Summary for CID 2723 Effect-based tools for monitoring and predicting the ecotoxicological effects of chemicals in the aquatic environment Ecotoxicology, ecophysiology, and mechanistic studies with rotifers The missing biomarker link: relationships between effects on the cellular energy allocation biomarker of toxicant-stressed Daphnia magna and corresponding population characteristics Assessment of ecological hazards and environmental fate of disinfectant quaternary ammonium compounds Biocides and novel antimicrobial agents for the mitigation of coronaviruses First comprehensive screening of lipophilic organic contaminants in surface waters of the megacity JakartaIndonesia Pharmaceuticals and personal care products (PPCPs) in the freshwater aquatic environment Ecotoxicity of disinfectant benzalkonium chloride and its mixture with antineoplastic drug 5-fluorouracil towards alga Pseudokirchneriella subcapitata Reregistration Eligibility Decision (R.E.D Facts Coast and speed of locomotion for rotifers Cosing, Annex V, List of Preservatives Allowed in Cosmetic Products Relative sensitivity of Daphnia magna and Brachionus calyciflorus to five pesticides Triclosan impairs swimming behavior and alters expression of excitation contraction coupling proteins in fathead minnow (Pimephales promelas) Swimming speed alteration of Artemia sp. and Brachionus plicatilis as a sub-lethal behavioural end-point for ecotoxicological surveys Lipid accumulation and allocation in Daphniid cladocera Crude oil exposure results in oxidative stress-mediated dysfunctional development and reproduction in the copepod Tigriopus japonicus and modulates expression of cytochrome P450 (CYP) genes Triclosan (TCS) and Triclocarban (TCC) cause lifespan reduction and reproductive impairment through oxidative stress-mediated expression of the defensome in the monogonont rotifer (Brachionus koreanus) The metabolism of p-chloro-m-xylenol (PCMX) in Sprague Dawley and Gunn Wistar rats Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources A new intertidal Brachionus and intrageneric phylogenetic relationships among Brachionus as revealed by allometry and CO1-ITS1 gene analysis Microbial actives with virucidal efficacy against SARS-CoV-2 and other β-and α-coronaviruses and implications for future emerging coronaviruses and other enveloped viruses Adverse effects of microplastics and oxidative stress-induced MAPK/Nrf2 pathway-mediated defense mechanisms in the marine copepod Paracyclopina nana Calcium-sensing receptor: a key target for extracellular calcium signaling in neurons The occurrence of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs in surface water in South Wales The removal of pharmaceuticals, personal care products, endocrine disruptors and illicit drugs during wastewater treatment and its impact on the quality of receiving waters Sorption and leaching of benzalkonium chlorides in agricultural soils Occurrences of benzalkonium chloride in streams near a pharmaceutical manufacturing complex in Korea and associated ecological risk Occurrence of preservatives and antimicrobials in Japanese rivers Aging extension and modifications of lipid metabolism in the monogonont rotifer Brachionus koreanus under chronic caloric restriction Multigenerational mitigating effects of ocean acidification on in vivo endpoints, antioxidant defense, DNA damage response, and epigenetic modification in an asexual monogonont rotifer Quaternary ammonium compounds in urban estuarine sediment environments -A class of contaminants in need of increased attention? Progress on the usage of the rotifer Brachionus plicatilis in marine ecotoxicology: a review New insights into the cellular mechanism of triclosaninduced dermal toxicity from a combined metabolomic and lipidomic approach Dietary pollutants induce oxidative stress, altering maternal antioxidant provisioning and reproductive output in the temperate sea urchin Evechinus chloroticus Determination of selected quaternary ammonium compounds by liquid chromatography with mass spectrometry. Part II. Application to sediment and sludge samples in Austria Effects of triclosan exposure on the energy budget of Ruditapes philippinarum and R. decussatus under climate change scenarios Transcriptional activation of lysosomal exocytosis promotes cellular clearance Benzalkonium chlorides: uses, regulatory status, and microbial resistance Biodegradation of chloroxylenol by Cunninghammella elegans IM 1785/21GP and Trametes versicolor IM 373: Insight into ecotoxicity and metabolic pathways Switching between swimming states in rotifers -case study Keratella cochlearis The genome of the marine rotifer Brachionus koreanus sheds light on the antioxidative defense system in response to 2-ethyl-phenanthrene and piperonyl butoxide Protecting cells by protecting their vulnerable lysosomes: identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress Degradation of benzyldimethylalkylammonium chloride by Aeromonas hydrophila sp. K Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown Disturbances in energy metabolism of Daphnia magna after exposure to tebuconazole Molecular diversity of myofibrillar proteins: gene regulation and functional significance Screening for contaminant hotspots in the marine environment of Kuwait using ecotoxicological and chemical screening techniques Rotifers in ecotixology: a review Acute toxicity bioassay using rotifers. I. A test for brackish and marine environments with Brachionus plicatilis Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates Benzalkonium chloride, benzethonium chloride, and chloroxylenol -Three replacement antimicrobials are more toxic than triclosan and triclocarban in two model organisms TCC: an R package for comparing tag count data with robust normalization strategies Human exposure and health risk assessment of an increasingly used antibacterial alternative in personal care products Identification of toxic substances in United Kingdom estuaries Swimming impairment and acetylcholinesterase inhibition in zebrafish exposed to copper or chlorpyrifos separately, or as mixtures Zooplankton feeding ecology: bacterivory by metazoan microzooplankton Available from. In: Guidelines on Hand Hygiene in Health Care: First Global Patient Safety Challenge Clean Care Is Safer Care. World Health Organization Disinfection By-Products in Drinking Water An interspecific comparison between morphology and swimming performance in cyprinids Human health risk assessment of chloroxylenol in liquid hand soap and dishwashing soap used by consumers and health-care professionals Occurrence and biodegradability studies of selected pharmaceuticals and personal care products in sewage effluent Studies on Brachionus (Rotifera): An example of interaction between fundamental and applied research Potential spreading risks and disinfection challenges of medical wastewater by the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral RNA in septic tanks of Fangcang Hospital Supplementary data to this article can be found online at https://doi. org/10.1016/j.marpolbul.2022.113396.