How Does Triclocarban Affect Sulfur Transformation in Anaerobic Systems?

Triclocarban (TCC), as a typical antimicrobial agent, accumulates at substantial levels in natural environments and engineered systems. This work investigated the impact of TCC on anaerobic sulfur transformation, especially toxic H2S production. Experimental findings revealed that TCC facilitated sulfur flow from the sludge solid phase to liquid phase, promoted sulfate reduction and sulfur-containing amino acid degradation, and largely improved anaerobic H2S production, i.e., 50-600 mg/kg total suspended solids (TSS) TCC increased the cumulative H2S yields by 24.76-478.12%. Although TCC can be partially biodegraded in anaerobic systems, the increase in H2S production can be mainly attributed to the effect of TCC rather than its degradation products. TCC was spontaneously adsorbed by protein-like substances contained in microbe extracellular polymers (EPSs), and the adsorbed TCC increased the direct electron transfer ability of EPSs, possibly due to the increase in the content of electroactive polymer protein in EPSs, the polarization of the amide group C═O bond, and the increase of the α-helical peptide dipole moment, which might be one important reason for promoting sulfur bioconversion processes. Microbial analysis showed that the presence of TCC enriched the organic substrate-degrading bacteria and sulfate-reducing bacteria and increased the abundances of functional genes encoding sulfate transport and dissimilatory sulfate reduction.

Mitigation of Triclocarban Inhibition in Microbial Electrolysis Cell-Assisted Anaerobic Digestion.

Triclocarban (TCC), as a widely used antimicrobial agent, is accumulated in waste activated sludge at a high level and inhibits the subsequent anaerobic digestion of sludge. This study, for the first time, investigated the effectiveness of microbial electrolysis cell-assisted anaerobic digestion (MEC-AD) in mitigating the inhibition of TCC to methane production. Experimental results showed that 20 mg/L TCC inhibited sludge disintegration, hydrolysis, acidogenesis, and methanogenesis processes and finally reduced methane production from traditional sludge anaerobic digestion by 19.1%. Molecular docking revealed the potential inactivation of binding of TCC to key enzymes in these processes. However, MEC-AD with 0.6 and 0.8 V external voltages achieved much higher methane production and controlled the TCC inhibition to less than 5.8%. TCC in the MEC-AD systems was adsorbed by humic substances and degraded to dichlorocarbanilide, leading to a certain detoxification effect. Methanogenic activities were increased in MEC-AD systems, accompanied by complete VFA consumption. Moreover, the applied voltage promoted cell apoptosis and sludge disintegration to release biodegradable organics. Metagenomic analysis revealed that the applied voltage increased the resistance of electrode biofilms to TCC by enriching functional microorganisms (syntrophic VFA-oxidizing and electroactive bacteria and hydrogenotrophic methanogens), acidification and methanogenesis pathways, multidrug efflux pumps, and SOS response.

Personal Care Products, Socioeconomic Status, and Endocrine-Disrupting Chemical Mixtures in Black Women.

Personal care products (PCPs) are sources of exposure to endocrine-disrupting chemicals (EDCs) among women, and socioeconomic status (SES) may influence these exposures. Black women have inequitable exposure to EDCs from PCP use, but no study has investigated how exposure to EDCs through PCPs may vary by SES, independent of race. Using data from the Study of Environment, Lifestyle, and Fibroids, a cohort of reproductive-aged Black women (n = 751), we quantified associations between PCPs and urinary biomarker concentrations of EDC mixtures (i.e., phthalates, phenols, parabens) within SES groups, defined using k-modes clustering based on education, income, marital status, and employment. Information about PCP use and SES was collected through questionnaires and interviews. We used principal component analysis to characterize the EDC mixture profiles. Stratified linear regression models were fit to assess associations between PCP use and EDC mixture profiles, quantified as mean differences in PC scores, by SES group. Associations between PCP use and EDC mixture profiles varied by SES group; e.g., vaginal powder use was associated with a mixture of phenols among lower SES women, whereas this association was null for higher SES women. Findings suggest that SES influences PCP EDC exposure in Black women, which has implications for public health interventions.

[Determination of triclocarban and triclosan in urine by QuEChERS extraction and ultra-performance liquid chromatography-tandem mass spectrometry].

Objective: To establish a method for the determination of triclocarban (TCC) and triclosan (TCS) in urine by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) after purification by QuEChERS. Methods: In May 2022, urine samples were extracted by acetonitrile, purified by QuEChERS, separated by Waters Acquity UPLC BEH C18 column (100 mm×2.1 mm, 1.7 µm), and eluated with water-acetonitrile as mobile phase gradient at a flow rate of 0.3 ml/min. The detection was conducted in negative ion mode (ESI(-)) and multiple reaction monitoring (MRM) scanning, it was quantified with a internal standard method, and the methodology was verified. Results: The linear ranges of TCC and TCS were 0.5-100.0 µg/L and 1.0-100.0 µg/L, and the correlation coefficients were 0.9997 and 0.9991, respectively. The limits of detection and quantitation of TCC and TCS were 0.17 and 0.33 µg/L, and 0.5 and 1.0 µg/L, respectively. The recoveries of TCC and TCS were 100.1%-102.8% and 96.7%-108.6%, and the relative standard deviations were 4.9%-6.7% and 4.1%-8.3%, respectively, at 2.0, 10.0 and 80.0 µg/L. Conclusion: QuEChERS-UPLC-MS/MS method is simple, rapid, sensitive and reproducible, and can be used for rapid and accurate simultaneous detection of TCC and TCS exposure levels in occupational population.

Complementary Biotransformation of Antimicrobial Triclocarban Obviously Mitigates Nitrous Oxide Emission toward Sustainable Microbial Denitrification.

Sustainable nitrogen cycle is an essential biogeochemical process that ensures ecosystem safety and byproduct greenhouse gas nitrous oxide reduction. Antimicrobials are always co-occurring with anthropogenic reactive nitrogen sources. However, their impacts on the ecological safety of microbial nitrogen cycle remain poorly understood. Here, a denitrifying bacterial strain Paracoccus denitrificans PD1222 was exposed to a widespread broad-spectrum antimicrobial triclocarban (TCC) at environmental concentrations. The denitrification was hindered by TCC at 25 µg L-1 and was completely inhibited once the TCC concentration exceeded 50 µg L-1. Importantly, the accumulation of N2O at 25 µg L-1 of TCC was 813 times as much as the control group without TCC, which attributed to the significantly downregulated expression of nitrous oxide reductase and the genes related to electron transfer, iron, and sulfur metabolism under TCC stress. Interestingly, combining TCC-degrading denitrifying Ochrobactrum sp. TCC-2 with strain PD1222 promoted the denitrification process and mitigated N2O emission by 2 orders of magnitude. We further consolidated the importance of complementary detoxification by introducing a TCC-hydrolyzing amidase gene tccA from strain TCC-2 into strain PD1222, which successfully protected strain PD1222 against the TCC stress. This study highlights an important link between TCC detoxification and sustainable denitrification and suggests a necessity to assess the ecological risks of antimicrobials in the context of climate change and ecosystem safety.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications.

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Thermodynamic Assessment of Triclocarban Dissolution Process in N-Methyl-2-pyrrolidone + Water Cosolvent Mixtures.

Solubility is one of the most important physicochemical properties due to its involvement in physiological (bioavailability), industrial (design) and environmental (biotoxicity) processes, and in this regard, cosolvency is one of the best strategies to increase the solubility of poorly soluble drugs in aqueous systems. Thus, the aim of this research is to thermodynamically evaluate the dissolution process of triclocarban (TCC) in cosolvent mixtures of {N-methyl-2-pyrrolidone (NMP) + water (W)} at seven temperatures (288.15, 293.15, 298.15, 303.15, 308.15, 313.15 and 318.15 K). Solubility is determined by UV/vis spectrophotometry using the flask-shaking method. The dissolution process of the TCC is endothermic and strongly dependent on the cosolvent composition, achieving the minimum solubility in pure water and the maximum solubility in NMP. The activity coefficient decreases from pure water to NMP, reaching values less than one, demonstrating the excellent positive cosolvent effect of NMP, which is corroborated by the negative values of the Gibbs energy of transfer. In general terms, the dissolution process is endothermic, and the increase in TCC solubility may be due to the affinity of TCC with NMP, in addition to the water de-structuring capacity of NMP generating a higher number of free water molecules.

Triclocarban evoked neutrophil extracellular trap formation in common carp (Cyprinus carpio L.) by modulating SIRT3-mediated ROS crosstalk with ERK1/2/p38 signaling.

Triclocarban (TCC), an antimicrobial ingredient in personal care products, is associated with immunosuppression and physiological dysfunctions of aquatic organisms. The aim of this study was to investigate whether TCC can induce common carp NETosis (neutrophil death by neutrophil extracellular trap (NET) release) and then to attempt to identify the potential molecular mechanisms. Herein, scanning electron microscopy and flow cytometric assays showed that revealed that TCC triggers DNA-containing web-like structures and increases extracellular DNA content. In the proteomic analysis, we observed that NET-related proteins, extracellular regulated protein kinase (Mapk1, Mapk14, Jak2) and apoptotic protein (caspase3) were significantly increased, and defender against cell death 1 (Dad1) was significantly decreased after TCC treatments. Meanwhile, we confirmed that TCC stress can trigger NETosis in common carp by activating the reactive oxygen species (ROS)/ERK1/2/p38 signaling. We think that the upregulated NDUFS1 expression is closely related to oxidative stress induced by TCC. Importantly, we discovered that SIRT3 expression was significantly decreased in the process of TCC-induced NETs. Importantly, pretreatment with the SIRT3 agonist honokiol (HKL) effectively suppressed TCC-induced NET release. In contrast, the SIRT3 antagonist 3-TYP escalated TCC-induced NET formation. Mechanistically, SIRT3 degradation serves as a potential mediator for regulating oxidative stress crosstalk between ERK1/2/p38 signals in the process of TCC-induced NET formation. These findings unveil new insights into the TCC-evoked health risk of fish and other aquatic organisms and suggest that SIRT3 is a potential pharmacological intervention target to alleviate TCC-induced common carp NETosis.

Bioelectrochemical catabolism of triclocarban through the cascade acclimation of triclocarban-hydrolyzing and chloroanilines-oxidizing microbial communities.

Chlorinated antimicrobial triclocarban (3,4,4'-trichlorocarbanilide, TCC) is an emerging refractory contaminant omnipresent in various environments. Preferential microbial hydrolysis of TCC to chloroanilines is essential for its efficient mineralization. However, the microbial mineralization of TCC in domestic wastewater is poorly understood. Here, the bioelectrochemical catabolism of TCC to chloroanilines (3,4-dichloroaniline and 4-chloroaniline) and then to CO2 was realized through the cascade acclimation of TCC-hydrolyzing and chloroanilines-oxidizing microbial communities. The biodegradation of chloroanilines was obviously enhanced in the bioelectrochemical reactors. Pseudomonas, Diaphorobacter, and Sphingomonas were the enriched TCC or chloroanilines degraders in the bioelectrochemical reactors. The addition of TCC enhanced the synergistic effect within functional microbial communities based on the feature of the phylogenetic ecological networks. This study provides a new idea for the targeted domestication and construction of functionally differentiated microbial communities to efficiently remove TCC from domestic wastewater through a green and low-carbon bioelectrochemical method.

Genetic bioaugmentation with triclocarban-catabolic plasmid effectively removes triclocarban from wastewater.

Triclocarban, one of the emerging pollutants, has been accumulating, and it is frequently detected in wastewater. Due to its toxicity and persistence, the efficient removal of triclocarban from wastewater systems is challenging. Genetic bioaugmentation with transferable catabolic plasmids has been considered to be a long-lasting method to clean up pollutants in continuous flow wastewater treatment systems. In this study, bioaugmentation with Pseudomonas putida KT2440, harboring the transferrable triclocarban-catabolic plasmid pDCA-1-gfp-tccA2, rapidly converted 50 µM triclocarban in wastewater into 3,4-dichloroaniline and 4-chloroaniline, which are further mineralized more easily. RT-qPCR results showed that the ratio of the copy number of pDCA-1-gfp-tccA2 to the cell number of strain KT2440 gradually increased during genetic bioaugmentation, suggesting horizontal transfer and proliferation of the plasmid. By using DNA stable isotope probing (SIP) and amplicon sequencing, OTU86 (Escherichia-Shigella), OTU155 (Citrobacter), OTU5 (Brucella), and OTU15 (Enterobacteriaceae) were found to be the potential recipients of the plasmid pDCA-1-gfp-tccA2 in the wastewater bacterial community. Furthermore, three transconjugants in the genera of Escherichia, Citrobacter, and Brucella showing triclocarban-degrading abilities were isolated from the wastewater. This study develops a new method for removing triclocarban from wastewater and provides insights into the environmental behavior of transferrable catabolic plasmids in bacterial community in wastewater systems.

Cosmetic Preservatives: Hazardous Micropollutants in Need of Greater Attention?

In recent years, personal care products (PCPs) have surfaced as a novel class of pollutants due to their release into wastewater treatment plants (WWTPs) and receiving environments by sewage effluent and biosolid-augmentation soil, which poses potential risks to non-target organisms. Among PCPs, there are preservatives that are added to cosmetics for protection against microbial spoilage. This paper presents a review of the occurrence in different environmental matrices, toxicological effects, and mechanisms of microbial degradation of four selected preservatives (triclocarban, chloroxylenol, methylisothiazolinone, and benzalkonium chloride). Due to the insufficient removal from WWTPs, cosmetic preservatives have been widely detected in aquatic environments and sewage sludge at concentrations mainly below tens of µg L-1. These compounds are toxic to aquatic organisms, such as fish, algae, daphnids, and rotifers, as well as terrestrial organisms. A summary of the mechanisms of preservative biodegradation by micro-organisms and analysis of emerging intermediates is also provided. Formed metabolites are often characterized by lower toxicity compared to the parent compounds. Further studies are needed for an evaluation of environmental concentrations of preservatives in diverse matrices and toxicity to more species of aquatic and terrestrial organisms, and for an understanding of the mechanisms of microbial degradation. The research should focus on chloroxylenol and methylisothiazolinone because these compounds are the least understood.

The Different Facets of Triclocarban: A Review.

In the late 1930s and early 1940s, it was discovered that the substitution on aromatic rings of hydrogen atoms with chlorine yielded a novel chemistry of antimicrobials. However, within a few years, many of these compounds and formulations showed adverse effects, including human toxicity, ecotoxicity, and unwanted environmental persistence and bioaccumulation, quickly leading to regulatory bans and phase-outs. Among these, the triclocarban, a polychlorinated aromatic antimicrobial agent, was employed as a major ingredient of toys, clothing, food packaging materials, food industry floors, medical supplies, and especially of personal care products, such as soaps, toothpaste, and shampoo. Triclocarban has been widely used for over 50 years, but only recently some concerns were raised about its endocrine disruptive properties. In September 2016, the U.S. Food and Drug Administration banned its use in over-the-counter hand and body washes because of its toxicity. The withdrawal of triclocarban has prompted the efforts to search for new antimicrobial compounds and several analogues of triclocarban have also been studied. In this review, an examination of different facets of triclocarban and its analogues will be analyzed.

Comparative Target Analysis of Chlorinated Biphenyl Antimicrobials Highlights MenG as a Molecular Target of Triclocarban.

Triclocarban (TCC), a formerly used disinfectant, kills bacteria via an unknown mechanism of action. A structural hallmark is its N,N'-diaryl urea motif, which is also present in other antibiotics, including the recently reported small molecule PK150. We show here that, like PK150, TCC exhibits an inhibitory effect on Staphylococcus aureus menaquinone metabolism via inhibition of the biosynthesis protein demethylmenaquinone methyltransferase (MenG). However, the activity spectrum (MIC90) of TCC across a broad range of multidrug-resistant staphylococcus and enterococcus strains was much narrower than that of PK150. Accordingly, TCC did not cause an overactivation of signal peptidase SpsB, a hallmark of the PK150 mode of action. Furthermore, we were able to rule out inhibition of FabI, a confirmed target of the diaryl ether antibiotic triclosan (TCS). Differences in the target profiles of TCC and TCS were further investigated by proteomic analysis, showing complex but rather distinct changes in the protein expression profile of S. aureus Downregulation of the arginine deiminase pathway provided additional evidence for an effect on bacterial energy metabolism by TCC.IMPORTANCE TCC's widespread use as an antimicrobial agent has made it a ubiquitous environmental pollutant despite its withdrawal due to ecological and toxicological concerns. With its antibacterial mechanism of action still being unknown, we undertook a comparative target analysis between TCC, PK150 (a recently discovered antibacterial compound with structural resemblance to TCC), and TCS (another widely employed chlorinated biphenyl antimicrobial) in the bacterium Staphylococcus aureus We show that there are distinct differences in each compound's mode of action, but also identify a shared target between TCC and PK150, the interference with menaquinone metabolism by inhibition of MenG. The prevailing differences, however, which also manifest in a remarkably better broad-spectrum activity of PK150, suggest that even high levels of TCC or TCS resistance observed by continuous environmental exposure may not affect the potential of PK150 or related N,N'-diaryl urea compounds as new antibiotic drug candidates against multidrug-resistant infections.

Bioaugmentation of triclocarban and its dechlorinated congeners contaminated soil with functional degraders and the bacterial community response.

Partial removal of haloaromatic antimicrobial triclocarban (TCC) during wastewater treatment caused the final introduction of residual TCC into soils. Bioaugmentation has been proposed for the biodegradation of TCC and its dechlorinated congeners 4,4'-dichlorocarbanilide (DCC) and carbanilide (NCC) in soil. The isolated TCC-degrading strain Ochrobactrum sp. TCC-2 and chloroanilines-degrading strain Diaphorobacter sp. LD72 were used to study the removal efficiency of TCC, DCC and NCC mixture and their chloroanilines intermediates, respectively. The potential degradation competition between TCC and its dechlorinated congeners, and the response of bacterial community during the bioremediation were also investigated. The biodegradation of DCC and TCC was significantly enhanced for soil with inoculums compared with sterilized and natural soils. Chloroanilines products could also be effectively removed. For the degradation of combined substrates in the aqueous medium, NCC had negative effect on the degradation of TCC and DCC, while TCC and DCC negatively influenced each other. The bioaugmentation with two degraders obviously changed the phylogenetic composition and function of indigenous soil microbiome. Importantly, the inoculated degraders could be maintained, suggesting their adaptability and potential application in bioaugmentation for such recalcitrant contaminants. This study offers new insights into the enhanced bioremediation of TCC and its dechlorinated congeners contaminated soils by the bioaugmentation of functional degraders and the structure and function response of the indigenous soil microbiome to the bioremediation process.

Disposition and metabolism of antibacterial agent, triclocarban, in rodents; a species and route comparison.

Triclocarban is a residue-producing antibacterial agent used in a variety of consumer products. These studies investigated the disposition and metabolism of [14C]triclocarban. In male rats following a single gavage administration of 50, 150, and 500 mg/kg, excretion was primarily via feces (feces, 85-86%; urine, 3-6%) with no apparent dose-related effect. In male rats, 29% of the administered dose was excreted in bile suggesting some of the fecal excretion is from the absorbed dose which was excreted to the intestine via bile. The tissue retention of radioactivity was low in male rats (24 h, 3.9%; 72 h, 0.1%). Disposition pattern following gavage administration of 50 mg/kg in female rats and male and female mice were similar to male rats. Plasma elimination half-life of triclocarban in rats following gavage administration was shorter (∼2 h) compared to that based on total radioactivity (≥9 h) which included all products of triclocarban. Absorption following a single dermal application of 1.5 or 3% was low (≤3%) in rodents. Hydroxylated and conjugated metabolites of triclocarban predominated in bile. In hepatocytes, clearance of triclocarban in mouse and human was similar and was faster than in rat.

Metabolic network and recovery mechanism of Escherichia coli associated with triclocarban stress.

Although the toxicity of triclocarban at molecular level has been investigated, the metabolic networks involved in regulating the stress processes are not clear. Whether the cells would maintain specific phenotypic characteristics after triclocarban stress is also needed to be clarified. In this study, Escherichia coli was selected as a model to elucidate the cellular metabolism response associated with triclocarban stress and the recovery metabolic network of the triclocarban-treated cells using the proteomics and metabolomics approaches. Results showed that triclocarban caused systematic metabolic remodeling. The adaptive pathways, glyoxylate shunt and acetate-switch were activated. These arrangements allowed cells to use more acetyl-CoA and to reduce carbon atom loss. The upregulation of NH3-dependent NAD+ synthetase complemented the NAD+ consumption by catabolism, maintaining the redox balance. The synthesis of 1-deoxy-D-xylulose-5-phosphate was suppressed, which would affect the accumulation of end products of its downstream pathway of isoprenoid synthesis. After recovery culture for 12 h, the state of cells returned to stability and the main impacts on metabolic network triggered by triclocarban have disappeared. However, drug resistance caused by long-term exposure to environmentally relevant concentration of triclocarban is still worthy of attention. The present study revealed the molecular events under triclocarban stress and clarified how triclocarban influence the metabolic networks.

A Review on the Fate of Legacy and Alternative Antimicrobials and Their Metabolites during Wastewater and Sludge Treatment.

Antimicrobial compounds are used in a broad range of personal care, consumer and healthcare products and are frequently encountered in modern life. The use of these compounds is being reexamined as their safety, effectiveness and necessity are increasingly being questioned by regulators and consumers alike. Wastewater often contains significant amounts of these chemicals, much of which ends up being released into the environment as existing wastewater and sludge treatment processes are simply not designed to treat many of these contaminants. Furthermore, many biotic and abiotic processes during wastewater treatment can generate significant quantities of potentially toxic and persistent antimicrobial metabolites and byproducts, many of which may be even more concerning than their parent antimicrobials. This review article explores the occurrence and fate of two of the most common legacy antimicrobials, triclosan and triclocarban, their metabolites/byproducts during wastewater and sludge treatment and their potential impacts on the environment. This article also explores the fate and transformation of emerging alternative antimicrobials and addresses some of the growing concerns regarding these compounds. This is becoming increasingly important as consumers and regulators alike shift away from legacy antimicrobials to alternative chemicals which may have similar environmental and human health concerns.

The fate of triclocarban in activated sludge and its influence on biological wastewater treatment system.

Triclocarban (TCC), a typical emerging contaminant, was abundantly released into environment and frequently detected in practical wastewater treatment plants. However it is also an important material when being added to personal skin care products as a antibacterial agent. In this work, the behavior of TCC in wastewater treatment process was investigated. Experiments showed that ~82% of influent TCC was removed by activated sludge adsorption and its adsorption isotherm was well fitted with Linear model and Freundich model. High levels of TCC had seriously impact on the settleability, dewaterability and extracellular polymetric substance (EPS) of activated sludge, even on effluent turbidity after a long-term exposure. Furthermore, the performance of biological wastewater treatment was damaged by TCC long-term exposure as well. The removal rates of total nitrogen and phosphorus decreased from 91.2 ± 2.1% to 72.6 ± 2.2% and from 94.7 ± 3.1% to 78.4 ± 2.3%, respectively, with TCC level increasing from 0 to 100 µg/L. Mechanism analysis showed that TCC exposure significantly inhibited the relevant biological processes, such as ammonia oxidation, denitrification, phosphorus release and uptake, which were closely relevant to nitrogen and phosphorus removal.

Structure and specificity of several triclocarban-binding single domain camelid antibody fragments.

The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well-studied, but considerably less work has been done to characterize their ability to bind haptens. A high-resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3-(4-chlorophenyl)-1-(3,4-dichlorophenyl)urea) with near-nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody-hapten interactions. Additional significant interactions are formed with a non-hypervariable loop, sometimes dubbed "CDR4". A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody-TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180° along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure-guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.

Developmental toxicity of triclocarban in zebrafish (Danio rerio) embryos.

Triclocarban (TCC), which is used as an antimicrobial agent in personal care products, has been widely detected in aquatic ecosystems. However, the consequence of TCC exposure on embryo development is still elusive. Here, by using zebrafish embryos, we aimed to understand the developmental defects caused by TCC exposure. After exposure to 0.3, 30, and 300 µg/L TCC from 4-hour postfertilization (hpf) to 120 hpf, we observed that TCC exposure significantly increased the mortality and malformation, delayed hatching, and reduced body length. Exposure to TCC also affected the heart rate and expressions of cardiac development-related genes in zebrafish embryos. In addition, TCC exposure altered the expressions of the genes involved in hormonal pathways, indicating its endocrine disrupting effects. In sum, our data highlight the impact of TCC on embryo development and its interference with the hormone system of zebrafish.