Emerging Disinfection By-products Research Articles

Untargeted metabolomics of human keratinocytes reveals the impact of exposure to 2,6-dichloro-1,4-benzoquinone and 2,6-dichloro-3-hydroxy-1,4-benzoquinone as emerging disinfection by-products

IntroductionThe 2,6-dichloro-1,4-benzoquinone (DCBQ) and its derivative 2,6-dichloro-3-hydroxy-1,4-benzoquinone (DCBQ-OH) are disinfection by-products (DBPs) and emerging pollutants in the environment. They are considered to be of particular importance as they have a high potential of toxicity and they are likely to be carcinogenic.ObjectivesIn this study, human epidermal keratinocyte cells (HaCaT) were exposed to the DCBQ and its derivative DCBQ-OH, at concentrations equivalent to their IC20 and IC50, and a study of the metabolic phenotype of cells was performed.MethodsThe perturbations induced in cellular metabolites and their relative content were screened and evaluated through a metabolomic study, using 1H-NMR and MS spectroscopy.ResultsChanges in the metabolic pathways of HaCaT at concentrations corresponding to IC20 and IC50 of DCBQ-OH involved the activation of cell membrane α-linolenic acid, biotin, and glutathione and deactivation of glycolysis/gluconeogenesis at IC50. The changes in metabolic pathways at IC20 and IC50 of DCBQ were associated with the activation of inositol phosphate, pertaining to the transfer of messages from the receptors of the membrane to the interior as well as with riboflavin. Deactivation of biotin metabolism was recorded, among others. The cells exposed to DCBQ exhibited a concentration-dependent decrease in saccharide concentrations. The concentration of steroids increased when cells were exposed to IC20 and decreased at IC50. Although both chemical factors stressed the cells, DCBQ led to the activation of transporting messages through phosphorylated derivatives of inositol.ConclusionOur findings provided insights into the impact of the two DBPs on human keratinocytes. Both chemical factors induced energy production perturbations, oxidative stress, and membrane damage.

Identification of disinfection by-product precursors by natural organic matter fractionation: a review

During disinfection of drinking water, natural organic matter reacts with chlorine to produce harmful disinfection by-products. The identification of precursors of disinfection by-products in natural organic matter is challenging because natural organic matter is very complex and poorly known. Therefore, scientists have focused on the fractionation of natural organic matter with membranes or resins to better understand how and which organic matter fractions react during chlorination. Here, we compared the reactivity of various organic fractions with disinfection by-products. For that we did a meta-analysis of 400 water samples published in 80 publications, with focus on chlorination time and dose, SUVA254 and the column capacity factor used during resin fractionation. SUVA254 refers to the ultraviolet absorbance at 254 nm divided by the organic matter concentration. We found that hydrophobic compounds have 10–20% higher reactivity to both trihalomethane and haloacetic acid formation compared to hydrophilic compounds in waters with SUVA254 above 2L/(mg∙m), while hydrophobic and hydrophilic compounds have equal reactivity in waters with low SUVA254. On the other hand, hydrophilic compounds are 20–80% more reactive towards emerging disinfection by-products, regardless of SUVA254. Chlorination time and dose do not influence the reactivity ratio between the different fractions. An increase in column capacity factor can shift the reactivity ratio from hydrophobic to hydrophilic fractions. Dead-end, stirred cell ultrafiltration membrane fractionation might not always produce sharply separated fractions, which is mainly due to fouling. Therefore, no clear correlation could be found between membrane fractions and all investigated disinfection by-product groups.

Disinfection byproducts in indoor swimming pool water: Detection and human lifetime health risk assessment

Quantification of regulated and emerging disinfection byproducts (DBPs) in swimming pool water, as well as the assessment of their lifetime health risk are limited in China. In this study, the occurrence of regulated DBPs (e.g., trihalomethanes, haloacetic acids) and emerging DBPs (e.g., haloacetonitriles, haloacetaldehydes) in indoor swimming pool water and the corresponding source water at a city in Eastern China were determined. The concentrations of DBPs in swimming pool water were 1-2 orders of magnitude higher than that in source water. Lifetime cancer and non-cancer risks of DBPs stemming from swimming pool water were also estimated. Inhalation and dermal exposure were the most significant exposure routes related to swimming pool DBP cancer and non-cancer risks. For the first time, buccal and aural exposure were considered, and were proven to be important routes of DBP exposure (accounting for 17.9%-38.9% of total risk). The cancer risks of DBPs for all swimmers were higher than 10−6 of lifetime exposure risk recommended by United States Environmental Protection Agency, and the competitive adult swimmers experienced the highest cancer risk (7.82 × 10−5). These findings provide important information and perspectives for future efforts to lower the health risks associated with exposure to DBPs in swimming pool water.

Molecular Insights into the Enhanced Formation of Halonitrophenols after the UV/Chloramine Process

Halonitrophenols (HNPs) are a group of emerging disinfection byproducts with high developmental toxicity. However, the formation and control of HNPs remain much uncertain, especially in the UV/chlor(am)ine process. In this study, we used Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to screen for the typical precursors of 2,4-dichloro-5-nitrophenol (DCNP). The results show that UV/chloramine treatment on natural organic matter can lead to the generation of compounds with higher O/C and lower double-bond equivalence (DBE), which were confirmed as the possible precursors of DCNP during subsequent chlorination. The impacts of four representative factors (i.e., precontact time, UV fluence, pH, and Cl2/N) on the formation of DCNP in the UV/chloramine process were investigated to optimize the parameters and to minimize HNP formation. Furthermore, we observed that DCNP formation was higher from aniline and acetaminophen pretreated with UV/chlorine than that with UV/chloramine, while that from tyrosine and benzylsulfonamide exhibited an opposite order because of the different -NH2 sites on compounds. We also proposed a formation pathway of DCNP from tyrosine to better clarify the formation mechanism of DCNP. Our study can provide various useful information to comprehensively understand the precursors, formation mechanisms, and control methods of HNPs.

Neurotoxicity and transcriptome changes in embryonic zebrafish induced by halobenzoquinone exposure

Halobenzoquinones (HBQs) are emerging disinfection byproducts (DBPs) with a widespread presence in drinking water that exhibit much higher cytotoxicity than regulated DBPs. However, the developmental neurotoxicity of HBQs has not been studied in vivo. In this work, we studied the neurotoxicity of HBQs on zebrafish embryos, after exposure to varying concentrations (0-8 µmol/L) of three HBQs, 2,5-dichloro-1,4-benzoquinone (2,5-DCBQ), 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), and 2,5-dibromo-1,4-benzoquinone (2,5-DBBQ) for 4 to 120 hr post fertilization (hpf). HBQ exposure significantly decreased the locomotor activity of larvae, accompanied by significant reduction of neurotransmitters (dopamine and γ-aminobutyric acid) and acetylcholinesterase activity. Furthermore, the expression of genes involved in neuronal morphogenesis (gfap, α1-tubulin, mbp, and syn-2α) were downregulated by 4.4-, 5.2-, 3.0-, and 4.5-fold in the 5 µmol/L 2,5-DCBQ group and 2.0-, 1.6-, 2.1-, and 2.3-fold in the 5 µmol/L 2,5-DBBQ group, respectively. Transcriptomic analysis revealed that HBQ exposure affected the signaling pathways of neural development. This study demonstrates the significant neurotoxicity of HBQs in embryonic zebrafish and provides molecular evidence for understanding the potential mechanisms of HBQ neurotoxicity.

Halonaphthoquinones: A group of emerging disinfection byproducts of high toxicity in drinking water

Aromatic halogenated disinfection byproducts (DBPs) have received particular attention in recent years due to their high toxicity. However, most relevant researches at present focused merely on halo-monocyclic DBPs, while halo-polycyclic DBPs were scarcely explored. In this study, a new group of halo-bicyclic DBPs termed as halonaphthoquinones (HNQs) was systematically studied. By coupling with vacuum centrifugal concentrator, a SPE-UPLC-MS/MS method with high accuracy and sensitivity was developed to detect five semi-volatile HNQs in drinking water, which achieved the detection limits in the range of 0.05–0.24 ng/L. Five HNQs were identified using this method with 100% detection frequency at concentrations up to 136.7 ng/L in drinking water originated from seven water treatment plants. The cytotoxicity of the five tested HNQs in CHO-K1 cells (IC50 from 3.17 to 13.18 μM) was comparable to the most toxic known carbonaceous DBP in drinking water, iodoacetic acid (IC50=2.95 μM). Meanwhile, the cytotoxicity of five tested HNQs were also higher than 2,6-dichloro-1,4-benzoquinone (IC50=21.73 μM) which is hundreds to thousands of times more toxic than regulated DBPs, indicating the significant toxicity risk of HNQ DBPs. To the best of our knowledge, this study presents the first analytical method for analysis of HNQ DBPs, and the first set of data on the occurrence and cytotoxicity of HNQ DBPs in drinking water. These findings are meaningful for probing deeply into the presence of varied halo-polycyclic DBPs in the aqueous environment.

Formation of chlorinated halobenzoquinones during chlorination of free aromatic amino acids

Chlorinated halobenzoquinones (HBQs) widely exist in drinking water as emerging disinfection byproducts (DBPs), which have attracted significant attention due to their wide occurrence and high toxicity. In this study, the formation of chlorinated HBQs from the three free aromatic amino acids, tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe), during chlorination was investigated, the formation pathways of chlorinated HBQs were explained based on the detected intermediates and influence factors. The results revealed that four chlorinated HBQs, including 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ), 2,3,5-trichloro-1,4-benzoquinone, 2,3,5,6-tetrachloro-1,4-benzoquinone and 2,6-dichloro-3-methyl-1,4-benzonquinone, were formed in chlorination of the three free aromatic amino acids, and 2,6-DCBQ was the dominant species among the formed chlorinated HBQs. Of the three free aromatic amino acids, Trp and Tyr presented relatively high yields of chlorinated HBQs than Phe. Moreover, ten intermediates were successfully detected (e.g., N,2-dichloroaniline from Trp, 2,4,6-trichlorophenol from Tyr) according to the isotope and fragment information obtained using high resolution mass spectrometry. The formation pathways of chlorinated HBQs from Trp and Tyr were proposed to include electrophilic addition, electrophilic substitution, oxidation, deacidification and dehydration reaction, and further validated using theoretical calculation. The yields of chlorinated HBQs during chlorination of the free aromatic amino acids were significantly affected by free chlorine dosage, pH and temperature.

Oxidative Stress Induced by Iodoacetamide (an Emerging Disinfection By-Product) on Hepg-2 Cells

Oxidative Stress Induced by Iodoacetamide (an Emerging Disinfection By-Product) on Hepg-2 Cells

Molecular composition of halobenzoquinone precursors in natural organic matter in source water

Halobenzoquinones (HBQs) are emerging disinfection byproducts generated during the reaction of chlorine disinfectant with natural organic matter (NOM) in source water. In this study, the correlations between molecular weight and HBQs generation of river NOM was evaluated. The compositional and functional characteristics of primary HBQs precursors were revealed by using Orbitrap mass spectrometry combined with molecular tagging. The NOM fraction larger than 50 kDa resulted in approximately 9 times more HBQs (50.9 ± 2.7 ng/mgC) than low molecular weight fractions. Significant correlations were found between the yields of HBQs and lignin-like and highly oxygen compounds in NOM, suggesting their critical roles in HBQs formation. Derivatizating the aldehydes, ketones, hydroxyl and carboxyl groups in NOM could reduce HBQs yields by 90.7%–100%. Unraveling the molecular characteristics of HBQs precursors in NOM would greatly benefit the prediction of HBQs yields of different source water, and develop more efficient disinfection byproduct control strategies.

Effect of silver nanoparticles and chlorine reaction time on the regulated and emerging disinfection by-products formation

Silver nanoparticles (AgNPs) are used in many industries for multiple applications that inevitably release AgNPs into surface water sources. The formation kinetics of disinfection by-products (DBPs) in the presence of AgNPs was investigated during chlorination. Experiments were carried out with raw water from a canal in Songkhla, Thailand, which analyzed the formation potential (FP) of trihalomethanes FP (THMFP), iodo-trihalomethanes FP (I-THMFP), haloacetonitriles FP (HANFP), and trichloronitromethane FP. Increased AgNP concentrations by 10–20 mg/L led to a higher specific formation rate of chloroform which is described by zero- and first-order kinetics. The increase in the specific formation of chloroform as increasing chlorine contact time could enhance both the THMFP rates and the maximum THMFP concentrations in all tested AgNPs. The AgNP content did not have a significant influence on I-THMFP and HANFP concentrations or speciation. The I-THMFP and HANFP increased in a short-chlorination time as mostly complete formation <12 h, and then the rate decreased as the reaction proceeded. The levels of THMs and many emerging DBPs are related to the presence of AgNPs in chlorinated water and chlorine reaction time. THMFP had a higher impact on integrated toxic risk value (ITRV) than I-THMFP and HANFP because of the chlorination of water with AgNPs. The chlorine reaction time was more effective for increasing the ITRV of THMFP than the level of AgNPs. Water treatment plants should control the DBPs that cause possible health risks from water consumption by optimizing water distribution time.

Simultaneous determination of six haloacetonitriles in finished water for drinking by purge and trap-gas chromatography-triple quadrupole mass spectrometry

目前卤乙腈作为我国非受监管的消毒副产物广泛存在于饮用出厂水中,可产生多种毒性,缺乏相关标准检测依据。研究建立了吹扫捕集-气相色谱-三重四极杆质谱同时测定饮用出厂水中氯乙腈、二氯乙腈、三氯乙腈、溴乙腈、溴氯乙腈、二溴乙腈的分析方法。吹扫捕集技术应用于卤乙腈的测定,实现了样品经采集后全程自动测定,有害试剂零消耗。同时吹扫捕集法相比固相微萃取法,样品制备的速度更快,成本更低。实验考察了样品6 h内目标组分的稳定性;比较了7#(2,6-二苯基对苯醚)、10#(2,6-二苯基对苯醚/硅胶/碳分子筛)、11#(疏水活性炭)、12#(疏水活性炭)捕集阱对目标组分响应的影响;考察了4种型号色谱柱(VF-5、Rxi-624、DB-VRX、HP-INNOWAX)对色谱峰形的影响。实验条件经优化,确定了吹扫捕集采用10#捕集阱,将25 mL水样于35 ℃吹扫11 min,于190 ℃解析1 min。气相色谱分流进样,分流比1:10,使用Rxi-624Sil MS色谱柱(60 m×0.25 mm×1.40 μm)程序升温分离,线速度30 cm/s,在MRM模式下检测,外标法定量。结果表明,6种卤乙腈的基质效应为0.85~1.09,在各自范围内线性良好,r>0.9991,方法检出限为0.8~120.0 ng/L,定量限为1.5~300.0 ng/L, 3水平平均加标回收率为84.2%~106%,相对标准偏差(RSD)为1.81%~10.7%。对38份出厂水样品进行测定,卤乙腈总检出率为92.1%,含量为0.0101~1.28 μg/L。该方法高效、灵敏、环保,为针对卤乙腈类新兴消毒副产物开展监测及健康风险评估提供了优质的技术选择。

Hydrolysis and Chlorination of 2,6-Dichloro-1,4-benzoquinone under conditions typical of drinking water distribution systems

Halobenzoquinones (HBQs) are emerging disinfection by-products (DBPs) that are postulated drivers of bladder carcinogenicity. Prior assessments of 2,6-dichloro-1,4-benzoquinone (DCBQ) occurrence in drinking water distribution systems have revealed a gradual decline with increasing distance from points of entry. While this signals a degradation pathway, there is limited quantitative data on rate of that degradation. A systematic evaluation of DCBQ hydrolysis was performed, resulting in a rate law that is first order in both hydroxide [OH−] and [DCBQ]. The impact of temperature on that rate was characterized according to the Arrhenius relationship. Under the conditions tested (pH~7.2, T = 20°C) chloramine did not significantly impact DCBQ concentrations. However, DCBQ was rapidly degraded in solutions containing free available chlorine (FAC). Kinetic analysis showed non-integer order with respect to FAC. Further investigation led to a model that invoked reaction with dichlorine monoxide (Cl2O) as well as FAC.

Transformation of emerging disinfection byproducts Halobenzoquinones to haloacetic acids during chlorination of drinking water

Halobenzoquinones (HBQs) are emerging disinfection byproducts of high toxicity and frequent occurrence in drinking water. HBQs are unstable and can be transformed to hydroxylated HBQs (OH-HBQs) and haloacetic acids (HAAs) in the presence of multiple reactive species, which are generated during photolysis of chlorine. In this study, we investigated the occurrence of HBQs in the effluent of 10 water treatment systems and household tap water across China, and demonstrated the kinetic and mechanism of 2,6-dichloro-1,4-benzoquinone (2,6-DCBQ) degradation to HAAs by the solar/chlorine processes. HBQs were detected in all samples at concentrations from 1.2 to 42.7 ng/L. The degradation of 2,6-DCBQ during solar/chlorine treatment followed the pseudo-first order kinetics. HOCl/OCl- and ClO. contributed to 47.9% and 50.2% of 2,6-DCBQ degradation, whereas the contributions of Cl. and .OH were <1.5%. The degradation rates of 2,6-DCBQ were stable in the presence of chloride, but decreased by 4.3% in the presence of 0.1 mgC/L natural organic matter (NOM). ClO. and Cl. directly attacked the aromatic ring of 2,6-DCBQ to generate HAAs, whereas .OH reacted with OH-HBQs, the intermediate products of 2,6-DCBQ to generate HAAs. Unraveling the mechanism of free radical mediated 2,6-DCBQ transformation would greatly benefit the development of effective HBQ elimination technologies at water treatment plants in the future.

The advances on the formation and toxic effects of haloacetaldehydes disinfection by-products in drinking water

Haloacetaldehydes (HALs), as emerging disinfection by-products in drinking water, are the third largest group by weight of identified disinfection by-products (DBPs) in drinking water. The formation of HALs is associated with the level of natural organic matter and halide in the source water, the treatment process of drinking water and the type of disinfectant. Recent studies have shown that HALs are more cytotoxic and genotoxic than regulated trihalomethanes and halo-acetic acids in drinking water. Currently, only a few countries and regions have set limit values for trichloroacetaldehyde with high detection rate in drinking water. However, there is growing evidence that unregulated HALs have a higher potential risk to human health compared to regulated HALs. This paper reviews the current research progress on the formation and transformation, cytotoxicity and genotoxicity of HALs in drinking water, and looks forward to the problems that should be paid attention in the future toxicological research of HALs in order to support the development of scientific drinking water standards.

Fabricating a novel ternary recyclable Fe3O4/graphene/sulfur-doped g-C3N4 composite catalyst for enhanced removal of ranitidine under visible-light irradiation and reducing of its N-nitrosodimethylamine formation potential

A novel ternary recyclable Fe3O4/graphene/sulfur-doped g-C3N4 (Fe3O4/GE/SCN) composite catalyst was synthesized and adopted in a visible-light driven catalytic system for the degradation of ranitidine, which is an important precursor of the emerging disinfection by-product of N-nitrosodimethylamine (NDMA). The addition of GE and Fe3O4 significantly improved the interface charge transfer of SCN, increased the light collection efficiency and decreased the photogenerated charge recombination efficiency. Considering both the ranitidine removal efficiency and catalyst recovery, the Fe3O4 mass fraction of 20% (20%-Fe3O4/GE/SCN) was recommended. Ranitidine (≤2 mg/L) was completely removed in 60 min under the conditions of an initial pH of 7.0 and a 20%-Fe3O4/GE/SCN dose of 1.0 g/L, and its degradation fitted well with the pseudo first-order kinetics model. Electron paramagnetic resonance analysis and trapping experiments confirmed that ·O2−, ·OH and h+ participated in the degradation of ranitidine. Ranitidine was removed through the pathways of demethylation and hydroxylation based on the analysis of the detected degradation intermediates, and 57.3% of the NDMA formation potential (FP) was reduced after the reaction. The visible-light driven 20%-Fe3O4/GE/SCN catalytic technology is a promising method not only for the control of NDMA FP but also the catalyst could be recovered and reused.

Ecotoxicological risk assessment of pentachlorophenol, an emerging DBP to plants: evaluation of oxidative stress and antioxidant responses

Chlorophenols are not only noticed in an effluvium of industries but also can emerge from the water treatment plants for domestic supply which poses a high threat for crop production and human health. Therefore, research on their risks to ecosystem and human health via ecotoxicological tests to derivate permissible environmental contaminant concentrations is necessary. The chlorophenols produced in the course of chlorination of potable water is an outcome of natural carboxylic acids/organic material and those chlorophenols occurred as emerging disinfection byproducts (EDBPs). Among chlorophenols, pentachlorophenol (PCP) has been recently identified as one of the important EDBPs. The main objective was to evaluate the PCP-induced genotoxicity and the oxidative damage in two plant species, i.e., Allium cepa and Vigna radiata. Genotoxicity of PCP was examined at three selected concentrations based on EC50 (half-maximal effective concentrations) values in both the plants along with the defense mechanism. EC50 value for A. cepa and V. radiata was 0.7mg/L and 35mg/L. Root length inhibition, DNA laddering, lipid peroxidation, H2O2 content, and antioxidant enzymatic assays evaluated revealed a dose-dependent response. PCP influenced defense enzyme glutathione peroxidase (GPX) and ascorbate peroxidase (APX) action in both plants and showed deprivement of catalase (CAT) with the increase of PCP concentrations. PCP-invaded toxicity management by these plants implied that A. cepa is more sensitive than V. radiata regarding PCP-induced toxicity.

Monohaloacetonitriles induce cytotoxicity and exhibit different mode of action in endocrine disruption

Haloacetonitriles are emerging disinfection by-products that can be detected in various aquatic environments. They are cytotoxic, genotoxic, mutagenic, and tumorigenic in vitro and in vivo, but their endocrine-disrupting potency remains unknown. In this study, we examined the agonistic and antagonistic estrogenic and androgenic activities of haloacetonitriles, as well as their cytotoxicity, using a yeast-based reporter assay. We also investigated the interactions of haloacetonitriles with human estrogen receptor alpha (hERα) through molecular docking. We observed that iodoacetonitrile (median lethal dose: 1.96 × 10−5 M) and bromoacetonitrile (median lethal dose: 1.97 × 10−5 M) had similar cytotoxicities, which are higher than that of chloroacetonitrile (median lethal dose: 7.16 × 10−5 M). We observed bromoacetonitrile and chloroacetonitrile elicited estrogenic activity with 10% effective concentrations of 3.30 × 10−9 M and 2.36 × 10−9 M, respectively. This finding indicates that bromoacetonitrile and chloroacetonitrile may mimic estrogen signaling through interaction with hERα. Consistent with that result, we identified bromoacetonitrile and chloroacetonitrile interacted with residues in the original estrogen recognition sites of hERα. Our results show that bromoacetonitrile and chloroacetonitrile affect the endocrine-disrupting potency mediated via estrogen receptors by using in vitro assay and molecular docking.

Efficient removal of halogenated phenols by vacuum-UV system through combined photolysis and OH oxidation: Efficiency, mechanism and economic analysis

In this study, efficient simultaneous degradation and dechlorination of the photo-recalcitrant emerging disinfection byproduct, 2-chlorophenol (2-CP), was achieved by vacuum-ultraviolet (VUV) system for the first time. Different from the conventional UV system, the combined action of direct photolysis and OH oxidation in VUV system led to a significantly higher removal efficiency for 2-CP. In UV system, 2-CP degradation rate constants was independent of the initial 2-CP concentration, and was increased with enhancing pH. To the contrary, in VUV system, higher initial concentration of 2-CP resulted in lower rate constant, and the degradation rates of 2-CP under both acidic and alkaline conditions were higher than that at the neutral pH. Moreover, humic acid could inhibit 2-CP degradation more prominently in VUV system than in UV system, owing to the scavenging effect of OH by it. The degradation pathways of 2-CP were proposed based on the identified main degradation products by GC–MS/MS. Furthermore, degradation of the other seven typical halogenated phenols by VUV irradiation in tap water, ultrafiltrated water and Mill-Q water were investigated to verify the feasibility of the system. Based on the systematic economic analysis, VUV process is economically feasible for the advanced treatment of tap water to remove halogenated phenols.

Evaluation of hypopigmentation in embryonic zebrafish induced by emerging disinfection byproduct, 3, 5-di-I-tyrosylalanine

Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 μM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 μM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.

Formation of N-nitrosodimethylamine precursors through the microbiological metabolism of nitrogenous substrates in water

N-nitrosodimethylamine (NDMA) as one emerging disinfection by-product has been investigated globally since 1990s. However, its main precursors are still unclear. We found that NDMA formation potential (NDMAFP) of various water samples increased firstly and then decreased gradually during incubation with microorganism. We hypothesized that NDMA precursors could be produced through metabolism of nitrogenous components and then gradually be biodegraded. To verify this hypothesis, six amino acids (AAs), peptone and ammonium were separately incubated with microorganism and NDMAFP was measured regularly. The average molar yield of the substrates to NDMAFP were 60–200 × 10−6 for the AAs, 350 × 10−6 for peptone under aerobic condition. The extracellular fraction with molecular weight (MW) less than 1 k Dalton contributed the majority to NDMAFP in the peptone experiment, followed by that with MW between 10 k and 0.22 μm and the intracellular materials. Dimethylamine and methylamine were detected during the experiments but their contribution to NDMAFP is quite limited. The results indicate that the nitrosamine precursors may not be the direct metabolite of AAs or peptones but the excretion of living bacteria or the components in dead bacteria body. Our results inferred that AA metabolism may give an NDMAFP of 0.12 nmol/L (maximum) or 0.09 nmol/L (average) in water under aerobic condition. This estimation of NDMAFP from AA metabolism can account for 38% (maximum) or 27% (average) of the median NDMAFP in waters of China (0.32 nmol/L) reported before.