Comparative Developmental Toxicity of New Aromatic Halogenated

Shengkun DongMartin A. PageElizabeth D. WagnerMichael J. Plewa. Environmental ...... Andrea Sapone , Donatella Canistro , Fabio Vivarelli , Moreno Pao...
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Comparative Developmental Toxicity of New Aromatic Halogenated DBPs in a Chlorinated Saline Sewage Effluent to the Marine Polychaete Platynereis dumerilii Mengting Yang and Xiangru Zhang* Environmental Engineering Program, Department of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR, China S Supporting Information *

ABSTRACT: Using seawater for toilet flushing may introduce high levels of bromide and iodide into a city’s sewage treatment works, and result in the formation of brominated and iodinated disinfection byproducts (DBPs) during chlorination to disinfect sewage effluents. In a previous study, the authors’ group has detected the presence of many brominated DBPs and identified five new aromatic brominated DBPs in chlorinated saline sewage effluents. The presence of brominated DBPs in chlorinated saline effluents may pose adverse implications for marine ecology. In this study, besides the detection and identification of another seven new aromatic halogenated DBPs in a chlorinated saline sewage effluent, their developmental toxicity was evaluated using the marine polychaete Platynereis dumerilii. For comparison, the developmental toxicity of some commonly known halogenated DBPs was also examined. The rank order of the developmental toxicity of 20 halogenated DBPs was 2,5-dibromohydroquinone > 2,6diiodo-4-nitrophenol ≥ 2,4,6-triiodophenol > 4-bromo-2-chlorophenol ≥ 4-bromophenol > 2,4-dibromophenol ≥ 2,6-dibromo4-nitrophenol > 2-bromo-4-chlorophenol > 2,6-dichloro-4-nitrophenol > 2,4-dichlorophenol > 2,4,6-tribromophenol > 3,5dibromo-4-hydroxybenzaldehyde > bromoform ≥ 2,4,6-trichlorophenol > 2,6-dibromophenol > 2,6-dichlorophenol > iodoacetic acid ≥ tribromoacetic acid > bromoacetic acid > chloroacetic acid. On the basis of developmental toxicity data, a quantitative structure−activity relationship (QSAR) was established. The QSAR involved two physical−chemical property descriptors (log P and pKa) and two electronic descriptors (the lowest unoccupied molecular orbital energy and the highest occupied molecular orbital energy) to indicate the transport, biouptake, and biointeraction of these DBPs. It can well predict the developmental toxicity of most of the DBPs tested.

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sensitive habitats.8 When the brominated/iodinated DBPs are discharged along with chlorinated saline sewage effluents into marine water, they are likely to exert potential adverse effects on marine organisms and thus need to be evaluated. Commonly known DBPs such as haloacetic acids and trihalomethanes have been reported to form in chlorinated (saline) sewage effluents.9−12 Recently, by using a powerful precursor ion scan (PIS) method with ultra performance liquid chromatography/electrospray ionization-triple quadrupole mass spectrometry (UPLC/ESI-tqMS), besides the detection of brominated haloacetic acids, four new brominated DBPs (2,6dibromo-4-nitrophenol, 2,4,6-tribromophenol, 3,5-dibromo-4hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzoic acid) and one new brominated DBP (5-bromosalicylic acid) have been identified in chlorinated saline secondary and primary sewage effluents, respectively.13 These newly identified DBPs in the chlorinated saline effluents are phenolic aromatic

INTRODUCTION Seawater has been used for toilet flushing on an extensive scale in Hong Kong since the 1950s, which has greatly reduced local freshwater demand.1 Seawater is also used for toilet flushing in the city of Avalon and several Pacific island nations, including the Marshall Islands and Kiribati.2,3 The shortage of freshwater may compel many coastal cities or nations to adopt the practice of using seawater for toilet flushing. Such a practice introduces high levels of sea salts, including bromide and iodide ions (65 mg/L as Br− and 60−120 μg/L as I− + IO3− in seawater), into sewage treatment systems. Chlorination could be the most costeffective method for disinfecting saline sewage effluents. During chlorination, chlorine or chloramines (from the reaction of chlorine with ammonia in the sewage effluents) can oxidize the bromide/iodide ions to hypobromous/hypoiodous acid, which may then react with organic matter in saline sewage effluents to unintentionally form brominated/iodinated disinfection byproducts (DBPs). Evidence has shown that brominated/ iodinated DBPs generally are significantly more cytotoxic and genotoxic than their chlorinated analogs.4−7 The receiving water body of chlorinated saline sewage effluents is coastal marine water, which generally covers many ecologically © 2013 American Chemical Society

Received: Revised: Accepted: Published: 10868

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phenylene diamine (DPD) ferrous titrimetric method.24 3,5Dichloro-4-hydroxybenzaldehyde (97%) was purchased from International Laboratory USA. 2,6-Diiodo-4-nitrophenol (97%) and 3,5-dibromo-4-hydroxybenzaldehyde (98%) were purchased from Alfa Aesar. 3,5-Dibromo-4-hydroxybenzoic acid (98%) was purchased from Indofine Chemical Company. 2,6Dichloro-4-nitrophenol (96%), 2,4-dibromophenol (99%), 2bromo-4-chlorophenol (≥99%), and 4-bromo-2-chlorophenol (99%) were purchased from Acros Organics. 2,6-Dibromo-4nitrophenol (98%), 2,4,6-triiodophenol (97%), 2,4,6-tribromophenol (99%), 2,4,6-trichlorophenol (98%), 2,6-dibromophenol (99%), 2,4-dichlorophenol (99%), 2,6-dichlorophenol (99%), 4-bromophenol (99%), 2,5-dibromohydroquinone (97%), bromoform (99%), tribromoacetic acid (99%), bromoacetic acid (≥99%), dibromoacetic acid (99%), 5-bromosalicylic acid (90%), iodoacetic acid (≥99%), chloroform (≥99%), chloroacetic acid (99%), and all other chemicals (reagent grade or higher) used in this study were purchased from SigmaAldrich. Sampling, Characterization, and Chlorination of a Saline Sewage Effluent. An undisinfected sewage effluent sample (24-h composite) from a saline secondary sewage treatment plant was collected with a Teflon container. At the time this study was conducted, chlorination had not been implemented in the plant. The sample was transferred to the laboratory in an ice cooler immediately and stored at 4 °C to minimize changes in the constituents. Prior to the experiment, the sewage effluent sample was brought back to room temperature and filtered through a 0.45-μm membrane (for better comparison of the DBP formation at different chlorine doses). Some chemical characteristics of the filtered sewage effluent sample were measured, including pH (7.78), dissolved organic carbon (5.97 mg/L as C), Br− (21.0 mg/L), I− (21.5 μg/L), and NH4+ (0.99 mg/L as N). Aliquots of the filtered saline sewage effluent sample were chlorinated by dosing 6, 10, and 30 mg/L NaOCl as Cl2. These chlorine doses represented low, moderate, and high chlorine dose levels. Chlorination was conducted in headspace-free amber glass bottles. After a 30-min contact time, each aliquot was dechlorinated with 105% of the requisite stoichiometric amount of 1.0 M Na2S2O3. These chlorine doses and the contact time were chosen mainly because they were capable of meeting the disinfection goal.13 Pretreatment of Chlorinated Sewage Effluent Samples. The chlorinated saline sewage effluent samples were pretreated for UPLC/ESI-tqMS analysis. The pretreatment procedure was based on a previous study.25 Briefly, a 4-L sample was adjusted to pH 0.5 with 7:3 (v/v) concentrated sulfuric acid/water and 400 g of Na2SO4 was added. The sample was then extracted with 400 mL of methyl tert-butyl ether (MtBE). After extraction, the MtBE layer was transferred to a rotary evaporator and concentrated to 2.0 mL. The 2.0 mL solution in MtBE was mixed with 20 mL of acetonitrile, and the mixture was rotoevaporated back to 2.0 mL. The 2.0 mL solution in acetonitrile was stored at 4 °C. Prior to UPLC/ESItqMS analysis, the 2.0 mL solution was diluted with ultrapure water to 4.0 mL. To determine whether there were any artifacts in the collected effluent or in the extraction and concentration, a control sample was prepared by repeating the same procedure with the filtered saline sewage effluent sample without chlorination. To improve the detection of certain DBPs, aliquots of the pretreated chlorinated saline sewage effluent samples (in 1:1 water/acetonitrile) were adjusted to different

compounds. Because they are fully brominated, we speculated that their monobromo/dibromo and chloro/iodo analogs might also form in the chlorinated saline effluents under the same or a different dose of chlorine. For instance, the monobromo/dibromo and trichloro/triiodo analogs of 2,4,6tribromophenol include 4-bromophenol, 2,4-dibromophenol, 2,4,6-trichlorophenol, and 2,4,6-triiodophenol; the monobromo and dichloro/diiodo analogs of 2,6-dibromo-4-nitrophenol include 2-bromo-4-nitrophenol, 2,6-dichloro-4-nitrophenol, and 2,6-diiodo-4-nitrophenol. 2,4,6-Tribromophenol has also been identified as a drinking water DBP and can be hydrolyzed to form 2,6-dibromohydroquinone.14,15 A review of the identification process found that these aromatic brominated DBPs newly identified by Ding et al.13 were initially detected using the UPLC/ESI-tqMS PIS mode at m/z 79 and 81. Although this mode can selectively detect nearly all polar known/unknown brominated DBPs in a water sample, it is not as sensitive as the UPLC/ESI-tqMS multiple reaction monitoring (MRM) mode. For the speculated DBPs, if their corresponding standard compounds are available, the UPLC/ ESI-tqMS MRM mode could be expected to sensitively detect/ confirm their presence in chlorinated saline sewage effluents. Aromatic halogenated DBPs could accumulate in marine organisms and thus be possibly more toxic than aliphatic halogenated DBPs. Quite a number of studies on the toxicity of drinking water DBPs have focused on mutagenicity, genotoxicity, and toxicogenomic analyses using Salmonella typhimurium, Chinese hamster ovary cells, or human cells.7,16−21 Few have investigated the toxicity of DBPs in chlorinated sewage effluents, especially chlorinated saline sewage effluents, to marine organisms. An in vivo assay based on the sensitive embryo-larval stages of a widely distributed marine polychaete Platynereis dumerilii has been designed for evaluating the developmental toxicity of marine contaminants.22,23 The developmental toxicity of the DBPs in chlorinated saline sewage effluents to the marine polychaete should provide intuitive insight into the adverse effects on the marine ecosystem. The objectives of this study were thus (1) to detect and identify more new aromatic halogenated DBPs that are structurally related to the aromatic brominated DBPs previously identified in chlorinated saline sewage effluents; (2) to quantitatively analyze and compare the developmental toxicity of the new aromatic halogenated DBPs and some commonly known halogenated DBPs using the embryos of the marine polychaete P. dumerilii; and (3) to develop a quantitative structure−activity relationship (QSAR) for the developmental toxicity of the tested DBPs to P. dumerilii. The results will aid in better understanding the biological impacts of chlorinated saline sewage DBPs on the marine ecology.

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MATERIALS AND METHODS Chemicals, Reagents, and Seawater. Ultrapure water (18.2 MΩ·cm) was obtained from a NANOpure Diamond purifier system (Barnstead). Seawater with a salinity of 3.5% was collected from Clear Water Bay, Hong Kong. It was filtered through a 0.22-μm membrane and autoclaved at 120 °C for 20 min. After cooling to 19 °C, the autoclaved seawater was aerated for approximately 1 h and then used for culturing the polychaete and conducting the developmental toxicity tests. A stock solution of sodium hypochlorite was purchased from Allied Signal and standardized using the N,N-diethyl-p10869

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Table 1. Occurrence of Halogenated Aromatic DBPs in the Chlorinated Saline Secondary Sewage Effluent

a b

Occurrences a, b, and c indicate the chlorinated saline secondary effluent samples with chlorine doses of 6, 10, and 30 mg/L as Cl2, respectively. Newly identified DBPs in this study.

basin containing 500 mL of the seawater for the developmental toxicity tests. Bioassay Method. Hutchinson et al.22 have developed a bioassay method that made use of the embryos of the polychaete. The method was adopted and modified (for reasons to be given in Results and Discussion) in this study. To initiate the bioassay of a DBP, the embryos at 12-h postfertilization (approximately 170 μm in diameter) were collected from the incubation glass basin using a 100-μm nylon mesh and transferred into a 60-mm Petri dish (with a working volume of 10 mL) that contained the required dilution of the DBP in the seawater. The embryos (approximately 15 embryos/mL) were allowed to develop for a further 12 h. Control samples were prepared by allowing the embryos to develop in the seawater for a further 12 h still. By 24-h postfertilization, normal embryos are expected to have reached the first larval (trochophore) stage, characterized by the presence of distinct ciliary bands, prominent fat droplets, and active swimming activity. Abnormal embryos lack some or all of these characteristics. The numbers of normal and total embryos in each DBP test solution were checked with a stereo microscope (magnification by 14 times), and the percent normal development was calculated. By plotting the curve of the percent normal development versus the DBP concentration (i.e., a concentration−response curve), the median effective concentration (EC50) of the DBP (i.e., the DBP concentration at which 50% of the embryos developed normally) was obtained. Preparation of Test Solutions of DBPs. The preparation of a series of DBP test solutions at different concentrations involved diluting appropriate aliquots of the stock solution with the seawater, followed by adjusting the pH to 8.1 (pH of the seawater) with 0.1 M HCl or 0.2 M NaOH. A preliminary bioassay was conducted to determine the critical range. The lowest and highest concentrations could induce a significant amount of abnormal development (the percent normal development at the concentration was 5% less than that of the seawater control sample) and 100% abnormal development,

pH values with acetic acid or ammonium hydroxide, which are volatile buffers compatible with ESI.26 UPLC/ESI-tqMS Analysis. The pretreated samples were analyzed using a UPLC/ESI-tqMS system (Waters). A pretreated sample (7.5 μL) was injected into the UPLC. The UPLC separation was carried out with an HSS T3 column (100 × 2.1 mm, 1.8 μm particle size, Waters). The eluent was composed of methanol and water. The composition of methanol/water (v/v) changed linearly from 5/95 to 90/10 in the first 8 min, and then returned in 0.10 min to 5/95, which was held for 2.9 min for re-equilibration. The flow rate was 0.40 mL/min and the column temperature was 35 °C. The setting of the parameters of the ESI-tqMS system followed a previous report.15 Based on a previous study,13 it was speculated that a series of aromatic halogenated DBPs might also form in the chlorinated saline sewage effluent. To confirm the formation of each DBP, the UPLC/ESI-tqMS MRM mode was applied to obtain the retention time (RT) and the corresponding isotopic abundance ratio of the molecular ion cluster. Then for the molecular ion cluster displaying the correct abundance ratio, the corresponding standard compound was purchased and analyzed under the MRM mode; the sample was spiked with the standard compound to confirm its presence. Finally, by spiking different levels of the standard compound to a sample, the concentration of the compound in the sample was determined with the MRM mode. Polychaete Culturing. Stock cultures of P. dumerilii were maintained using Dorresteijn’s procedure.27 Briefly, the polychaete was cultured on a diet of algae, fish flakes, and chopped spinach, and held in the prepared seawater at 19 ± 1 °C. Breeding was controlled by photoperiod manipulation giving 16 h of light (light intensity approximately 300 lx) followed by 8 h of absolute darkness. Mature males and females were transferred from the culturing tanks and allowed to spawn naturally in approximately 50 mL of the seawater. After fertilization success (routinely >95%, as indicated by the first cellular cleavage) was confirmed, the developing embryos were transferred to an incubation glass 10870

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Table 2. Comparison of the P. dumerilii Embryo Developmental Toxicity, log P, pKa, ELUMO, and EHOMO of DBPs EC50a (μM)

compound 2,6-diiodo-4-nitrophenol 2,6-dibromo-4-nitrophenol 2,6-dichloro-4-nitrophenol 2,4,6-triiodophenol 2,4,6-tribromophenol 2,4,6-trichlorophenol 2,4-dibromophenol 2,6-dibromophenol 2-bromo-4-chlorophenol 4-bromo-2-chlorophenol 2,4-dichlorophenol 2,6-dichlorophenol 4-bromophenol 3,5-dibromo-4-hydroxybenzaldehyde 2,5-dibromohydroquinone bromoform iodoacetic acid tribromoacetic acid bromoacetic acid 3,5-dibromo-4-hydroxybenzoic acid 5-bromosalicylic acid dibromoacetic acid chloroform chloroacetic acid a

2.02 8.45 1.12 2.04 1.95 7.40 8.31 1.13 9.43 7.55 1.73 1.37 7.75 2.19 9.12 7.30 3.67 3.86 1.40 >4.37 >4.61 >1.80 >3.20 >9.82

× × × × × × × × × × × × × × × × × × × × × × × ×

1

10 101 102 101 102 102 101 103 101 101 102 103 101 102 100 102 103 103 104 103 103 104 104 104

R2

log P

pKa

ELUMO

EHOMO

0.98 0.96 0.97 0.98 0.98 0.99 0.95 0.95 0.97 0.95 0.96 0.94 0.99 0.97 0.98 0.97 0.96 0.96 0.97 NAb NAb NAb NAb NAb

4.243 3.688 3.197 5.014 4.183 3.446 3.293 3.293 3.047 3.047 2.802 2.802 2.403 3.010 2.813 1.790 0.845 1.711 0.430

3.85 3.67 3.81 6.47 6.34 6.59 7.86 6.89 7.98 7.92 8.05 7.02 9.34 4.72 7.90 NAb 3.18 0.22 2.73

−1.419 −1.451 −1.439 −0.590 −0.621 −0.501 −0.299 −0.378 −0.277 −0.230 −0.196 −0.258 0.020 −0.887 −0.563 −0.747 −0.537 −1.204 −0.274

−10.244 −10.217 −10.174 −9.570 −9.503 −9.390 −9.330 −9.443 −9.275 −9.288 −9.230 −9.373 −9.190 −9.744 −9.082 −11.071 −11.043 −11.232 −11.301

EC50 values were obtained with the four-parameter logistic equation by regression analysis: Max − Min

% Normal development = Min + 1 +

(

Concentration EC50

−Hillslope

)

where Min, Max, EC50, and Hillslope are the four parameters. Min, Max, and Hillslope indicate bottom of the curve, top of the curve, and slope of the curve at its midpoint, respectively. bNA indicates “not available”.

aromatic halogenated DBPs detected in the chlorinated saline sewage effluent, of which seven were identified as wastewater DBPs for the first time, including 4-bromophenol, 2,4dibromophenol, 2,4,6-triiodophenol, 2,6-dichloro-4-nitrophenol, 2,6-diiodo-4-nitrophenol, 3,5-dichloro-4-hydroxybenzaldehyde, and 2,5-dibromohydroquinone. Figure S1 in the Supporting Information (SI) shows the UPLC/ESI-tqMS MRM chromatograms and corresponding spectrum of 4bromophenol, the chlorinated saline secondary effluent sample (with a chlorine dose of 10 mg/L as Cl2), and the chlorinated saline secondary effluent sample mixed with 4-bromophenol. The RT (6.06 min) and the mass spectrum of the peak in the chlorinated saline secondary effluent sample were the same as those of the standard compound of 4-bromophenol, confirming the formation of 4-bromophenol as a new wastewater DBP. The UPLC/ESI-tqMS MRM chromatograms and corresponding spectra of other newly identified DBPs are also shown in the SI and Figures S2−S7. By following the procedure described in the SI and Figure S8, the concentrations of the seven newly identified aromatic DBPs in the chlorinated saline secondary effluent sample with the chlorine dose of 6 mg/L as Cl2 were measured to be as follows: 4-bromophenol 1.62 μg/L, 2,4-dibromophenol 0.58 μg/L, 2,4,6-triiodophenol 0.40 ng/L, 2,6-dichloro-4-nitrophenol 0.13 μg/L, 2,6-diiodo-4-nitrophenol 1.13 ng/L, 3,5-dichloro-4-hydroxybenzaldehyde 28.9 ng/L, and 2,5-dibromohydroquinone 0.20 μg/L. It needs emphasizing that it was not easy to detect 4bromophenol and 2,4-dibromophenol in the pretreated

respectively. Based on the critical range, a series of DBP test solutions at different concentrations (with pH adjusted to 8.1) were prepared and another bioassay was conducted. A concentration−response curve was obtained for the DBP. The concentrations of the DBP test solutions were expressed in micromole per liter to facilitate comparison.

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RESULTS AND DISCUSSION Newly Identified DBPs in the Chlorinated Saline Secondary Effluent. As aforementioned, four aromatic brominated DBPs including 2,4,6-tribromophenol, 2,6-dibromo-4-nitrophenol, 3,5-dibromo-4-hydroxybenzaldehyde, and 3,5-dibromo-4-hydroxybenzoic acid have been identified in a chlorinated saline secondary sewage effluent.13 It was speculated that their monobromo/dibromo and chloro/iodo analogs might also form in the chlorinated saline effluent under the same or a different dose of chlorine. It was noticed that the aromatic brominated DBPs identified by Ding et al.13 were initially detected using the UPLC/ESI-tqMS PIS mode at m/z 79 and 81. Despite the fact that the UPLC/ESI-tqMS PIS mode can selectively detect nearly all known and unknown halogenated DBPs, the UPLC/ESI-tqMS MRM mode can be 1 order of magnitude more sensitive in detecting structurally known DBPs like the speculated ones. As expected, a number of new aromatic halogenated DBPs were detected in the chlorinated saline secondary sewage effluent by the UPLC/ESItqMS MRM mode. For those with standard compounds available, their structures were confirmed. Table 1 lists eleven 10871

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2,4,6-trichlorophenol were considered as “putative” wastewater DBPs and also included in the developmental toxicity tests. Bioassay Method Modification. Initially, the bioassay method developed by Hutchinson et al.22 was directly adopted in this study, but the results were not satisfactory. We noticed that this method evaluated the developmental toxicity to the polychaete embryos aged 12-h postfertilization at the end of a 7-h period of exposure. According to Hutchinson et al.,22 normal embryos should have reached the trochophore stage by 19-h postfertilization, in which case they should exhibit distinct ciliary bands, prominent fat droplets, and active swimming activity. However, during the counting time slot (usually 1−2 h), the number of embryos having reached the trochophore stage kept increasing. As a result, the percent normal development for a sample that was counted later was higher than that of a duplicate sample that was counted earlier. The relative standard deviation of the percent normal development for duplicate samples was as high as 30%. Therefore, it was likely impossible to evaluate the comparative toxicity of samples with subtle disparity by applying the method directly. In the course of investigating the aforementioned problem with the method, it was found that not all of the polychaete embryos were developed enough to show the characteristics of the trochophore stage by 19-h postfertilization. Some of them reached the trochophore stage at a much later time (SI Figure S10). Many embryos in the seawater started to swim at 16-h postfertilization. It was not until 24-h postfertilization that the percent normal development stopped increasing and approached a relatively stable period. Dorresteijn and Fischer29 also investigated the development of P. dumerilii, and suggested that the trochophore larva hatches from the egg jelly by approximately 20−24-h postfertilization. Hence, we modified the method by counting the percent normal development after a 12-h exposure period (24-h postfertilization), when all the embryos in the seawater control samples had reached the trochophore stage. The improved method, which gave a much lower relative standard deviation of the percent normal development of 2,6-diiodo-4nitrophenol ≥ 2,4,6-triiodophenol > 4-bromo-2-chlorophenol ≥ 4-bromophenol > 2,4-dibromophenol ≥ 2,6-dibromo-4nitrophenol > 2-bromo-4-chlorophenol > 2,6-dichloro-4-nitrophenol > 2,4-dichlorophenol > 2,4,6-tribromophenol > 3,5dibromo-4-hydroxybenzaldehyde > bromoform ≥ 2,4,6-trichlorophenol > 2,6-dibromophenol > 2,6-dichlorophenol >

chlorinated sewage effluent samples even with the UPLC/ESItqMS MRM. A major factor affecting the sensitivity of the detection was found to be pH. Aliquots of the pretreated chlorinated saline secondary effluent samples (in 1:1 water/ acetonitrile) were adjusted to different pH values with acetic acid or ammonium hydroxide. The results showed that the peak of 4-bromophenol or 2,4-dibromophenol in the MRM chromatogram was most intense when the pretreated chlorinated sewage effluent samples were adjusted to pH 2.5 by adding 1% acetic acid. The pKa values of 4-bromophenol and 2,4-dibromophenol are 9.34 and 7.86, respectively (Table 2; pKa values for all compounds used in this study were obtained from the SciFinder database). At a relatively high pH value, the two compounds may be partially ionized to the corresponding phenolate anions, which may result in the formation of dimers in the eluent (water/methanol). However, such dimers could be in a transitional state and undergo alternate dissociation and formation as they move in the column, leading to the formation of “flattened” dimer peaks and the diminution of monomer peaks. After the pH of the pretreated samples was lowered by adding 1% acetic acid, 4bromophenol and 2,4-dibromophenol should have been present in their neutral forms, and the formation of their dimers might be inhibited (SI Figure S9). This explains the intense peak in the MRM chromatograms of the acidified samples. For 2,4,6-tribromophenol, due to steric effect, it might not form a dimer in the eluent and thus was readily detected by the MRM. It should be noted that two aromatic iodinated DBPs, 2,6diiodo-4-nitrophenol and 2,4,6-triiodophenol, were detected in the chlorinated saline secondary effluent sample with a chlorine dose of 6 mg/L as Cl2, but not in the samples with chlorine doses of 10 and 30 mg/L as Cl2. The ammonia concentration in the collected saline secondary effluent sample was 0.99 mg/L as N. The chlorine dose for the breakpoint chlorination can be calculated to be 7.5 mg/L as Cl2. The actual breakpoint should somewhat exceed this value due to the presence of readily reducing substances in the effluent sample. Thus, for the saline secondary effluent sample with a chlorine dose of 6 mg/L as Cl2, the chlorine should exist mainly in the form of monochloramine; whereas for the saline secondary effluent samples with chlorine doses of 10 and 30 mg/L as Cl2, the chlorine residual should be present mainly as free chlorine. Both free chlorine and monochloramine can oxidize iodide to hypoiodous acid, which then reacts with organic matter to form iodinated DBPs, but free chlorine can further oxidize hypoiodous acid to iodate,28 leading to the formation of less iodinated DBPs in chlorination than in chloramination. Consequently, the two iodinated aromatic DBPs were detected only in the chlorinated saline secondary effluent sample with the relatively low chlorine dose. A recent study has shown that, for four groups of new aromatic halogenated DBPs in chlorinated drinking water, decreasing the bromide concentration shifts the brominated species to the corresponding bromochloro-mixed species and then to the corresponding fully chlorinated species.15 While 4bromophenol and 2,4-dibromophenol were identified for the first time as wastewater DBPs in the chlorinated saline sewage effluent, it is not surprising that other bromo-/chloro-phenols would also form during chlorination of wastewater effluents containing relatively low bromide concentrations. Therefore, 2,6-dibromophenol, 2-bromo-4-chlorophenol, 4-bromo-2chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, and 10872

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Thus, it seems to be well established that iodinated/brominated DBPs are significantly more toxic than their chlorinated analogs. Third, nitrogenous DBPs induced remarkably higher developmental toxicity than their carbonaceous analogs or even carbonaceous brominated analogs. For instance, 2,6dibromo-4-nitrophenol was 13.4 and 2.3 times more developmentally toxic than 2,6-dibromophenol and 2,4,6-tribromophenol, respectively; and 2,6-dichloro-4-nitrophenol was 12.2 and 6.6 times more developmentally toxic than 2,6-dichlorophenol and 2,4,6-trichlorophenol, respectively. In vitro mammalian cell cytotoxicity and genotoxicity assays have indicated substantially higher toxicity of nitrogenous DBPs than carbonaceous DBP analogs.31 Fourth, isomers of a DBP might exhibit different levels of developmental toxicity. 2,4-Dihalophenols exhibited considerably higher developmental toxicity than the corresponding 2,6dihalophenols, e.g., 2,4-dibromophenol was 13.6 times more toxic than 2,6-dibromophenol, and 2,4-dichlorophenol was 7.9 times more toxic than 2,6-dichlorophenol. By contrast, 2bromo-4-chlorophenol and 4-bromo-2-chlorophenol exhibited similar levels of developmental toxicity. Richardson et al.7 have found that the E isomer of 3-bromo-3-iodopropenoic acid was more toxic than the Z isomer; e.g., in Chinese hamster ovary cell chronic cytotoxicity analysis, the E isomer was 1.43 times more cytotoxic than the Z isomer; and in single-cell gel electrophoresis, the E isomer was genotoxic but the Z isomer was not. Fifth, the number of halogens in a DBP might affect the developmental toxicity of the DBP. The rank order of the developmental toxicity of brominated phenols was 4bromophenol > 2,4-bromophenol > 2,4,6-tribromophenol, while the rank order of brominated acetic acids was tribromoacetic acid > bromoacetic acid > dibromoacetic acid. Sixth, 2,5-dibromohydroquinone exhibited the highest developmental toxicity among all the DBPs tested. QSAR of the DBPs. QSARs have been widely used to predict toxicity from chemical structure and corresponding physicochemical properties (known as molecular descriptors).32,33 For environmental end points, the most frequently used descriptor is the logarithm of the octanol−water partition coefficient (log P); complex descriptors including the electronic, quantum mechanical, and topological properties become more important nowadays.34 Log P is a measure of lipophilicity which correlates with cell permeability and is an important factor in toxicity. Table 2 lists the log P values, which are estimated with the KOWWIN program (version 1.68) developed by the U.S. Environmental Protection Agency. A higher log P value implies that the corresponding compound may possess a higher cellular uptake efficiency and thus is capable of inflicting more harm on organisms. This was found to be true for most groups of DBPs tested. In particular, aromatic and aliphatic DBPs have log P values varying in the ranges of 2.40−5.01 and 0.43−1.79, respectively, so aromatic DBPs were found to be generally more developmentally toxic than aliphatic DBPs. Xia et al.35 have investigated the mitochondrial dysfunction induced by the intake of diesel exhaust particulate matter and reported that the aromatic fraction exerted more toxic effects than the aliphatic fraction to RAW 264.7 cellsthe aromatic fraction induced mitochondrial swelling, yet the aliphatic fraction failed to perturb the mitochondrial function. Sikkema et al.36 have shown that the (cytoplasmic) membrane is the primary site of

Figure 1. Concentration−response curves of the developmental toxicity of 19 DBPs to P. dumerilii embryos.

iodoacetic acid ≥ tribromoacetic acid > bromoacetic acid > chloroacetic acid (“≥” was used when the difference of the EC50 values of two compounds was less than 5% of the larger EC50 value). Notably, the EC50 values of these DBPs were obtained from different batches of embryos, and the percentages of normal development of seawater control samples from different batches of embryos were different, but this basically had no impact on the EC50 value of a DBP, e.g., the EC50 values of 2,4,6-tribromophenol from three batches of embryos were 195.4, 193.8, and 198.5 μM, with a relative standard deviation of 1.2%. Based on the EC50 values of the tested DBPs (Table 2), some important points could be made. First, aromatic halogenated DBPs generally presented higher developmental toxicity than aliphatic halogenated DBPs. The EC50 values of the aromatic and aliphatic halogenated DBPs generally ranged from 103 to 100 and from 104 to 102 μM, respectively. The comparative toxicity of commonly known aliphatic halogenated DBPs has been well studied, but the comparative toxicity of aromatic halogenated DBPs is only being evaluated for the first time now. Second, iodinated DBPs presented higher developmental toxicity than their brominated analogs, which in turn presented higher developmental toxicity than their chlorinated analogs. For the three groups of halogenated DBPs tested, the rank order of the developmental toxicity was 2,4,6-triiodophenol > 2,4,6-tribromophenol > 2,4,6-trichlorophenol; 2,6-diiodo-4nitrophenol > 2,6-dibromo-4-nitrophenol > 2,6-dichloro-4nitrophenol; and iodoacetic acid > bromoacetic acid > chloroacetic acid. The same rank order, iodoacetic acid > bromoacetic acid > chloroacetic acid, has been found for cytotoxicity and genotoxicity in human cells, mutagenicity in Salmonella typhimurium, genotoxicity in Chinese hamster ovary cells, and teratogenicity in mouse embryos.16,18,19,30 The rank order, diiodoacetic acid > dibromoacetic acid > dichloroacetic acid has also been observed for cytotoxicity in Chinese hamster ovary cells.7 Echigo et al.6 have demonstrated that the activity inducing chromosomal aberrations of the mixture of brominated DBPs per unit total organic halogen (TOX) was roughly 2−6 times higher than that of chlorinated DBPs per unit TOX. 10873

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as the toxicity descriptors in this study, and their values can be calculated with MOPAC 2012 software. After the addition of pKa, or ELUMO−EHOMO, or both, to eq 1, regression analysis (with JMP10 statistic software) was performed again:

toxic action of phenolic compounds, which could change membrane functioning and influence protein-to-lipid ratios in the membrane. For the three groups of DBPs tested (trihalogenated phenols, 2,6-dihalogenated-4-nitrophenols, and monohaloacetic acids), iodinated DBPs presented higher developmental toxicity than their brominated analogs, which in turn presented higher developmental toxicity than their chlorinated analogs; their log P values follow the rank order of iodinated DBPs > brominated analogs > chlorinated analogs. The plot of the developmental toxicity against log P is shown in SI Figure S12a. By conducting a regression analysis of log EC50−1 versus log P of the DBPs, a QSAR can be derived (eq 1). log EC50−1 = 0.5742log P − 4.1437

log EC50−1 = 0.5227log P + 0.0641pK a − 4.3554

(2)

2

where R = 0.6858. log EC50−1 = 0.3626log P − 0.3982E LUMO + 0.4391 E HOMO + 0.5652

(3)

2

where R = 0.6843. log EC50−1 = 0.3535log P + 0.7243pK a − 1.7101E LUMO

(1)

− 1.4132E HOMO − 22.5945

where R2 = 0.6542. This QSAR illustrates the anticipated importance of the hydrophobicity parameter. SI Figure S12b plots the observed toxicity against the toxicity predicted by eq 1. However, from a prediction standpoint, eq 1 is not satisfactory, since there are obvious outliers in this figure. Therefore, other additional physicochemical descriptors were sought to obtain a better correlation. The parameter pKa has been reported to be important in the prediction of toxicity in a QSAR study.37 An organic weak acid has two molecular forms (ionized and nonionized) in aquatic solutions. A significant degree of ionization will occur when the pH is greater than the pKa of an acid. It is suggested that the ionized and nonionized forms have different toxic responses, and that the latter may play an important role in toxicity.38 In particular, it is believed that the toxicity decreases with increasing ionization by decreasing the biouptake of extremely ionizable compounds.39 Molecular toxicology studies have indicated that organic compounds exert active effects on living organisms primarily during the interactions between the compounds and the target molecules.40 From a chemical perspective, the nucleophilic sites in peptides, proteins, and nucleic acids are important biological targets for electrophilic chemicals.32 It has been reported that a nitro substituent on an aromatic ring has a strong electronwithdrawing effect, which may result in a number of potential electrophilic reactions in vivo.41,42 The electrophilic−nucleophilic interactions can occur through various mechanisms including nucleophilic substitution, Schiff’s base formation, and the Michael addition, where the reactions are not specific and can produce various adverse outcomes.37,43 A quantum chemical method has already been proven to be very useful in elucidating the reactivity of a molecule, which is closely linked to its frontier molecular orbitals, including the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO). The LUMO energy (ELUMO) is related to the relative electrophilicity, and a lower value suggests that the corresponding molecule tends to accept electrons, thus possessing a higher potential to be reduced.44 Moreover, it has been reported that ELUMO is essentially related to molecular reactions with nucleophiles, whereas a higher HOMO energy (EHOMO) corresponds to more reactive reactions with electrophiles.45 The quantum chemical method made us speculate that the toxicity of an electrophilic DBP to a nucleophile site and the toxicity of a nucleophilic DBP to an electrophile site may increase with decreasing ELUMO and increasing EHOMO. Hence, ELUMO and EHOMO were considered

(4)

2

where R = 0.8650. With the introduction of either pKa or ELUMO−EHOMO to eq 1, the R2 value increased slightly. However, with the introduction of both pKa and ELUMO−EHOMO to eq 1, the R2 value increased dramatically and the data points became less spread out (SI Figure S12c−e). The EC50 values predicted from eq 4 were closer to the observed ones than those predicted from eqs 1−3 (SI Table S1), indicating that all of the descriptors (ELUMO, EHOMO, and pKa) play important roles in the developmental toxicity. It should be pointed out that for eqs 1−4, 2,5dibromohydroquinone was excluded as it significantly deviated from the trends. For this compound (log P = 2.8126, pKa = 7.9, ELUMO = −0.563, and EHOMO = −9.082), the log EC50−1 value predicted from eq 4 was −2.081, which was lower than the observed one (−0.960), so its highest toxicity cannot be explained by its hydrophobic-dependent penetration, pKa, or the electrophilic reactivity. It has been demonstrated that the ease with which hydroxyphenols can be metabolized or oxidized to quinones makes them more toxic than expected. 46,47 Recently, it has been reported that some haloquinones can bind to oligodeoxynucleotides through Hbonding modes48 and can generate intracellular reactive oxygen species in T24 bladder cancer cells.49 It seems that once 2,5dibromohydroquinone entered the cells of the embryos, it was metabolized or oxidized to 2,5-dibromoquinone. Consequently, its cell permeability was determined mainly by its log P and pKa and its interaction with biological targets was determined mainly by the ELUMO (−2.200) and EHOMO (−10.857) of 2,5dibromoquinone. For such a combination, the log EC50−1 value predicted from eq 4 was 3.227, much higher than the observed one (−0.960). As the extent of 2,5-dibromohydroquinone’s conversion to 2,5-dibromoquinone in the embryos during the test was unknown, the developmental toxicity of 2,5dibromohydroquinone was essentially a combination of the toxicities of both compounds. The analytical and toxicological data provided through this study can help prioritize future research on the ecological effects of wastewater DBPs. Aromatic halogenated DBPs generally presented significantly higher developmental toxicity than aliphatic halogenated DBPs. In particular, two of the seven newly identified DBPs in the chlorinated saline sewage effluent, 2,5-dibromohydroquinone and 2,6-diiodo-4-nitrophenol, were hundreds to thousands of times more toxic than the commonly known aliphatic DBPs, suggesting that more attention should be paid to the emerging aromatic halogenated DBPs. Although 10874

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M. J. Occurrence and mammalian cell toxicity of iodinated disinfection byproducts in drinking water. Environ. Sci. Technol. 2008, 42 (22), 8330−8338. (8) Hong Kong 2030: Planning Vision and Strategy − Strategic Environmental Assessment; No. CE25/2001; Planning Department of the Government of the Hong Kong SAR: Hong Kong, 2007; http:// www.epd.gov.hk/epd/SEA/eng/file/FinalSEAReport.pdf. (9) Yang, X.; Shang, C.; Huang, J. C. DBP formation in breakpoint chlorination of wastewater. Water Res. 2005, 39 (19), 4755−4767. (10) Sun, Y. X.; Wu, Q. Y.; Hu, H. Y.; Tian, J. Effect of bromide on the formation of disinfection by-products during wastewater chlorination. Water Res. 2009, 43 (9), 2391−2398. (11) Krasner, S. W.; Westerhoff, P.; Chen, B.; Rittmann, B. E.; Amy, G. Occurrence of disinfection byproducts in United States wastewater treatment plant effluents. Environ. Sci. Technol. 2009, 43 (21), 8320− 8325. (12) Krasner, S. W.; Westerhoff, P.; Chen, B.; Rittmann, B. E.; Nam, S. N.; Amy, G. Impact of wastewater treatment processes on organic carbon, organic nitrogen, and DBP precursors in effluent organic matter. Environ. Sci. Technol. 2009, 43 (8), 2911−2918. (13) Ding, G.; Zhang, X.; Yang, M.; Pan, Y. Formation of new brominated disinfection byproducts during chlorination of saline sewage effluents. Water Res. 2013, 47 (8), 2710−2718. (14) Zhai, H.; Zhang, X. Formation and decomposition of new and unknown polar brominated disinfection byproducts during chlorination. Environ. Sci. Technol. 2011, 45 (6), 2194−2201. (15) Pan, Y.; Zhang, X. Four groups of new aromatic halogenated disinfection byproducts: Effect of bromide concentration on their formation and speciation in chlorinated drinking water. Environ. Sci. Technol. 2013, 47 (3), 1265−1273. (16) Kargalioglu, Y.; McMillan, B. J.; Minear, R. A.; Plewa, M. J. Analysis of the cytotoxicity and mutagenicity of drinking water disinfection by-products in Salmonella typhimurium. Teratog., Carcinog. Mutagen. 2002, 22 (2), 113−128. (17) Cemeli, E.; Wagner, E. D.; Anderson, D.; Richardson, S. D.; Plewa, M. J. Modulation of the cytotoxicity and genotoxicity of the drinking water disinfection byproduct iodoacetic acid by suppressors of oxidative stress. Environ. Sci. Technol. 2006, 40 (6), 1878−1883. (18) Plewa, M. J.; Wagner, E. D.; Jazwierska, P. Halonitromethane drinking water disinfection byproducts: Chemical characterization and mammalian cell cytotoxicity and genotoxicity. Environ. Sci. Technol. 2004, 38 (1), 62−68. (19) Attene-Ramos, M. S.; Wagner, E. D.; Plewa, M. J. Comparative human cell toxicogenomic analysis of monohaloacetic acid drinking water disinfection byproducts. Environ. Sci. Technol. 2010, 44 (19), 7206−7212. (20) Itoh, S.; Gordon, B. A.; Callan, P.; Bartram, J. Regulations and perspectives on disinfection by-products: Importance of estimating overall toxicity. J. Water Supply Res. Technol. 2011, 60 (5), 261−274. (21) Neale, P. A.; Antony, A.; Bartkow, M. E.; Farré, M. J.; Heitz, A.; Kristiana, I.; Tang, J. Y. M.; Escher, B. I. Bioanalytical assessment of the formation of disinfection byproducts in a drinking water treatment plant. Environ. Sci. Technol. 2012, 46 (18), 10317−10325. (22) Hutchinson, T. H.; Jha, A. N.; Dixon, D. R. The Polychaete Platynereis dumerilii (Audouin and Milne-Edwards): A new species for assessing the hazardous potential of chemicals in the marine environment. Ecotoxicol. Environ. Saf. 1995, 31 (3), 271−281. (23) Palau-Casellas, A.; Hutchinson, T. H. Acute toxicity of chlorinated organic chemicals to the embryos and larvae of the marine worm Platynereis dumerilii (Polychaeta: Nereidae). Environ. Toxicol. Water Qual. 1998, 13 (2), 149−155. (24) APHA, AWWA, and WEF. Standard Methods for the Examination of Water and Wastewater, 19th ed.; Washington, DC, 1995. (25) Zhang, X.; Talley, J. W.; Boggess, B.; Ding, G.; Birdsell, D. Fast selective detection of polar brominated disinfection byproducts in drinking water using precursor ion scans. Environ. Sci. Technol. 2008, 42 (17), 6598−6603.

concentrations of aromatic halogenated DBPs in the chlorinated saline sewage effluent were lower than those used in the bioassays for the “acute” (12-h exposure) developmental toxicity, exposure to and accumulation of these aromatic DBPs present in receiving marine water may persist throughout marine organisms’ life, which may still have chronic adverse effects on marine organisms. Also, due to the presence of a countless number of DBPs in the chlorinated saline sewage effluent, combination effects may occur even if DBPs are present at levels below their individual no-observed-effect concentrations. In consideration of the complexity of chlorinated saline sewage effluents, the overall toxicity of the effluents (containing DBP mixtures) needs to be evaluated and compared. In addition, the bioassay method for developmental toxicity analysis, which was modified and improved in this study, provides a sensitive metric for adverse biological responses and may be used for nongenotoxic end points.

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ASSOCIATED CONTENT

S Supporting Information *

Additional details, Figures S1−S12, and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +852-2358-8479; fax: +852-2358-1534; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (project 622808). We are grateful to Dr. Adriaan Dorresteijn of the University of Mainz, Germany, for providing parental P. dumerilii. We thank Guoyu Ding, Rui Yu, and Lin Ai for culturing the polychaete and assisting in the toxicity tests.

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REFERENCES

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