Monitoring the concentrations of nonsteroidal anti-inflammatory drugs and cyclooxygenase-inhibiting activities in the surface waters of the Tone Canal and Edo River Basin.

Environmental pollution by pharmaceuticals has become a major problem in many countries worldwide. However, little is known about the concentrations of pharmaceuticals in water sources in Japan. The objective of this study was to clarify variations in the concentrations of seven nonsteroidal anti-inflammatory drugs (NSAIDs) and in cyclooxygenase(COX)-inhibiting activities in river water and domestic wastewater collected from the Tone Canal and the Edo River Basin in Japan. Total NSAID concentrations were higher in the Tone Canal than in the Edo River, and the highest concentration was observed at the domestic wastewater inflow point located in the Tone Canal (concentration averages of salicylic acid, ibuprofen, felbinac, naproxen, mefenamic acid, diclofenac, and ketoprofen in wastewater samples were 55.3, 162.9, 39.7, 11.8, 30.8, 259.7, and 48.3 ng L(-1), respectively). Gas chromatography-tandem mass spectrometry showed that wastewater samples collected during cooler seasons contained higher levels of COX-inhibiting activity. COX-inhibiting activities were highly correlated with NSAID concentrations (particularly for ketoprofen and diclofenac); however, other COX inhibitors, such as NSAIDs that were not examined in this study and/or other chemicals with COX-inhibiting activity, could exist in the water samples because the concentrations of NSAIDs obtained from the water samples did not account for the total COX-inhibiting activities observed. Therefore, COX inhibition assays may be helpful for evaluating the aquatic toxicity of COX inhibitors. In this study, we demonstrated that COX inhibitors in surface water may influence aquatic organisms more than was expected based on NSAID concentrations. Thus, further studies examining other COX inhibitors in the aquatic environment are necessary.

In vitro cyclooxygenase inhibition assay for evaluating ecotoxicity of the surface water and domestic wastewater in the Tone Canal, Japan.

Cyclooxygenase (COX) plays an important role in eicosanoid metabolism. Nonsteroidal anti-inflammatory drugs (NSAIDs) function as COX inhibitors and are frequently detected in the aquatic environment. Here, we measured the in vitro COX-inhibiting activity of the surface water and domestic wastewater in the Tone Canal, Japan. The concentrations of several NSAIDs in the some samples were also determined using gas chromatography-tandem mass spectrometry for confirming the validity of the assay. The target compounds were extracted from the samples using a solid-phase extraction cartridge. A dose-response relationship between the inhibiting activity and sample volume were observed in the wastewater sample. The higher COX-inhibiting activities were observed in the wastewater sample, as compared with the samples of the surface water in the canal. These inhibiting activities reflected the trends of NSAIDs distribution in the canal. However, the inhibiting activities of the water samples could not be entirely explained by the NSAIDs that were selected for instrumental analysis in this study. Other compounds that were not measured by instrumental analysis in this study might contribute to the inhibiting activities. Therefore, the COX-inhibiting assay would be effective for evaluating inclusive ecotoxicity in the aquatic environment.

[Formation mechanism and chemical safety of nonintentional chemical substances present in chlorinated drinking water and wastewater].

This paper reviews the formation mechanism and chemical safety of nonintentional chemical substances (NICS) present in chlorine-treated water containing organic contaminants. Undesirable compounds, i.e., NICS, may be formed under certain conditions when chlorine reacts with organic matter. The rate and extent of chlorine consumption with organics are strongly dependent on their chemical structures, particularly whether double bonds or sulfur and nitrogen atoms occur in the molecules. Organothiophosphorus pesticides (P=S type) are easily oxidized to their phosphorus compounds (P=O type) in chlorinated water containing HOCl as little as 0.5 mg/l, resulting in an increase in cholinesterase-inhibitory activity. Chlorination of phenols in water also produces a series of highly chlorinated compounds, including chlorophenols, chloroquinones, chlorinated carboxylic acids, and polychlorinated phenoxyphenols (PCPPs). In some of these chloroquinones, 2,6-dichloroalkylsemiquinones exhibit a strong mutagenic response as do positive controls used in the Ames test. 2-phenoxyphenols in these PCPPs are particularly interesting, as they are present in the chlorine-treated phenol solution and they are also precursors (predioxins) of the highly toxic chlorinated dioxins. Polynuclear aromatic hydrocarbons (PAHs) were found to undergo chemical changes due to hypochlorite reactions to give chloro-substituted PAHs, oxygenated (quinones) and hydroxylated (phenols) compounds, but they exhibit a lower mutagenic response. In addition, field work was performed in river water and drinking water to obtain information on chemical distribution and their safety, and the results are compared with those obtained in the model chlorination experiments.

Simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) products and human saliva by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric (GC-MS) method using selected ion monitoring (SIM) is described for the simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) (PVC) products. The method consists of the following four procedures; (1). liquid-liquid extraction with ethyl acetate from the acidified aqueous homogenates of the PVC products, (2). esterification with trimethylsilyldiazomethane (TMSD) and methanol, (3). clean-up using Florisil column chromatography and (4). quantitative determination of methylated phthalate monoesters by GC-MS using SIM. The methylated monoesters show a characteristic mass fragment pattern at m/z 163, 149 and 91. The calibration curves for the monoesters were linear from 0.05 to 10 ng (injection volume 1 micro l). Overall recoveries ranged from 86.6 to 94.3%. The limits of detections for these methylated derivatives were in the range of 2.0-5.0 ng/g (S/N=3). This method was applied to phthalate monoesters in PVC toy products. Mono-n-butyl phthalate and mono-2-ethylhexyl phthalate were found at levels of 6.42-11.62 micro g/g and 30.50-41.81 micro g/g, respectively. No monoethyl phthalate, mono-n-hexyl phthalate and monobenzyl phthalate were found in the toy products. The method was also applied to these compounds in human saliva.

Development of a headspace GC/MS analysis for carbonyl compounds (aldehydes and ketones) in household products after derivatization with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine.

Carbonyl compounds (aldehydes and ketones) are suspected to be among the chemical compounds responsible for Sick Building Syndrome and Multiple Chemical Sensitivities. A headspace gas chromatography/mass spectrometry (GC/MS) analysis for these compounds was developed using derivatization of the compounds into volatile derivatives with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBOA). For GC/MS detection, two ionization modes including electron impact ionization (EI) and negative chemical ionization (NCI) were compared. The NCI mode seemed to be better because of its higher selectivity and sensitivity. This headspace GC/MS (NCI mode) was employed as analysis for aldehydes and ketones in materials (fiber products, adhesives, and printed materials). Formaldehyde was detected in the range of N.D. (not detected) to 39 microg/g; acetaldehyde, N.D. to 4.1 microg/g; propionaldehyde, N.D. to 1.0 microg/g; n-butyraldehyde, N.D. to 0.10 microg/g; and acetone, N.D. to 3.1 microg/g in the samples analyzed.

A simple and reproducible testing method for dialkyl phthalate migration from polyvinyl chloride products into saliva simulant.

We describe a method of mechanical agitation to determine rates of dialkyl phthalate migration from polyvinyl chloride (PVC) products into saliva simulant. The method consists of rotary shaking of a sample with 30 mL of saliva simulant (pH 7.0) at 35 degrees C in a 50 mL glass tube at 300 rpm for 15 min, then measuring the amount of dialkyl phthalate in the saliva simulant by HPLC with a UV detector. The migration rates of diisononyl phthalate (DINP), di-2-ethylhexyl phthalate (DEHP) and di-n-butyl phthalate (DBP) from PVC plates containing about 45% (w/w) plasticizer (molded in our laboratory) were identical. However, the migration rates from molded plates containing 13% (w/w) DBP were almost double those of DINP and DEHP at the same ratios. In addition, the amounts of DINP that migrated in vitro after rotary shaking for 15 min were equivalent to those in vivo determined in saliva from volunteers who chewed plates for 60 min. The migration rates of dialkyl phthalates from 11 commercially available toys ranged from 15.6 to 85.2 micrograms/cm2/h [relative standard deviation (RSD), 3 to 12%].

Effect of hypochlorite oxidation on cholinesterase-inhibition assay of acetonitrile extracts from fruits and vegetables for monitoring traces of organophosphate pesticides.

A reproducible method for monitoring traces of cholinesterase (ChE) inhibitors in acetonitrile extracts from fruits and vegetables is described. The method is based on hypochlorite oxidation and ChE inhibition assay. Four common representative samples of produce were selected from a supermarket to investigate the effect of different matrices on pesticides recoveries and assay precision. The samples were extracted with acetonitrile to prepare them for ChE inhibition assays: if necessary, clean-up was performed using dispersive solid-phase extraction for gas chromatography-mass spectrometry (GC/MS) analyses. Chlorine was tested as an oxidising reagent for the conversion of thiophosphorus pesticides (P=S compounds) into their P=O analogues, which have high ChE-inhibiting activity. Chlorine consumption of individual acetonitrile extracts was determined and was strongly dependent on the individual types of fruits and vegetables. After treating the acetonitrile extracts with an excess hypochlorite at 25°C for 15 min, the ChE-inhibiting activities and detection limits for each chlorine-treated pesticide solution were determined. Matrix composition did not interfere significantly with the determination of the pesticides. Enhanced anti-ChE activities leading to low detection limits (ppb levels) were observed for the chlorine-treated extracts that were spiked with chlorpyrifos, diazinon, fenitrothion, and isoxathion. This combination of oxidative derivatisation and ChE inhibition assays was used successfully to monitor and perform semi-quantitative determination of ChE inhibitors in apple, tomato, cucumber, and strawberry samples.

Enhancement of anti-cholinesterase activity of aqueous samples by hypochlorite oxidation for monitoring traces of organophosphorus pesticides in water.

A reproducible method for monitoring traces of cholinesterase (ChE) inhibitors in aqueous samples is described: the method is based on chemical oxidation and a ChE inhibition assay. Chlorine was tested as an oxidizing reagent for conversion of various thiophosphorus pesticides (P=S compounds) into their P=O analogs, which have higher ChE-inhibiting activity. After treating buffered pesticide solutions (pH 6.0) with chlorine (final concentration less than 10 mg/l) of at 25°C for 15 min, the ChE-inhibiting activities and detection limits for each pesticide were determined. Greater ChE-inhibiting activities, leading to lower detection limits (ppb levels), were observed for the chlorine-treated solutions fortified azinphos methyl, diazinon, isoxathion and ronnel etc. No changes in the ChE-inhibiting activities were observed for carbamate pesticide solutions tested before and after chlorination, but an additive effect showed against ChE when these compounds were mixed with paraoxon in water. This combination of oxidative derivatization and ChE inhibition assay was applied successfully to the detection and determination of ChE inhibitors in natural and drinking water samples.

Chemical fate and mutagenic formation potentials of phenothiazine and related compounds during water chlorination.

The reactions of hetero-tricyclic aromatic hydrocarbons (H-TCAHs) with hypochlorite in an aqueous solution were investigated under conditions that simulate wastewater disinfection. H-TCAH-hypochlorite reaction products were determined by gas chromatographic-mass spectrometric (GC-MS) analyses. For 20 µM, 10H-phenothiazine, 10H-phenoxazine, and phenoxathiin reacted rapidly with active chlorine in neutral pH (7.0), but no phenazine-hypochlorite reaction was observed over pH values of 5-9 for 1 hr. The 10H-phenothiazine-hypochlorite reaction began by oxidation with active chlorine to form its dioxides, followed by chloro-substitution in water. The extent of the reactions depended on the chlorine dose, solution pH and compound structures. Ames assays for the chlorination byproducts of 10H-phenothiazine and 10H-phenoxazine also showed to be weak mutagenicity in TA98 and TA100 strains without S9 mix, but no chlorination byproducts of phenoxathiin exhibited any mutagenicity in both tester strains with and without S9 mix.

Suppression of PCDD/Fs formation because of the presence of DEHP during the model slow combustion of 2,4,6-trichlorophenol.

The thermal reactions of 2,4,6-T(3)CP in the presence and absence of DEHP in a dry air stream was investigated using a silica flow reactor at a residence time of 10s and a temperature range from 450 degrees C to 850 degrees C. Two isomers of T(4)CDDs (1,3,6,8- and 1,3,7,9-T(4)CDDs) were the most abundant products during the combustion of 2,4,6-T(3)CP alone and were observed at temperatures ranging from 550 degrees C to 800 degrees C. In the presence of DEHP, we observed a remarkable decrease in the yields of T(4)CDDs during the combustion of 2,4,6-T(3)CP. The suppression ratio of the T(4)CDDs formation was more than 90% in the case of the co-combustion with 10% DEHP in molar ratio. Other PCDD/Fs except for 2,7-/2,8-DCDD and 2,8-DCDF also decreased upon the combustion of 2,4,6-T(3)CP in the presence of DEHP. During the co-combustion of 2,4,6-T(3)CP and DEHP, the residual ratio of 2,4,6-T(3)CP increased slightly and formations of lower chlorinated phenols were observed. The suppression of the T(4)CDDs was strongly dependent on the DEHP ratio in the starting material. The prospective pathways of the suppressions of the T(4)CDDs formations during the combustion of 2,4,6-T(3)CP in the presence of DEHP were proposed.

Mutagenic potentials of Amberlite XAD-2-resin extracts obtained from river and drinking waters in the Northwest district of Chiba, Japan.

Amberlite XAD-2 resin extracts of river and drinking water sampled from the Northwest district of Chiba Prefecture in each month during the period from January to December 2008 were investigated to characterize and determine their mutagenic potentials and polycyclic aromatic hydrocarbon (PAH) levels. The extracts from the river water were shown to be mutagenic in Salmonella typhimurium TA98 (a flameshift mutagen) without S9 mix, with higher mutagenic responses in summer and early fall seasons. While the drinking water extracts exhibited weak mutagenicity in both the TA98 and TA100 strains (a base-pair substitution mutagen) without S9 mix, with high mutagenic responses in fall and early winter seasons. GC/MS determinations of the water concentrates showed some seasonal scatter in PAH levels in river water. In contrast, comparatively high concentrations of PAHs were observed for drinking water samples collected during warmer seasons. Statistical studies revealed that there is a lower correlation between the levels of flameshift mutagenicity and the concentrations of PAH in the river water concentrations, but a higher correlation between them in the drinking water samples.

Formation of polybrominated and polychlorinated ethylphenoxyethylphenols (PXEPEPs) during aqueous chlorination of 4-ethylphenol solutions in the presence of bromide ions.

This investigation was undertaken to determine the effect of the bromide concentration on the formation of polyhalogenated ethylphenoxyethylphenols (PXEPEPs), including predioxins, during the chlorination of 4-ethylphenol in solution. An aqueous solution of 4-ethylphenol was treated with hypochlorite in the presence of various concentrations of bromide ions. The changes in the compositions of the halogenated products in hexane extracts of the chlorinated solution were analyzed by gas chromatograph (GC) and a flame ionization detector (FID) and mass spectrometry (MS). 4-ethylphenol was shown to from several halogenated compounds, including PXEPEPs, as by-products of chlorination. The number of substituted chlorine or bromine atoms ranged from 0 to 4. The formation of bromine-substituted PXEPEPs was observed in the presence of 0.1 equivalents of bromide ions per mole of 4-ethylphenol. The number of substituted bromine atoms increased with the amount of co-existing bromide ions. In the presence of more than one equivalent of bromide ions per mole of 4-ethylphenol, the number of bromine atoms substituted in the PXEPEPs increased, whereas the number of chlorine atoms substituted in the PXEPEPs decreased. GC-MS total ion chromatograms confirmed the formation of polybrominated and polychlorinated predioxins during the aqueous chlorination of 4-ethylphenol in the presence of bromide ions. However, at ten equivalents of bromide ions per mole of 4-ethylphenol, no predioxins were observed in the hexane extract obtained from the aqueous 4-ethylphenol solution after being treated with chlorine. The formation of PXEPEPs during the chlorination of 4-ethylphenol in the presence of bromide ions was also influenced by the reaction pH.

Influence of experimental conditions on the formation of PCDD/Fs during the thermal reactions of 2,4,6-trichlorophenol.

In order to obtain information on thermochemical reactions of chlorophenols, which are well known as dioxin precursors, occurring during the combustion of municipal solid wastes, the combustion of 2,4,6-trichlorophenol (2,4,6-T(3)CP) in an air stream was investigated over a temperature range of 500-800 degrees C for a residence time of 1-20s using a quartz flow reactor. Gas chromatographic/mass spectrometric (GC/MS) analysis of the gaseous products and residues showed that 2,4,6-T(3)CP began to decompose at 500 degrees C and produced several compounds, with 1,3,6,8- and 1,3,7,9-tetrachlorodibenzo-p-dioxins (T(4)CDD) as the major products. In addition, more than fifty organic products were observed in our experimental conditions. The yields of polychlorinated benzenes, phenols, dibenzofurans, and dibenzo-p-dioxins formed during the combustion of 2,4,6-T(3)CP were plotted as a function of temperature and residence time. Furthermore, it was found that the yields and the compositions of these gaseous products were strongly dependent on the residence time in the flow reactor.

Monitoring of triclosan in the surface water of the Tone Canal, Japan.

Triclosan, which is used widely as an antibacterial agent, was ubiquitously found in the water samples collected from the Tone Canal, Chiba, Japan. The concentrations of triclosan ranged from 11 to 31 ng/L. Higher concentrations of triclosan were observed in water samples collected from downstream, as compared to in the water samples collected upstream. The daily monitoring of a selected point from where the domestic wastewater inflow occurs showed that the triclosan levels in the water samples ranged from 55 to 134 ng/L; the levels of the loading amounts peaked from 10:00 to 12:00.

Chemical fate and changes in mutagenic activity of antibiotics nitrofurazone and furazolidone during aqueous chlorination.

Reactions of nitrofuran antibiotics (nitrofurazone (NFZ) and frazolidone (FZD)) with hypochlorite in aqueous solution were investigated under the conditions that simulate wastewater disinfection. The chlorination byproducts were determined by high performance liquid chromatography. At the levels of 5 microM, NFZ reacted rapidly with free chlorine in neutral pH (7.0), while the FZD-hypochlorite reaction was reasonably slow under the same pH. Nevertheless, the strong mutagenic parents disappeared completely after the hypochlorite reactions, and the chlorination byproducts were observed to exert a weak mutagenic effect on Salmonella typhimurium TA100 without S9-mix. The extent of the reactions depended on the chlorine dose, solution pH and compound structures.

Monitoring of cholinesterase-inhibiting activity in water from the Tone canal, Japan, as a biomarker of ecotoxicity.

The cholinesterase (ChE)-inhibiting activity of water and the concentrations of representative inhibitors were monitored in the Tone canal, Japan, during April to December 2006. The ChE-inhibiting activity, measured by using horse serum as enzyme source, increased from late April to early June, and from September to October. Although the trends in the ChE-inhibiting activity of the samples were consistent with concentration changes of organophosphorus pesticides, ChE-inhibiting activity was not observed in samples replicated on the basis of the chemical concentrations detected. The water samples were treated with chlorine to enhance the ChE-inhibiting activity by conversion of thiophosphate pesticides to phosphate pesticides. The ChE-inhibiting activity increased in almost all the chlorine-treated samples, although organophosphorus pesticides were either not detected or detected in traces in the samples by gas chromatographic-mass spectrometric analysis. These results suggested that assay of ChE-inhibiting activity is important for evaluating the ecotoxicity of environmental water, because toxicological investigations based solely on inhibitor concentrations may underestimate the contamination. Furthermore, the combined method of oxidation by chlorination and the ChE assay is very effective for screening and monitoring of organophosphorus pesticides in environmental water.