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The HIWATE (Health Impacts of long-term exposure to disinfection byproducts in drinking WATEr) project was a systematic analysis that combined the epidemiology on adverse pregnancy outcomes and other health effects with long-term exposure to low levels of drinking water disinfection byproducts (DBPs) in the European Union. The present study focused on the relationship of the occurrence and concentration of DBPs with in vitro mammalian cell toxicity. Eleven drinking water samples were collected from five European countries. Each sampling location corresponded with an epidemiological study for the HIWATE program. Over 90 DBPs were identified; the range in the number of DBPs and their levels reflected the diverse collection sites, different disinfection processes, and the different characteristics of the source waters. For each sampling site, chronic mammalian cell cytotoxicity correlated highly with the numbers of DBPs identified and the levels of DBP chemical classes. Although there was a clear difference in the genotoxic responses among the drinking waters, these data did not correlate as well with the chemical analyses. Thus, the agents responsible for the genomic DNA damage observed in the HIWATE samples may be due to unresolved associations of combinations of identified DBPs, unknown emerging DBPs that were not identified, or other toxic water contaminants. This study represents the first to integrate quantitative in vitro toxicological data with analytical chemistry and human epidemiologic outcomes for drinking water DBPs.

The U.S. Environmental Protection Agency's "Four Lab Study" involved participation of researchers from four national Laboratories and Centers of the Office of Research and Development along with collaborators from the water industry and academia. The study evaluated toxicological effects of complex disinfection byproduct (DBP) mixtures, with an emphasis on reproductive and developmental effects that have been associated with DBP exposures in some human epidemiologic studies. This paper describes a new procedure for producing chlorinated drinking water concentrate for animal toxicology experiments, comprehensive identification of >100 DBPs, and quantification of 75 priority and regulated DBPs. In the research reported herein, complex mixtures of DBPs were produced by concentrating a natural source water with reverse osmosis membranes, followed by addition of bromide and treatment with chlorine. By concentrating natural organic matter in the source water first and disinfecting with chlorine afterward, DBPs (including volatiles and semivolatiles) were formed and maintained in a water matrix suitable for animal studies. DBP levels in the chlorinated concentrate compared well to those from EPA's Information Collection Rule (ICR) and a nationwide study of priority unregulated DBPs when normalized by total organic carbon (TOC). DBPs were relatively stable over the course of the animal studies (125 days) with multiple chlorination events (every 5-14 days), and a significant portion of total organic halogen was accounted for through a comprehensive identification approach. DBPs quantified included regulated DBPs, priority unregulated DBPs, and additional DBPs targeted by the ICR. Many DBPs are reported for the first time, including previously undetected and unreported haloacids and haloamides. The new concentration procedure not only produced a concentrated drinking water suitable for animal experiments, but also provided a greater TOC concentration factor (136×), enhancing the detection of trace DBPs that are often below detection using conventional approaches.

An occurrence study was conducted to measure five iodo-acids (iodoacetic acid, bromoiodoacetic acid, (Z)-3-bromo-3-iodo-propenoic acid, (E)-3-bromo-3-iodo-propenoic acid, and (E)-2-iodo-3-methylbutenedioic acid) and two iodo-trihalomethanes (iodo-THMs), (dichloroiodomethane and bromochloroiodomethane) in chloraminated and chlorinated drinking waters from 23 cities in the United States and Canada. Since iodoacetic acid was previouslyfound to be genotoxic in mammalian cells, the iodo-acids and iodo-THMs were analyzed for toxicity. A gas chromatography (GC)/negative chemical ionization-mass spectrometry (MS) method was developed to measure the iodo-acids; iodo-THMs were measured using GC/high resolution electron ionization-MS with isotope dilution. The iodo-acids and iodo-THMs were found in waters from most plants, at maximum levels of 1.7 microg/L (iodoacetic acid), 1.4 microg/L (bromoiodoacetic acid), 0.50 microg/L ((Z)-3-bromo-3-iodopropenoic acid), 0.28 microg/L ((E)-3-bromo-3-iodopropenoic acid), 0.58 microg/L ((E)-2-iodo-3-methylbutenedioic acid), 10.2 microg/L (bromochloroiodomethane), and 7.9 microg/L (dichloroiodomethane). Iodo-acids and iodo-THMs were highest at plants with short free chlorine contact times (< 1 min), and were lowest at a chlorine-only plant or at plants with long free chlorine contact times (> 45 min). Iodide levels in source waters ranged from 0.4 to 104.2 microg/L (when detected), but there was not a consistent correlation between bromide and iodide. The rank order for mammalian cell chronic cytotoxicity of the compounds measured in this study, plus other iodinated compounds, was iodoacetic acid > (E)-3-bromo-2-iodopropenoic acid > iodoform > (E)-3-bromo-3-iodo-propenoic acid > (Z)-3-bromo-3-iodo-propenoic acid > diiodoacetic acid > bromoiodoacetic acid > (E)-2-iodo-3-methylbutenedioic acid > bromodiiodomethane > dibromoiodomethane > bromochloroiodomethane approximately chlorodiiodomethane > dichloroiodomethane. With the exception of iodoform, the iodo-THMs were much less cytotoxic than the iodo-acids. Of the 13 compounds analyzed, 7 were genotoxic; their rank order was iodoacetic acid >> diiodoacetic acid > chlorodiiodomethane > bromoiodoacetic acid > E-2-iodo-3-methylbutenedioic acid > (E)-3-bromo-3-iodo-propenoic acid > (E)-3-bromo-2-iodopropenoic acid. In general, compounds that contain an iodo-group have enhanced mammalian cell cytotoxicity and genotoxicity as compared to their brominated and chlorinated analogues.

The haloacetamides, a class of emerging nitrogenous drinking water disinfection byproduct (DBPs), were analyzed for their chronic cytotoxicity and for the induction of genomic DNA damage in Chinese hamster ovary cells. The rank order for cytotoxicity of 13 haloacetamides was DIAcAm > IAcAm > BAcAm > TBAcAm > BIAcAm > DBCAcAm > CIAcAm > BDCAcAm > DBAcAm > BCAcAm > CAcAm > DCAcAm > TCAcAm. The rank order of their genotoxicity was TBAcAm > DIAcAm approximately equal to IAcAm > BAcAm > DBCAcAm > BIAcAm > BDCAcAm > CIAcAm > BCAcAm > DBAcAm > CAcAm > TCAcAm. DCAcAm was not genotoxic. Cytotoxicity and genotoxicity were primarily determined by the leaving tendency of the halogens and followed the order I > Br > > Cl. With the exception of brominated trihaloacetamides, most of the toxicity rank order was consistent with structure-activity relationship expectations. For di- and trihaloacetamides, the presence of at least one good leaving halogen group (I or Br but not Cl) appears to be critical for significant toxic activity. Log P was not a factor for monohaloacetamides but may play a role in the genotoxicity of trihaloacetamides and possible activation of dihaloacetamides by intracellular GSH and -SH compounds.

This study examined the hypothesis that chlorine dioxide bleaching used in pulp and paper production causes the formation of reproductive-endocrine disrupting compounds from plant sterols. This was tested by conducting a laboratory simulation of the chlorine dioxide oxidation of two plant sterols, beta-sitosterol and stigmasterol. Oxidation products of the plant sterol beta-sitosterol were purified and identified and found to be cholestan-24-ethyl-3-one, 4-cholestene-24-ethyl-3-one, and 4-cholestene-24-ethyl-3,6-dione. The first two compounds were found in a number of pulp and paper effluents and biosolids. The sterols and their oxidation products were tested in vitro using bioassays for androgenicity and estrogenicity. A 28 d in vivo bioassay was employed to examine masculinization in female mosquitofish. In vitro bioassays revealed little estrogenic activity in the parent sterols or in mixtures of their oxidation products. Androgenic activity as measured by the androgen receptor binding bioassay was in the order of 19-96 microg/g testosterone equivalents but with no increase or decrease with chlorine dioxide oxidation. The mosquitofish bioassay did not show significant masculinization for any of the preparations tested. A number of androstane steroids were identified in the sterols tested, however, those compounds could only account for a small fraction of the androgenic activity in the sterols. It was clear that the parent sterols were not themselves acting as androgens in the bioassays used. This study indicated that chlorine dioxide oxidation of sterols produced predominantly oxidized sterols that were not likely to act through androgenic or estrogenic mechanisms.

Iodoacid drinking water disinfection byproducts (DBPs) were recently uncovered in drinking water samples from source water with a high bromide/iodide concentration that was disinfected with chloramines. The purpose of this paper is to report the analytical chemical identification of iodoacetic acid (IA) and other iodoacids in drinking water samples, to address the cytotoxicity and genotoxicity of IA in Salmonella typhimurium and mammalian cells, and to report a structure-function analysis of IA with its chlorinated and brominated monohalogenated analogues. The iodoacid DBPs were identified as iodoacetic acid, bromoiodoacetic acid, (Z)- and (E)-3-bromo-3-iodopropenoic acid, and (E)-2-iodo-3-methylbutenedioic acid. IA represents a new class (iodoacid DBPs) of highly toxic drinking water contaminants. The cytotoxicity of IA in S. typhimurium was 2.9x and 53.5x higher than bromoacetic acid (BA) and chloroacetic acid (CA), respectively. A similar trend was found with cytotoxicity in Chinese hamster ovary (CHO) cells; IA was 3.2x and 287.5x more potent than BA and CA, respectively. This rank order was also expressed in its genotoxicity with IA being 2.6x and 523.3x more mutagenic in S. typhimurium strain TA100 than BA and CA, respectively. IA was 2.0x more genotoxic than BA and 47.2x more genotoxic than CA in CHO cells. The rank order of the toxicity of these monohalogenated acetic acids is correlated with the electrophilic reactivity of the DBPs. IA is the most toxic and genotoxic DBP in mammalian cells reported in the literature. These data suggest that chloraminated drinking waters that have high bromide and iodide source waters may contain these iodoacids and most likely other iodo-DBPs. Ultimately, it will be important to know the levels at which these iodoacids occur in drinking water in order to assess the potential for adverse environmental and human health risks.

Halonitromethanes are drinking water disinfection byproducts that have recently received a high priority for health effects research from the U.S. Environmental Protection Agency (EPA). Our purpose was to identify and synthesize where necessary the mixed halonitromethanes and to determine the chronic cytotoxicity and the acute genotoxicity of these agents in mammalian cells. The halonitromethanes included bromonitromethane (BNM), dibromonitromethane (DBNM), tribromonitromethane (TBNM), bromochloronitromethane (BCNM), dibromochloronitromethane (DBCNM), bromodichloronitromethane (BDCNM), chloronitromethane (CNM), dichloronitromethane (DCNM), and trichloronitromethane (TCNM). Low- and high-resolution gas chromatography/mass spectrometry (GC/MS) was used to identify the mixed chloro-bromonitromethanes in finished drinking waters, and analytical standards that were not commercially available were synthesized (BDCNM, DBCNM, TBNM, CNM, DCNM, BCNM). The rank order of their chronic cytotoxicity (72 h exposure) to Chinese hamster ovary (CHO) cells was DBNM > DBCNM > BNM > TBNM > BDCNM > BCNM > DCNM > CNM > TCNM. The rank order to induce genomic DNA damage in CHO cells was DBNM > BDCNM > TBNM > TCNM > BNM > DBCNM > BCNM > DCNM > CNM. The brominated nitromethanes were more cytotoxic and genotoxic than their chlorinated analogues. This research demonstrated the integration of the procedures for the analytical chemistry and analytical biology when working with limited amounts of sample. The halonitromethanes are potent mammalian cell cytotoxins and genotoxins and may pose a hazard to the public health and the environment.

Using gas chromatography/mass spectrometry (GC/MS), we investigated the formation of disinfection byproducts (DBPs) from high bromide waters (2 mg/L) treated with chlorine or chlorine dioxide used in combination with chlorine and chloramines. This study represents the first comprehensive investigation of DBPs formed by chlorine dioxide under high bromide conditions. Drinking water from full-scale treatment plants in Israel was studied, along with source water (Sea of Galilee) treated under carefully controlled laboratory conditions. Select DBPs (trihalomethanes, haloacetic acids, aldehydes, chlorite, chlorate, and bromate) were quantified. Many of the DBPs identified have not been previously reported, and several of the identifications were confirmed through the analysis of authentic standards. Elevated bromide levels in the source water caused a significant shift in speciation to bromine-containing DBPs; bromoform and dibromoacetic acid were the dominant DBPs observed, with very few chlorine-containing compounds found. Iodo-trihalomethanes were also identified, as well as a number of new brominated carboxylic acids and 2,3,5-tribromopyrrole, which represents the first time a halogenated pyrrole has been reported as a DBP. Most of the bromine-containing DBPs were formed during pre-chlorination at the initial reservoir, and were not formed by chlorine dioxide itself. An exception wasthe iodo-THMs, which appeared to be formed by a combination of chlorine dioxide with chloramines or chlorine (either added deliberately or as an impurity in the chlorine dioxide). A separate laboratory study was also conducted to quantitatively determine the contribution of fulvic acids and humic acids (from isolated natural organic matter in the Sea of Galilee) as precursor material to several of the DBPs identified. Results showed that fulvic acid plays a greater role in the formation of THMs, haloacetic acids, and aldehydes, but 2,3,5-tribromopyrrole was produced primarily from humic acid. Because this was the first time a halopyrrole has been identified as a DBP, 2,3,5-tribromopyrrole was tested for mammalian cell cytotoxicity and genotoxicity. In comparison to other DBPs, 2,3,5-tribromopyrrole was 8x, 4.5x, and 16x more cytotoxic than dibromoacetic acid, 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone [MX], and potassium bromate, respectively. 2,3,5-Tribromopyrrole also induced acute genomic damage, with a genotoxic potency (299 microM) similar to that of MX.