The Healthy Men Study: An Evaluation of Exposure to Disinfection By-Products in Tap Water and Sperm Quality

Luben, Thomas J., Olshan, Andrew F., Herring, Amy H., Jeffay, Susan, Strader, Lillian, Buus, Rebecca M., Chan, Ronna L., Savitz, David A., Singer, Philip C., Weinberg, Howard S., Perreault, Sally D., Environmental Health Perspectives

Disinfection by-products (DBPs) form when chlorine or other disinfectants react with organic matter during preparation of drinking water. There are many classes of DBPs, including trihalomethanes (THMs) and haloacetic acids (HAAs). The relative concentrations of these DBPs, as well as the proportional distributions of individual chemicals within these classes, vary based on source water characteristics and treatment methods and on distribution system characteristics. The U.S. Environmental Protection Agency (EPA) and others have evaluated the potential adverse health effects of DBPs in both toxicologic and epidemiologic research. Several epidemiologic studies have suggested a possible association between DBPs and adverse pregnancy outcomes (Klotz and Pyrch 1999; Savitz et al. 1995, 2005, 2006; Toledano et al. 2005; Waller et al. 1998; Wright et al. 2003). To date, only one study has addressed DBPs and male reproductive health wherein exposure to DBPs was associated with decrements in sperm motility (Fenster et al. 2003).

Animal studies have consistently demonstrated an association between oral exposure to HAAs and adverse effects in the male reproductive system (Christian et al. 2002; Linder et al. 1994a, 1994b, 1995, 1997a, 1997b; Veeramachaneni et al. 2000). Testicular toxicity, including acute spermatotoxicity (Linder et al. 1994b), impaired reproductive competence and sperm quality (Linder et al. 1995), delayed spermiation and distorted sperm motility and morphology (Linder et al. 1997a), histopathologic changes in testis and epididymis (Linder et al. 1997b), transient subfertility (Luft et al. 2000), and altered sperm production and epididymal tubule changes (Christian et al. 2002), has been demonstrated when rodents were exposed to high doses (in the milligrams per kilogram range) of selected individual HAA species, with dibromoacetic acid being the most studied.

These rodent toxicology studies suggest that HAAs in drinking water, especially the brominated species, could pose a risk to the male reproductive system in humans, and that an evaluation of similar outcomes in human semen (sperm numbers, morphology, and motility) would be appropriate. Continued or intermittent exposure of men to DBPs in drinking water, even at levels below those that are acutely toxic to rodents, may have the potential to produce testicular toxicity as evidenced by altered semen quality.

We conducted a cohort study to address this possibility by evaluating semen quality in men with well-characterized exposures to DBPs. We were able to integrate this study with a cohort study of drinking-water DBPs and pregnancy loss (Savitz et al. 2006). Recruitment for both studies involved couples in three geographic locations in the United States, with our study adding home semen collection in a subcohort. We examined exposure to THMs and HAAs based on the concentration of these two classes of DBPs (as well as the four individual species of THMs and nine individual species of HAAs) and to TOX (total organic halides) measured weekly in the tap water from distribution systems serving the three study sites. These study sites were selected specifically to provide a reference site with low overall DBPs and two sites with relatively high levels of DPBs but differing with respect to brominated versus chlorinated species. Based on the concentrations of four regulated THMs (THM4), nine HAAs (HAA9), and total organic halides (TOX) measured weekly in tap water sampled from the distribution systems (Savitz et al. 2006), we calculated mean levels of DBPs over the 90 days preceding semen sampling. We then used questionnaire data to estimate the daily volume of tap water ingested and the frequency and duration of bathing and showering for each participant; we used these data to create exposure indices estimating exposure to DBPs via the ingestion, inhalation, and dermal routes of exposure. In the statistical analyses, we considered exposure to combined DBPs in accordance with current regulations [i.e., THM4 and HAA5 (sum of five regulated haloacetic acids)], as well as HAA9, to capture maximum exposures to HAAs. Because brominated HAAs may be more potent male reproductive toxicants, we also considered the composite concentration of brominated HAAs (HAA-Br) and THMs (THM-Br).

Methods

Study design and subject recruitment. The basic study design of this study, "The Healthy Men Study" (HMS), has been described previously (Olshan et al. 2007). The University of North Carolina School of Public Health's Institutional Review Board approved the study protocol, and all study participants gave written informed consent. Briefly, the HMS identified male partners of women who participated in a prospective study of drinkingwater DBPs and spontaneous abortion (the "Right From the Start" (RFTS) study (Savitz et al. 2005, 2006). Men were prospectively identified from the RFTS study and recruited from the three RFTS study sites. The sampling strategy included one group of men from a study site with very low levels of all targeted DBPs (low-DBP site), one group from a site with moderate levels of chlorinated DBPs and lower levels of brominated DBPs (chlorinated DBP site), and one group from a site with moderate levels of brominated DBPs and lower levels of chlorinecontaining species (brominated DBP site). The term "moderate" is used here to describe exposures that approach but are still below the limits established by the U.S. EPA for regulated DBPs (i.e., 80 [micro]g/L THM4 and 60 [micro]g/L HAA5) (U.S. EPA 1998).

Questionnaire. A computer-assisted telephone interview was administered to each participant by experienced interviewers, with responses entered directly into a computerized database. The average duration of the interview was approximately 40 min. Questions covered the following topics: general lifestyle, health, reproductive history, environment, diet, stress, occupational exposures, hobbies, and demographic factors, as well as drinkingwater consumption and water exposures. The section of the interview regarding drinkingwater consumption and water exposures included questions about the quantity (volume) of hot and cold drinks consumed at home and at work, use of household water filters and filter types, frequency and duration of bathing and showering, and frequency and duration of pool and Jacuzzi use. Participants were provided with a diagram of small, medium, and large glasses, with the relative volume of each listed in cups, pints, quarts, and ounces to facilitate the accurate estimation of daily personal consumption at home and at work. Participants also gave the street address of their home, workplace, and other locations where they spent a significant amount of time in order for us to determine if these locations were within the water utility service area.

Semen collection and analyses. Participants were asked to provide a single semen sample using a special kit designed to allow the man to collect a semen specimen in the privacy of his own home and at a time convenient to him (Royster et al. 2000). Before sending the kit to the participant, study staff confirmed by telephone the participant's mailing address, gave brief instructions on how to use the kit, and asked the participant if he had experience doing a similar collection in the past. Verbal instructions included the importance of doing the collection after (2-7) days of abstinence from sexual activity. The kit was mailed to the participant during the week before his anticipated collection date. Participants in the low DBP site and the brominated DBP site were instructed to open the box as soon as they received it because the package mailed to those sites also contained ice packs that would need to be frozen at least 24 hr before collection and packaged with the sample before mailing.

The instructions accompanying the kit included photographs and instructions on how to properly collect the specimen, package and prepare it for shipping, and call to arrange the courier pickup. Because the package was delivered to the laboratory by overnight carrier for residents of the low-and brominated-DBP sites, participants from these sites were instructed to collect the specimen only on Monday, Tuesday, or Wednesday, between 0500 and 1700 hours. Participants in the chlorinated DBP site were told to collect the specimen between 0800 and 1200 hours so the courier could pick up the package and deliver it to the U.S. EPA laboratory within 1.5 hr of collection. Upon receipt of samples from the chlorinated DBP site, aliquots were removed for immediate processing, and the remainder was stored overnight in the refrigerator to mimic overnight shipping. A second set of aliquots was processed the next morning for comparison with the fresh sample.

When the initial specimen volume was very low (< 0.5 mL), the man reported spillage or incomplete sample collection, shipping was delayed or the sample was not packaged correctly, or the participant's abstinence interval was too far out of the suggested (2-7) day range, participants were asked to provide a second or third specimen. This affected 10% (n = 20) of participants, of which all 20 complied with the repeat collections.

All samples were received by a single laboratory at the U.S. EPA. Immediately upon receipt, semen volume was measured, and aliquots were removed for determination of sperm concentration by IVOS-IDENT (Hamilton Thorne Research, Beverly, MA; Zinaman et al. 1996), from which total sperm count was calculated. Additional aliquots were used to prepare smears that were airdried and stored for later analyses of sperm morphology [World Health Organization (WHO) 1999]. Sperm motility, which declines over time and is therefore not a reliable measure for shipped semen, was not included in the statistical analysis. However, sperm motility and viability (using propitium iodide as a vital stain) were monitored. All samples retained motile and viable sperm, an indication that the sample had been collected and shipped according to instructions.

Additional aliquots (0.1 mL) were frozen and stored at -70[degress]C for later analysis of chromatin integrity by the sperm chromatin structure assay (SCSA; Evenson and Jost 2000) and for chromatin maturity by chromomycin A3 (CMA) staining (Sakkas et al. 1995). These measures were included because some DBPs are considered to be carcinogenic and might, therefore, damage sperm DNA. For the SCSA, aliquots were shipped on liquid nitrogen to SCSA Diagnostics (Brookings, SD) for analysis according to established methods …

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Publication information: Article title: The Healthy Men Study: An Evaluation of Exposure to Disinfection By-Products in Tap Water and Sperm Quality. Contributors: Luben, Thomas J. - Author, Olshan, Andrew F. - Author, Herring, Amy H. - Author, Jeffay, Susan - Author, Strader, Lillian - Author, Buus, Rebecca M. - Author, Chan, Ronna L. - Author, Savitz, David A. - Author, Singer, Philip C. - Author, Weinberg, Howard S. - Author, Perreault, Sally D. - Author. Journal title: Environmental Health Perspectives. Volume: 115. Issue: 8 Publication date: August 2007. Page number: 1169+. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2007 Gale Group.

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