Environmental Research 135 (2014) 276–284

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Environmental Research

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journal homepage: www.elsevier.com/locate/envres

Use of urinary trichloroacetic acid as an exposure biomarkerof disinfection by-products in cancer studies$,$$

Lucas A. Salas a,b,c, Esther Gracia-Lavedan a,b,c, Fernando Goñi c,d,e, Victor Moreno f,g,h,Cristina M. Villanueva a,b,c,i,n

a Centre for Research in Environmental Epidemiology (CREAL), Doctor Aiguader 88, 08003 Barcelona, Spainb Universitat Pompeu Fabra (UPF), Barcelona, Spainc CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spaind Basque Laboratory of Health, Gipuzkoa, Spaine BioDonostia Health Research Institute, Spainf Catalan Institute of Oncology (ICO), Spaing Bellvitge Biomedical Research Institute (IDIBELL), Spainh University of Barcelona (UB), Spaini IMIM (Hospital del Mar Medical Research Institute), Spain

a r t i c l e i n f o

Article history:Received 7 July 2014Received in revised form19 September 2014Accepted 25 September 2014

Keywords:AcetatesBiological markersEnvironmental exposureTrichloroacetic acidTrihalomethanes

x.doi.org/10.1016/j.envres.2014.09.01851/& 2014 Elsevier Inc. All rights reserved.

cs committee approval: The study was approveearch Center (IMIM-IMAS).ding resources: This study was funded byde Investigaciones Sanitarias – FIS, InstitutFIS PI11-226). Lucas A. Salas received a C

ia (Grant: 529/2011). Parc de Salut Mar Biobing facilities for to the project. MARBiobancarlos III FEDER (RD09/0076/00036).esponding author at: Centre for Research in, Doctor Aiguader 88, 08003 Barcelona, Spainail address: cvillanueva@creal.cat (C.M. Villan

a b s t r a c t

Urinary trichloroacetic acid (TCAA) has been proposed as a valid exposure biomarker for ingesteddisinfection by-products (DBP) for reproductive studies. However, it has never been used in epidemio-logic studies on cancer. We investigate the performance of urinary TCAA as a biomarker of DBP exposurein the framework of an epidemiologic study on cancer.

We conducted home visits to collect tap water, first morning void urine, and a 48 h fluid intake diaryamong 120 controls from a case-control study of colorectal cancer in Barcelona, Spain. We measuredurine TCAA and creatinine, and 9 haloacetic acids and 4 trihalomethanes (THM) in tap water. LifetimeTHM exposure was estimated based on residential history since age 18 plus routine monitoring data.Robust linear regressions were used to estimate mean change in urinary TCAA adjusted by covariates.

Among the studied group, mean age was 74 years (range 63–85) and 41 (34%) were females. Meantotal tap water consumption was 2.2 l/48 h (standard error, 0.1 l/48 h). Geometric mean urine TCAAexcretion rate was 17.3 pmol/min [95%CI: 14.0–21.3], which increased 2% for a 10% increase in TCAAingestion and decreased with total tap water consumption (�17%/l), water intake outside home (�32%),plasmatic volume (�64%/l), in smokers (�79%), and in users of non-steroidal anti-inflammatory drugs(�50%). Urinary TCAA levels were not associated with lifetime THM exposure.

In conclusion, our findings support that urine TCAA is not a valid biomarker in case-control studies ofadult cancer given that advanced age, comorbidites and medication use are prevalent and aredeterminants of urine TCAA levels, apart from ingested TCAA levels. In addition, low TCAA concentrationsin drinking water limit the validity of urine TCAA as an exposure biomarker.

& 2014 Elsevier Inc. All rights reserved.

d by the Ethics Committee of

the Spanish Health Ministryo de Salud Carlos III, Spainolciencias Ph.D. Scholarship,ank (MARBiobanc) providedis supported by Instituto de

Environmental Epidemiology.ueva).

1. Introduction

Water disinfection is central in public health to preventinfections from waterborne pathogens. Nevertheless, disinfectionprocesses produce undesired toxicants known as disinfection by-products (DBP), which constitute a widespread environmentalexposure in developed countries. Trihalomethanes (THM) andhaloacetic acids (HAA) are the most prevalent DBP classes result-ing from chlorination (Krasner et al., 2006). Disinfection by-products have been linked to an increased risk of bladder cancer(Costet et al., 2011) while evidence of the association with other

L.A. Salas et al. / Environmental Research 135 (2014) 276–284 277

cancer sites is mixed (Cantor, 2010). Exposure assessment inepidemiological studies is challenging and has traditionally beenbased on personal information from questionnaires and historicalenvironmental data from routine monitoring.

Urinary trichloroacetic acid (TCAA) has been proposed as avalid exposure biomarker of ingested DBP since half life (48–120 h) is longer than consecutive exposure events and urine levelsare associated with ingested levels (Bader et al., 2004; Froese et al.,2002; Zhang et al., 2009). Pharmacokinetics of TCAA is knownfrom occupational studies on trichloroethylene 1.1.1-trichlor-oethane and perchloroethylene, which are metabolized to TCAA(Chiu et al., 2009; Chiu and Ginsberg, 2011; Clewell et al., 2000;Covington et al., 2004). TCAA is distributed in the plasma com-partment exclusively, with approximately 50% bounded to plas-matic proteins, and is excreted by the kidney (Covington et al.,2004). In consequence, changes in plasma volume (e.g. use ofdiuretics, cardiac failure), diseases that generate chronic malnour-ishment and altered levels of plasma proteins (e.g. chronicobstructive pulmonary disease-COPD, anemia, cancer), impairedrenal function (e.g. diabetes mellitus, systemic arterial hyperten-sion, use of nonsteroidal anti-inflamatory drugs-NSAIDs, tobaccouse), or competition for binding protein areas (e.g. warfarin) mayalter TCAA kinetics. In addition, consumption of dairy productsmay affect the ingested levels of TCAA due to chemical reactionswith milk proteins (Ebina and Nagai, 1979).

The use of urinary TCAA as a biomarker of DBP exposure inepidemiological studies is limited to a few studies on reproductiveoutcomes (Costet et al., 2012; Smith et al., 2013; Xie et al., 2011;Zeng et al., 2014a; Zhou et al., 2012). Methodological aspectsrequire further development prior to a general use (Savitz, 2012).Urinary TCAA has never been used in epidemiological studies oncancer in adults, where the population has older age, presence ofcomorbidities and use several medications (Chapman et al., 2012).

The primary aim of this work was to assess the validity ofurinary TCAA as an exposure biomarker of ingested TCAA indrinking water in a study on cancer. We specifically evaluate ifage, the use of medications, and the presence of comorbidities isassociated with the performance of urinary TCAA for exposureassessment.

2. Materials and methods

2.1. Study population

This study is part of a population-based case-control study of colorectal cancer,which in turn is part of a larger multicase-control study conducted in Spain in2007-2012 (MCC-Sp, www.mccspain.org). A subset of controls was re-contacted inJuly-December 2012 to conduct the present study. Study subjects were selectedbased on age at recruitment (460 years), availability of lifetime estimates of THMexposure and residence in Barcelona metropolitan area. A total of 257 subjectsfulfilled these criteria. The MCC-Sp project and the present nested biomarker studyhave been approved by the Investigation Review Boards of the hospitals andinstitutions involved. All the participants signed a written informed consent if theyagreed to participate in the present analyzes.

2.2. Telephone contacts

Potential participants were telephoned to the number provided at recruitment.Participation consisted of a home visit to collect a tap water sample, a urine sampleand completion of a fluid consumption diary the 48 h prior to the visit. For thosewho accepted, a courier was sent with instructions and information of the study,the fluid consumption diary, and a container for urine collection. Once received, theparticipants were telephoned to clarify instructions and to schedule the home visit.

2.3. Questionnaires

A self-administered questionnaire was designed to ascertain fluid consumptionduring the 48 h before the collection of urine and tap water samples. The amount of

tap water, bottled water, coffee, tea or herbal drinks and soups consumed wasrequested by indicating the number of glasses, cups, mugs or bowls consumed inthe morning, afternoon and evening. Free space was left to add other beverages(spirits, sodas, juices, liquid yogurts, etc). Serving sizes were measured on site usinga ruler to measure height and diameter (narrowest, widest) of the glasses, cups,and mugs used. The serving size was assumed one centimeter under the border andvolume was calculated using a formula for truncated cones. The questionnaire wasverified during the home visit, and we asked if any of the reported hot beverageswere prepared using bottled water.

A five minute questionnaire was administered by fieldworkers during the homevisit to ascertain consumption of milk and dairy products (including proportion ofwater-milk mixtures), processing of tap water (filtering, boiling, cooling, storage),the consumption of water or water-based beverages outside the home, swimmingin pools and exposure to chlorinated solvents or dry cleaning products (trichlor-oethylene, perchloroethylene), paints, or industrial solvents during the previousweek, the time of the last voiding before urine collection and the time of urinecollection.

Personal, socio-demographic, lifestyle, diet, family history and medical historyvariables were available from the MCC-Sp questionnaire. These interviews wereconducted at recruitment by trained interviewers in primary care centers. Avalidated self-administered food frequency questionnaire was also collected. Thefull questionnaire is available at the study website: www.mccspain.org.

2.4. Urine and tap water samples

First morning urine void was collected in 500 ml polyethylene graduated flasksprovided by the researchers, with instructions to collect the very first urine void inthe morning after waking up. The participants were asked to avoid showeringbefore urine collection. Volume was measured using the bottle graduation plusmillimeters between graduation marks. Two 10 ml aliquots were collected on siteusing urine Monovettes stabilizer-free tubes (Sarstedt, France). Tubes werelabeled, placed in a self-sealed plastic bag and transported with icepacks. Unfilteredcold tap water from the kitchen or bathroom faucets was allowed to flush thesystem for 1 min. A 40 ml amber glass vial with 100 ml of sodium thiosulfate 10%and 35 ml of 4 N HCl was used to collect tap water for THMmeasurements. A 100 mlamber glass vial with 250 ml of ammonia chloride 4% (parts/volume) was used tocollect tap water samples for HAA measurements. Vials were totally filled avoidingbubble formation and quencher loss. Teflon-faced screw cap without headspacewas used for sealing. Samples were refrigerated with icepacks. Five bottled mineralwater samples were collected randomly during the field work as field blanks usingthe same vials of tap water. Water samples were stored at 4 °C and analyzed forTHM and HAA within 72 h of collection.

2.5. Water analyses

The experimental analysis of THM followed a modified form of StandardMethod 6232B (APHA, 1998). Four THM (chloroform-TCM, bromodichloro-methane-BDCM, dibromochloromethane-DBCM, and bromoform-TBM) were mea-sured through salted liquid–liquid extraction with n-pentane and quantification bygas chromatography (Agilent 6890, Santa Clara, CA, USA) with a HP-5 capillarycolumn (length 50 m, diameter 0.32 mm, and film thickness 1.05 mm, Agilent) andelectron capture detector. Nine HAA (monochloroacetic acid-MCAA, dichloroaceticacid-DCAA, trichloroacetic acid-TCAA, monobromoacetic acid-MBAA, dibromoace-tic acid-DBAA, bromochloroacetic acid-BCAA, bromodichloroacetic acid-BDCAA,dibromochloroacetic acid- DBCAA, and tribromoacetic acid-TBAA) were analyzedusing a modified form of USEPA Method 552.3 (Domino et al., 2003). Thisprocedure involved salted liquid–liquid extraction with tert-butyl methyl ether atpHr0.5, followed by methyl derivatization and gas chromatography (Agilent6890) with a HP-5 MS capillary column (length 30 m, diameter 0.25 mm, and filmthickness x 0.25 mm, Agilent) and negative chemical ionization mass spectrometrydetection (NCI-MS). Limit of quantification (LQ) were 0.5 mg/l for MCAA, 0.4 for thefour THM, 0.3 for TBAA, 0.05 mg/l for TCAA and DBAA, and 0.1 for the other HAA.Experimental analyses were conducted in the Basque Health Laboratory inGipuzkoa (Spain), certified for THM analysis in tap water.

2.6. Urinary TCAA analyses

Urine samples were stored at �20 °C in the MarBiobank (Barcelona, Spain) andshipped in dry ice to the laboratory. TCAA concentrations were measured in 5 mlurine samples in the Health and Safety Laboratory (Buxton, UK), using solid phaseextraction followed by liquid chromatography tandem mass spectrometry (LC-MS-MS). One milliliter (ml) of urine was diluted with 2 ml of pH7 buffer and 100 ml ofdeuterated TCAA solution (1 ppm) was added to all the tubes. Calibrations wereprepared in the range 0–100 mg/l. External quality control samples from G-EQUAS(www.g-equas.de) were prepared. Strata X-AW cartridges (30 mg/1 ml) were usedfor extraction after conditioning with 1 ml of methanol and 1 ml pH7 buffer. Thesample was loaded and washed with 1 ml of water and then 1 ml of methanol.Cartridges were dried and the TCAA eluted using 1 ml 1%HCl in methanol.

L.A. Salas et al. / Environmental Research 135 (2014) 276–284278

LC-MS-MS (API3200, ABSciex) used electrospray ionization with negative polarity.Instrument conditions were: curtain gas 40 psi, collision gas 6 psi, ion spray voltage�4500 V, temperature 300 °C, ion source gases 30 and 40 psi, declusteringpotential �15 V, entrance potential –3 V, collision energy –10 V, and collision cellexit potential �4 V. The HPLC column was 150�4.6 mm Hydro-RP (Phenomenex),flow rate 0.5 ml/min. Mobile phase was isocratic with 30% methanol and 70%20 mM ammonium acetate (pH5). Injection volume was 25 ml. Using multiplereaction monitoring (MRM), TCAA was monitored using the transition 160.8/116.8and the internal standard transition was 161.8/117.7. Analytical precision was below15.5%. The limit of detection (LoD) was 3 nmol/l (E0.5 mg/l) and the recoveryproportion ranged from 75% to 104.8% (mean¼84.9%, n¼14). Urinary creatininewas determined using an automated alkaline picrate method (Cocker et al., 2011).The LoD was 0.5 mmol/l.

2.7. Ingestion of trichloroacetic acid

The volume of water ingested during the 48 h before urine collection wascalculated based on the consumption of tap water, bottled water, coffee, tea, herbaldrinks and soups reported in the 48 h fluid record. Number of glasses, cups, andmugs were multiplied by serving sizes to calculate the volume. Milk proportionwassubtracted from the total volume. The volume (in liters) of plain tap water wasmultiplied by the level of TCAA measured in the tap water. A 70% reduction wasassumed for filtered water, and a 39% reduction was assumed for water-based hotbeverages (coffee, tea, herbal drinks, soups) (Kim and Weisel, 1998; Kim et al.,1999). Concentration in bottled water was imputed from the average of TCAAmeasurements of the field blanks. The levels of ingested TCAA were converted tomoles using the molecular weight (163.39 gr/mol).

2.8. Lifetime trihalomethane levels

Residential history, including complete address of all residences where studysubjects had lived since age 18 was available from the main questionnaire of MCC-Sp. Trihalomethane levels in the study municipalities were estimated back to 1940.Annual average THM levels were calculated by zip code using available measure-ments. Historical data on water source and the available THM measurements wereused to impute THM levels at municipality (zip code) level for years in the pastwhen THM measurements were absent. Available THM measurements wereaveraged and imputed to the past when water source and treatment wereunchanged. Proportion of surface water was used as a weight to this average inthe event of changes in water source. Before chlorination started, THM levels wereassumed to be zero. Residential histories and estimated THM levels were mergedby zip code and year to estimate average THM levels from age 18 to the age ofinterview in participants.

2.9. Trihalomethane ingestion estimates

Three THM ingestion estimates (mg/day) were calculated: lifetime (since 18years), last five years, and last year before recruitment. Estimated ingested levelswere the product of the daily volume of cold water ingested times the residentiallevels of THM assigned to that year (according to the water source: municipal,bottled or private well). Bottled water trihalomethane levels were assigned zero.Private wells′ trihalomethane levels were assigned 3.2 mg/l, based on 56 samples ofwell water in Spain (MCC unpublished data).

2.10. Covariables

Age, sex, municipality, education level (elementary or less, high school, anduniversity or more), tobacco smoking (classified as never, former and currentsmokers) and comorbidities (type 2 diabetes mellitus, arterial hypertension,hypercholesterolemia, gout, urolithiasis, COPD, anemia) were available from theMCC-Sp questionnaire and were considered as potential confounders. Medicationswere grouped by pharmacologic groups (Appendix A, supporting information:Tables A1 and A2). Body surface area in square meters was calculated using theMosteller's formula, square root of the product of the weight (kilograms) and theheight (centimeters) divided by 3600 (Mosteller, 1987). Plasma volume wasestimated at 2 l/m2 of body surface, as a proxy of the volume of distribution. Foursubjects did not provide information about height and one did not provideinformation of weight. Plasma volume was imputed for these subjects based ontheir sex.

2.11. Statistical analysis

Creatinine was used to normalize the TCAA concentrations (analyte concentra-tion divided by creatinine concentration, mmol/mol). TCAA excreted in a urine voidwas calculated by multiplying the concentration of TCAA in the urine (mmol/l) bythe total volume of the urine void (in liters). This variable (expressed in picomoles,pmol) was divided by the depuration time (minutes between the first morning

urine void and the previous urination) to calculate excretion rate. Agreementbetween excretion rate and creatinine adjusted TCAA were compared using BlandAltman plot and Lin's concordance correlation. Bivariate comparisons of geometricmeans were performed using a t-test (two groups) or ANOVA test (more than twogroups) of the log-transformed variables assuming a log-normal distribution of thevariables (White and Thompson, 2003). Urinary TCAA levels were log transformedfor regression analyses. Association between urinary TCAA depuration and covari-ables were calculated using simple and multiple linear regressions through Huber–White sandwich adjustment to reduce influence outliers. Beta coefficients wereexponentiated to be interpreted as geometric mean ratios relative to the referencegroup. Water zones were defined statistically using a k-means cluster analysis ofthe four THM and the nine HAA measured in tap water. Interactions were tested asproduct terms in the regression. Interaction p-value was calculated using Wald testin robust regression. Levels under the LoD were imputed half the LoD. Spearmanrank correlation coefficients were calculated between urinary TCAA and historicalTHM levels.

3. Results

Among the 257 eligible subjects, 120 (46.6%) participated.Reasons for no participation included death (19, 7.4%), unable tocontact – wrong telephone number or never answered the call,(59, 23%)–, and refusal (59, 23%). Mean age and sex ratio weresimilar in the included and excluded subjects (70 years at the firstinterview, 1.9:1 male to female ratio). Compared with the excludedsubjects, we recruited less subjects than expected in the Cornellàde Llobregat area (p-value¼0.02). Finally, 10% fewer people withelementary studies or less were recruited compared to the non-participating subjects (p-value¼0.009).

Among participants, 41 (34%) were women, and mean age was74 years old (range: 63–85) at the time of urine collection(Table 1). As only seven participants lived in Cornellà de Llobregatthey were pooled with Hospitalet participants as they shared thesame water source. Among former and current smokers, mostsubjects were males (male to female ratio 9:1). Only sevenparticipants reported not suffering from any comorbidity andthree subjects attended swimming pools in the previous week.

Total THM levels in tap water samples ranged between un-quantifiable to 84.5 mg/l. Three water zone clusters were identifiedstatistically according to concentrations and speciation of THMsand HAAs, i.e. proportion of brominated and chlorinated species(Table 2). Water zone 1 (n¼56) showed lowest levels of totalTHMs and HAAs, and highest proportion of brominated species.Water zone 2 (n¼51) showed highest concentrations of totalTHMs and high proportion of brominated species. Finally, waterzone 3 (n¼13) showed highest proportion of chloroform andchlorinated HAAs species. TCM and MCAA were, respectively, theTHM and HAA with higher proportion of undetected levels (34%and 93%, respectively). In contrast, TCAA was detected in all thewater samples. Average lifetime THM levels were higher thancurrently measured levels. Lifetime THM ranged from 50 to136.7 mg/l, 2 to 50 times the current measured levels. Spearmancorrelations between historical and currently measured levelswere �0.51 for total THM (p-value o0.001), 0.53 for TCM (p-value o0.001), �0.48 for BDCM (p-value o0.001), �0.18 forDBCM (p-value¼0.05), and �0.17 for TBM (p-value¼0.07). Similarresults were obtained with THM ingestion estimates at thedifferent cut-offs (lifetime, last 5 years, and last year), data notshown. Calculated ingestion of TCAA ranged from 0.8 to261.4 nmol/48 h (equivalent to 0.1–42 mg/48 h). Estimated TCAAingestion changed in the three areas according to the concentra-tion of TCAA in water (mean levels per water zone: 9.3, 32.8 and87.9 nmol/l, respectively).

Mean total fluid, total tap water and bottled water consumedthe previous 48 h were, respectively, 4.1, 2.3 and 1 l (Table 3).Water consumption habits were similar by sex. Statistically sig-nificant differences in water consumption habits were observed by

Table 1General characteristics of the study population.

Male n¼79 Female n¼41 Total n¼120 Min Maxmean7SE mean7SE mean7SE

Age (years) 7070.6 69.471.0 73.670.5 63.1 85.3Anthropometry

Weight (kg) 7871 6872 7571 50 106Height (cm) 16871 15871 16571 145 184Body surface area (m2) 1.970.02 1.770.02 1.870.02 1.5 2.3

n (%) n (%) n (%)Municipality of residence

Badalona 20(25.3) 4(9.8) 24(20.0)Barcelona 31(39.2) 21(51.2) 52(43.3)Hospitalet/ Cornellà de Llobregat 28(35.4) 16(39) 44(36.7)

Socioeconomic statusrPrimary school 48(60.0) 30(75.0) 78(65.0)High school 24(30.0) 5(12.5) 29(24.2)ZUniversity 8(10.0) 5(12.5) 13(10.8)

Tobacco smokingNever smokers 25(31.3) 34(85.0) 59(49.2)Former smokers 47(58.8) 4(10.0) 51(42.5)Current smokers 8(10.0) 2(5.0) 10(8.3)

ComorbiditiesType 2 diabetes 20(25.3) 6(14.6) 26(21.7)Arterial hypertension 46(58.2) 15(36.6) 61(50.8)Hipercholesterolemia 38(48.1) 20(48.8) 58(48.3)Gout 15(19.0) 0(0.0) 15(12.5)Urolithiasis 20(25.3) 5(12.2) 25(20.8)Chronic obstructive pulmonary disease (COPD) 9(11.4) 5(12.2) 14(11.7)Anemia 6(7.6) 10(24.4) 16(13.4)

MedicationsAngiotensin converting enzyme inhibitors, Angiotensin receptor antagonists, Potassium sparing diuretic 32(40.5) 10(24.4) 42(35)Loop or thiazide diuretics 14(17.7) 5(12.2) 19(15.8)Uricosurics 11(13.9) 0(0.0) 11(9.2)Warfarinic anticoagulants 2(2.5) 0(0.0) 2(1.7)Nonsteroidal anti-inflammatory drugs 4(5.1) 7(17.1) 11(9.2)Statins 30(38) 11(26.8) 41(34.2)Acetylsalycilic Acid (low dose) 13(16.5) 3(7.3) 16(13.3)

Abbreviations: SE: Standard error of mean.

L.A. Salas et al. / Environmental Research 135 (2014) 276–284 279

water zones and municipalities. Tap water consumption washighest in the water zone 2 (mean 1.5 vs. 0.9 l in zone 1 and 3,p-value¼0.02) and in Badalona (1.7 vs. 0.8 l in Barcelona and 1.2 lin Hospitalet/Cornellà de Llobregat, p-value¼0.008). Consumptionof bottled water was lowest in Badalona (0.3 vs. 1.1 l in Barcelonaand 1.2 l in Hospitalet/Cornellà, p-value¼0.006). Filters were usedby 18% of the subjects, and 26% consumed water outside home inthe 48 h previous to urine sampling (Table 3).

Urinary TCAA levels ranged from non detectable in 13 subjects(o3 nmol/l) to 3.5 mmol/l of urine, equivalent to o0.49 mg/l to0.57 mg/l (Table 4). Creatinine adjusted TCAA levels ranged fromo0.2 mmol/mol to 0.76 mmol/mol. Among those above the limit ofdetection, and excluding the highest measurement, the creatinineadjusted urinary TCAA levels ranged from 0.4 to 14.7 mmol/mol. Ofthem, 13 subjects (10.8%) had creatinine measurements below3 mmol/l. The TCAA excretion rate ranged between o0.15 pmol/min and 4.93 TCAA nmol/min.

Simple robust linear regression showed no statistically signifi-cant association between urine TCAA levels and historical THMexposure (o0.1% increase, p-value¼0.61). Significant associationswere observed for smoking (average 56% reduction in currentcompared to never smokers), plasmatic volume (average 32%reduction per liter increase), tap water consumption (average

15% reduction per liter of water consumed) and water zone(average 40% reduction in water zone 2 compared to water zone1). A significant interaction was found between sex and age(average 40% reduction of urinary TCAA excretion rate in femalesper year over 74 years of age, compared to a male of 74 years ofage). Medications and comorbidities (use of antihypertensivemedication, or diabetes) were not associated with urine TCAAlevels (Table 5). Several covariables were excluded due to a limitedsample size (COPD, warfarin use). Multiple robust linear regressionshowed a 2% increase in urinary TCAA for a 10% increase iningested TCAA (Table 5) and the interaction between sex and agebecame non-statistically significant. Urinary TCAA decreased 79%in current versus never smokers, 64%/l increase in plasmaticvolume, 50% among users of NSAIDs, and 17%/l of tap waterconsumed. Subjects that consumed fluids outside home had 32%less TCAA level compared to those who only consumed fluids athome. Subjects from Hospitalet/Cornellà de Llobregat showed 63%higher levels of urinary TCAA excretion rate compared subjects inBarcelona, and subjects in water zone 2 had 42% less TCAAcompared to those in water zone 1. The model explained 42.7%of the urinary TCAA excretion rate (crude R2 42.7%, adjusted R2

33.5%).

Table 2Characteristics of tap water and historical THM estimates in the study area.

Exposure Water zone 1 Water zone 2 Water zone 3 Total Min Max noLoDn¼56 n¼51 n¼13 n¼120GM(SD) GM(SD) GM(SD) GM(SD)

Tap water spot samples (lg/l)Total trihalomethanes 17.8(1.9) 41.0(1.2) 36.5(1.2) 27.4(1.9) oLoD 84.5

Chloroform 0.5(3.6) 2.4(4.3) 20.5(1.4) 1.4(5.8) o0.4 31.8 41Bromodichloromethane 1.0(1.9) 3.0(1.9) 8.6(1.3) 2.0(2.6) o0.4 11.5 2Dibromochloromethane 4.1(2.2) 10.0(1.3) 3.8(1.4) 6.0(2.1) o0.4 23.4 3Bromoform 10.6(2.5) 22.5(1.3) 1.3(3.7) 11.6(3.1) o0.4 43.7 4

Total haloacetic acids 11.3(1.8) 22.8(1.3) 26.2(1.1) 16.7(1.8)Monochloroacetic acid 0.2(1.1) 0.2(1.2) 0.4(1.8) 0.3(1.3) o0.4 0.9 112Dichloroacetic acid 0.5(2.3) 1.7(2.5) 8.3(1.3) 1.2(3.4) o0.2 12.4 1Trichloroacetic acid 0.5(2.4) 1.7(2.9) 8.2(1.3) 1.1(3.7) o0.2 13.3 0Monobromoacetic acid 0.5(1.9) 0.7(1.8) 0.2(1.8) 0.5(2.0) o0.2 1.9 12Dibromoacetic acid 4.5(2.7) 7.7(1.5) 1.4(2.2) 5.0(2.5) o0.2 12.4 3Bromochloroacetic acid 1.1(2.1) 2.3(1.5) 2.8(1.2) 1.7(2.0) o0.2 5.3 3Bromodichloroacetic acid 0.3(2.3) 1.0(2.0) 2.5(1.4) 0.6(2.9) o0.2 4.1 14Dibromochloroacetic acid 0.8(2.2) 1.8(1.3) 0.7(1.5) 1.1(2.0) o0.2 3.4 3Tribromoacetic acid 1.9(2.0) 3.5(1.3) 0.3(2.7) 2.0(2.5) o0.4 6.6 11

Historical lifetime levels (lg/l)Total THM 99.7(1.4) 76.9(1.3) 70.7(1.3) 86.0(1.4) 50 136.8

Chloroform 19.1(1.2) 21.5(1.2) 23.6(1.2) 20.6(1.2) 13.2 30.6Bromodichloromethane 28.4(1.3) 23.9(1.2) 22.0(1.2) 25.7(1.3) 13.7 36.9Dibromochloromethane 19.9(1.6) 13.9(1.5) 12.0(1.6) 16.1(1.6) 7.0 30.0Bromoform 24.3(3.0) 9.5(4.3) 6.8(3.9) 14.2(4.0) 0.7 54.7

Calculated ingestion 48 hIngested TCAA (nmol/48 h) 5.6(2.4) 16.8(3.3) 48.3(3.3) 11.2(3.6) 0.8 261.4

Historical ingestion levels (last year) (lg/day)a n¼31 n¼37 n¼11 n¼79Total THM 72.0(2.2) 79.4(2.0) 45.2(2.9) 70.6(2.2) 0.0 293.6

Chloroform 8.1(3.0) 13.2(3.0) 11.0(3.8) 10.6(3.1) 0.0 99.2Bromodichloromethane 9.7(2.3) 12.1(1.9) 8.2(2.9) 10.5(2.2) 0.0 38.6Dibromochloromethane 13.6(2.7) 12.0(2.8) 7.2(3.4) 11.8(2.8) 0.0 69.4Bromoform 20.6(6.3) 11.6(9.2) 7.6(7.6) 13.7(7.8) 0.0 168.0

p-Value of heterogeneity for all the compounds between water zones was o0.001.Abbreviations: GM(SD): geometric mean (standard deviation of geometric mean), LoD: limit of detection.

a Geometric means calculated over non-zero values.

Table 3Reported fluid intake and filter use in the study population.

Questionnaireinformation

n¼120 Percentage of totalfluids

Min Max

Fluid intake/48 h (ml) mean7SE

Milk 386758 9 0 6390Bottled water 10137111 25 0 6464Hot tap watera 1083769 26 0 3372Plain tap water 11657126 28 0 5600Tap water-based fluidsb 22487145 55 0 6910Total fluidsc 41277146 100 1344 9108

n (%)Filter use 22(18)Non-residential waterintake

31(26)

Abbreviations: SE: standard error of mean.a Includes coffee, tea, herbal drinks, and soups subtracting milk volume.b Includes (a) plus plain tap water.c Includes (b) plus milk volume and others fluids (spirits, yogurts and sodas).

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As sensitivity analyses we excluded the highest extreme value(potential outlier), excluded the subjects with creatinineo3 mmol/l, included participant's weight instead of plasma vo-lume, and compared. Models using urine TCAA levels (either

adjusted and unadjusted by creatinine) showed the same associa-tion between ingested and urinary levels TCAA (Table 6) comparedto the model using urinary TCAA excretion rate (Table 5). However,these alternative models did not fulfill all the linear regressionassumptions: the models were either misspecified or they hadomitted variables. We compared results excluding missing valuesagainst the multiple imputed model without changes in theestimations.

4. Discussion

The study population had a mean age of 74 years old and a highprevalence of comorbidities (94%). Trichloroacetic acid in tapwater samples showed low concentrations (geometric mean of1.1 mg/l) and urine TCAA adjusted for creatinine was on average2.9 mmol/mol or 4.2 mg/g (geometric mean). In this study popula-tion, urinary TCAA levels are affected by several individual factorsincluding age, sex, tobacco consumption, plasmatic volume, use ofmedications (NSAIDs) and amount of water consumed, apart fromingested TCAA. Lifetime THM exposure and THM ingestion esti-mates did not correlate with urinary TCAA levels.

The arithmetic mean of urine TCAA levels we observe is compar-able to other reports in the literature. However, given the rightskewed distribution, the geometric means are much lower thanprevious studies. Arithmetic mean of urine TCAA excretion rate was

Table 4Urinary trichloroacetic acid (TCAA) levels in the study population.

Urine levels n TCAA(mmol/l)

Creatinine adjusted TCAA(mmol/mol)

TCAA- excretion rate(pmol/min)

Time of depuration (h) Creatinine (mmol/l)

GM (SD) GM (SD) GM (SD) GM (SD) GM (SD)

Totala 120 16.7(3.0) 2.9(2.9) 17.3(3.2) 4.6(1.8) 5.9(1.7)Min 1.5 0.1 0.7 0.5 1.4Max 3515 764.1 4933.3 30 16.5noLoD 13 13 13 0 0

Male 79 19.1(3.0) 2.8(3.0) 20.5(3.2) 4.7(1.8) 6.9(1.6)Female 41 13.0(2.9) 3.0(2.7) 12.4(3.0) 4.4(1.8) 4.3(1.6)

p-Value of heterogeneityb 0.07 0.6 0.03 0.6 o0.001

Water zone 1 56 19.8(2.4) 3.6(2.2) 20.7(2.6) 4.5(1.9) 5.5(1.6)Water zone 2 51 12.3(2.8) 1.9(2.5) 12.4(2.9) 4.7(1.7) 6.4(1.7)Water zone 3 13 26.8(5.9) 4.9(6.1) 28.8(6.8) 5.0(1.6) 5.4(1.7)

p-Value of heterogeneityb 0.02 o0.001 0.02 0.8 0.3

Barcelona 52 14.6(4.0) 2.5(3.9) 14.3(4.4) 4.8(1.9) 5.9(1.8)Hospitalet/Cornellà de Llobregat 44 18.4(2.1) 3.4(2.0) 19.9(2.3) 4.4(1.7) 5.5(1.6)Badalona 24 18.9(2.5) 2.9(2.2) 20.0(2.6) 4.8(1.5) 6.5(1.5)

p-Value of heterogeneityb 0.5 0.3 0.3 0.7 0.4

Abbreviations: GM(SD): geometric mean (standard deviation of geometric mean); LoD: limit of detection.a All values under LoD were replaced by LoD/2 for mean calculations.b p-Value of heterogeneity based on t-test or ANOVA of the log-transformed variables.

Table 5Geometric mean change in Urinary TCAA excretion rate (pmol/min) in the study population.

Unadjusted models Adjusted modelGeometric mean ratio (95%CI) p-Value R2(%) Geometric mean ratio (95%CI) p-Value

Male Ref 6.80 RefFemale 0.64 (0.41, 0.99) 0.04 0.36 (0.22, 0.59) o0.01Age, yrs (74 yrs centered), Male 1.02 (0.97, 1.06) 0.47 1.00 (0.95, 1.05) 0.97Age, yrs (74 yrs centered), Female 0.60 (0.39, 0.94) 0.03 0.33 (0.20, 0.55) o0.01

Interaction p-value 0.05 0.14

r Primary school Ref 4.50 RefHigh school 1.80 (1.10, 2.97) 0.02 1.72 (1.04, 2.81) 0.04University 1.26 (0.63, 2.50) 0.51 1.30 (0.79, 2.13) 0.30

Never smokers Ref 3.70 RefFormer smokers 0.97 (0.63, 1.51) 0.89 0.62 (0.38, 0.99) 0.04Current smokers 0.44 (0.20, 0.97) 0.04 0.21 (0.10, 0.44) o0.001

Plasmatic volume (l) 0.68 (0.38, 1.24) 0.21 1.40 0.36 (0.19, 0.67) o0.01NSAIDs consumption (yes/no) 0.53 (0.26, 1.10) 0.09 2.50 0.50 (0.26, 0.93) 0.03Tap water consumption (l) 0.85 (0.75, 0.98) 0.02 4.40 0.83 (0.71, 0.97) 0.02Ingestion of fluids outside home 0.79 (0.48, 1.27) 0.32 0.80 0.68 (0.46, 0.99) 0.04

10% Increase in ingested TCAA 1.00 (0.99, 1.02) 0.85 1.40 1.02 (1.00, 1.04) 0.02

Barcelona Ref 2.00 RefHospitalet/Cornellà de Llobregat 1.39 (0.87, 2.24) 0.17 1.63 (1.15, 2.34) 0.01Badalona 1.40 (0.79, 2.48) 0.25 1.35 (0.84, 2.18) 0.21

Water zone 1 Ref 6.60 RefWater zone 2 0.60 (0.39, 0.93) 0.02 0.58 (0.38, 0.89) 0.01Water zone 3 1.39 (0.69, 2.79) 0.35 0.92 (0.37, 2.34) 0.87

Intercept Adjusted R2(%)1.22 33.50

Abbreviations: NSAIDs: nonsteroid anti-inflammatory drugs; Ref: reference category; 95% CI: 95% Confidence interval.

L.A. Salas et al. / Environmental Research 135 (2014) 276–284 281

66 pmol/min while the geometric mean was 17.3 pmol/min, approxi-mately one third of the global concentration (50.6 pmol/min) re-ported by Weisel et al. (1999) or half the concentration of the less

exposed group (38.9 pmol/min). The geometric mean levels of urineTCAA that we observe (2.9 mmol/mol) are lower than previousstudies reporting 4.9 and 4.2 mmol/mol, respectively (Smith et al.,

Table 6Geometric mean change in urinary TCAA levels with and without urine creatinine adjustment in the study population (models adjusted for all the covariables).

Urine TCAA levels (lmol/l)p-Value

Urine TCAA-per creatinine mol (lmol/mol)p-ValueGeometric mean ratio (95%CI) Geometric mean ratio (95%CI)

Male Ref RefFemale 0.44 (0.27, 0.73) o0.01 0.74 (0.46, 1.17) 0.20Age, yrs (74 yrs centered), Male 0.98 (0.94, 1.03) 0.47 1.01 (0.97, 1.06) 0.59Age, yrs (74 yrs centered), Female 0.97 (0.91, 1.03) 0.32 0.95 (0.90, 1.01) 0.07

interaction p-value 0.25 0.06

r Primary school Ref RefHigh school 1.30 (0.80, 2.12) 0.29 1.50 (0.97, 2.32) 0.07University 1.00 (0.59, 1.7) 1.00 1.02 (0.60, 1.74) 0.95

Never smokers Ref RefFormer smokers 0.74 (0.47, 1.17) 0.20 0.72 (0.46, 1.11) 0.14Current smokers 0.30 (0.13, 0.71) o0.01 0.32 (0.15, 0.69) o0.01

Plasmatic volume (l) 0.41 (0.23, 0.75) o0.01 0.40 (0.24, 0.66) o0.001NSAIDs consumption (yes/no) 0.57 (0.33, 0.97) 0.04 0.53 (0.34, 0.82) 0.01Tap water consumption (l) 0.9998 (0.9996, 0.9999) 0.01 0.9998 (0.9996, 0.9999) o0.01Ingestion of fluids outside home 0.70 (0.49, 1.02) 0.06 0.66 (0.47, 0.92) 0.02

10% Increase in ingested TCAA 1.02 (0.999, 1.04) 0.07 1.02 (1.004, 1.04) 0.02

Barcelona Ref RefHospitalet/Cornellà de Llobregat 1.40 (0.98, 2.00) 0.06 1.64 (1.19, 2.27) o0.01Badalona 1.21 (0.77, 1.90) 0.41 1.32 (0.87, 2.00) 0.19

Water zone 1 Ref RefWater zone 2 0.61 (0.41, 0.91) 0.02 0.61 (0.43, 0.86) 0.01Water zone 3 0.96 (0.37, 2.47) 0.93 1.09 (0.44, 2.69) 0.85

Intercept Adjusted R2(%) Intercept Adjusted R2(%)821.22 25.20 116.02 33.70

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2013; Weisel et al., 1999). In contrast, a large biobank study, n¼402including 20–59 years old subjects, reported a mean level of1.8 mmol/mol (Calafat et al., 2002), half the concentration we ob-served. In a study among 611 pregnant women (Costet et al., 2012),only 41 were above the limit of detection (LoDE61.2 nmol/l), whichhampers the comparisonwith our study (LoD 3 nmol/l). Comparisonsbetween studies are limited due to different population character-istics, laboratory methods, normalization using creatinine, excretionrates and limits of detection. The average proportion of excretioncompared to the calculated ingestion was about 21%, which isconsistent with experimental studies in younger populations wherethe excretion percentage compared to ingestion ranged between 15%and 71% (Froese et al., 2002; Zhang et al., 2009). This is explained bythe long half life of the compound, ranging between 2.1 and 6.3 days(Bader et al., 2004).

Total tap water ingestion was only moderately related to urineTCAA excretion rate (R2¼4.4%). These findings are not consistentwith experimental studies, where TCAA ingestion and total wateringestion have been related to urinary TCAA levels (Kim et al.,1999; Zhang et al., 2009). The difference may partly rely on thevariability of ingested TCAA levels. While ingested levels aremaintained constant in experimental studies, intake is affectedby water handling variables (e.g. filtering or boiling) in real lifeconditions (Chowdhury et al., 2010). Ultimately, this means thatcalculated TCAA ingestion and total water ingested variablesmeasure different things, and thus they can be included in thesame model. On the other hand, consumption of bottled water wasnot related with urinary TCAA levels in our data. As in previousstudies, urinary TCAA excretion rate was not related to tap waterTCAA levels. However, in contrast to previous studies (Smith et al.,

2013; Weisel et al., 1999), cold tap water intake at home did notincrease excretion of urine TCAA. Moreover, in our data, theingestion of water outside home reduced TCAA excretion rate,probably because of dilution. However, in contrast to previousstudies it did not modify the association between total wateringestion and urinary TCAA levels (Kim et al., 1999; Smith et al.,2013). Finally, as almost 100% of our population was retired, wecannot compare our results with Smith et al. (2013) findings whofound a potential differential intake due to employment statusdifferences.

Several physiological and lifestyle variables were related tourine TCAA excretion rate. Plasma volume, which corresponds tothe distribution compartment, explained 5.1% of the variance ofthe model. It is also a proxy of the renal compartment and thehemodynamics, i.e. volume filtered by the kidneys (Faupel-Badgeret al., 2007). We found that age and sex interaction was associatedwith TCAA levels in the unadjusted model, which was notobserved in the multivariable model, probably due to insufficientsample size. This age-sex interaction is consistent with previousphysiological evidence (Silbiger and Neugarten, 2008), and tophysiologically-based kinetic model-PBKM considering extremeage groups for THM exposure (Haddad et al., 2006). As seen inother study, former and current smokers had reduced urine TCAAexcretion (Zeng et al., 2014b), which is also consistent with studiesshowing that smoking modifies glomerular filtration rate andincreases chronic kidney disease (Miyatake et al., 2010). Therelationship with NSAIDs may be related to an altered glomerularfiltration, however, we did not find this relationship with angio-tensin enzyme converter inhibitors, or angiotensinogen receptorblockers (Harris, 2013). The inability to exclude all potential

L.A. Salas et al. / Environmental Research 135 (2014) 276–284 283

sources of TCAA exposure is a limitation in our study. Although werequested information about occupational and non-occupationalexposure to TCAA and/or to TCAA precursors, many consumerproducts may contain traces of TCAA (i.e. paints, herbicides).Second, we were unable to obtain water samples before the urinecollection, leading to potential measurement error in the esti-mated ingested TCAA levels. Finally, due to the observationalcharacter of the study we were unable to oversample somecovariables of interest such as the warfarin users (n¼3) andswimming pool users (n¼7).

To our knowledge this is the first study measuring urinaryTCAA in the framework of an epidemiological study on cancer, andthe first to measure urinary TCAA in population older than 60years old. A 48 h record was used to ascertain drinking waterintake. This approach has the advantage of being prospective, self-administered, and the customary consumption pattern is lessaffected by the interviewer, and less prone to recall bias comparedto recall questionnaires. The measurement of the serving sizes is astrength of this study since the use of standard serving sizes(glasses, cups, etc.) is a source of inaccuracy in previous studies(Mons et al., 2007). Using a ruler to calculate the serving sizevolume was less burdensome in the field than carrying andcalibrating a balance. The ruler method was highly correlated(r¼0.85) when compared to weight (assuming 1 mg ofwater¼1 ml of water) in our pilot validation. The collection oftotal urine void was an advantage since 10.8% of urine samples hadless than 3 mmol creatinine/l. This proportion is higher than a 5%expected in population-based studies (Cocker et al., 2011). Thesesamples should have been excluded if using the TCAA-creatinineadjusted measures because of dilution.

Under the conditions observed in our study, the utility ofurinary TCAA as a biomarker of exposure to DBPs in adult cancerstudies shows several limitations. First, remarkably low levels ofTCAA in drinking water samples probably undermine the perfor-mance of urine TCAA as a marker of exposure. Second, weobserved long-term changes in THM levels (lower current levelscompared to historical estimates) that reflect treatment improve-ments, and that urine TCAA is unable to capture. Third, we showthat the biomarker is affected by several physiological/pathologicalvariables that are more prevalent in aging populations such as thestudy populations of case-control studies of adult cancer. Finally,the extent of biomarker distortion in cancer wasting is uncertain(e.g. hypoalbuminemia/edema associated or not with organdysfunction).

In conclusion, our findings support that urine TCAA is not avalid biomarker in case-control studies of adult cancer given thatadvanced age, comorbidites and medication use are prevalent andare determinants of urine TCAA levels, apart from ingested TCAAlevels. In addition, low TCAA concentrations in drinking waterlimit the validity of urine TCAA as an exposure biomarker.

Acknowledgements

Authors thank our fieldworkers Lourdes Arjona, Jeroen de Bont,and Estela Carrasco. We also thank the Health and Safety Labora-tory (Buxton, UK) for processing the urine samples. We acknowl-edge Manolis Kogevinas, Kate Jones and Mark Nieuwenhuijsen fortheir feedback on the earliest drafts. We thank the Parc de SalutMar Biobank (MARBiobanc) and Núria Somoza for their support tothe project.

Appendix A. Suplementary Information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.envres.2014.09.018.

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