Measurement of Low Levels of Arsenic Exposure: A Comparison of Water and Toenail Concentrations

Margaret R. Karagas1, Tor D. Tosteson1, Joel Blum2, Bjoern Klaue2, Julia E. Weiss1, Virginia Stannard1, Vickie Spate3 and J. Steven Morris3

1 Departments of Community and Family Medicine, Dartmouth Medical School, Hanover, NH.
2 Department of Earth Sciences, Dartmouth College, Hanover, NH.
3 Research Reactor Center, University of Missouri, Columbia, MO.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A study was conducted to evaluate toenail arsenic concentrations as a biologic marker of drinking water arsenic exposure. Study subjects were controls in a US population-based case-control study of nonmelanoma skin cancer, randomly selected from drivers' license records (those <65 years of age) and Medicare enrollment files (those >=65 years of age). Between 1994 and 1997, a total of 540 controls were interviewed and toenail samples of sufficient weight were collected from 506 (93.7%) of these. Beginning in 1995, a sample of tap water was taken from the participants' homes; a total of 217 (98.6%) water samples were obtained from the 220 subjects interviewed. Arsenic determinations were made from toenail samples using neutron activation analysis. Water samples were analyzed using hydride-generation magnet sector inductively coupled mass spectrometry. Among 208 subjects with both toenail and water measurements, the correlation (r) between water and nail arsenic was 0.65 (p < 0.001) among those with water arsenic concentrations of 1 µg/liter or higher and 0.08 (p = 0.31) among those with concentrations below 1 µg/liter (overall r = 0.46, p < 0.001). Our data suggest that toenail samples provide a useful biologic marker for quantifying low-level arsenic exposure.

arsenic; epidemiologic studies; metals, heavy


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Arsenic is a metalloid element present throughout the earth's crust. An excess of cancers, cardiovascular diseases, diabetes, and other health conditions has been found in populations highly exposed to inorganic arsenic through drinking water contamination, occupational exposure, or medicinal sources (1GoGoGo–4Go). Most humans are exposed to trace amounts of arsenic in food and water. However, the effects of inorganic arsenic at lower levels of exposure are as yet uncertain. Risk assessment models based on extrapolations from more highly exposed populations suggest that levels as low as 2 µg/liter may be carcinogenic (5Go). A methodological challenge to investigating the effects of low dose exposure is the ability to precisely quantify exposure on an individual level. This may, in part, explain why ecologic group assessments of arsenic exposure and cancer risk in the United States have been inconclusive (6GoGo–8Go). Moreover, the relevant time period of exposure for many diseases of interest, such as cancer, may be several years if not decades.

Arsenic is rapidly cleared from the bloodstream and excreted via the kidneys (1Go). For this reason, blood and urine can provide only short-term measures of arsenic exposure. Because of arsenic's affinity for sulfhydryl groups of keratin, arsenic accumulates in scleroproteins such as the hair and nails (9Go, 10Go). The normal growth rate for toenails is 3–12 months, and there is evidence that toenail measures of arsenic are reproducible over a period of several years (11Go). Toenail clippings are easy to collect (i.e., noninvasively) and can be analyzed for arsenic along with other trace elements (e.g., selenium, mercury, zinc) (12Go). In a small pilot study, we found that toenail concentrations were highly correlated with well-water concentrations, particularly among those with detectable water arsenic concentrations (1 µg/liter or greater) (13Go). Because of the importance of this issue to our case-control study, and because of a greatly improved method for assessing water concentrations, we conducted a more definitive study in a random sample of individuals from our study population.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study subjects
Subjects selected for the study were controls enlisted in a nonmelanoma skin cancer case-control study of New Hampshire residents aged 25–74 years described elsewhere (14Go). Briefly, controls were frequency matched on age and sex to the combined distribution of the skin cancer cases (basal cell and squamous cell). For those less than 65 years of age, we selected controls from annual computer tapes of state residents holding a driver's license provided by the New Hampshire Department of Transportation. Controls 65 years of age or older were chosen from annual computerized Medicare enrollment files of residents maintained by the Health Care Financing Administration. Enrollment (cases and controls) was limited to English-speaking individuals with working phone numbers. Those selected were sent a letter informing them of the general aims and requirements of the study (but not the specific hypotheses). The letter was followed by a telephone call from the study interviewer to clarify any questions and schedule an in-person interview. The present study did not include the 21 individuals selected for our previously reported pilot investigation (13Go).

Interview
Those who agreed to take part in the study underwent an extensive in-person interview, covering residential and medical history and lifestyle factors (e.g., use of tobacco). Participants were asked several questions relating to their household water supply including the type of water supply that served their household, classified as public water, shared well, or a private well or spring serving 15 or fewer households and less than 25 individuals. Those who used a private water system were asked to specify the type of system (i.e., artesian, shallow, or spring). For all subjects, the interviewer asked the duration of their use of the current water supply, the number of glasses of water per day they consumed in foods or beverages from this water source, and whether they used water filters (e.g., what type and for how long). Prior to the interview, subjects were mailed the instructions and materials to collect toenail clipping samples. A toenail clipper was included as an incentive. At the interview, those who had not saved a toenail sample were asked to mail it in later using a self-addressed stamped envelope provided.

Interviews took place between 1994 and 1997, and in 1995 we began collecting tap water samples from each subject's home. Strict precautions were taken to avoid contamination as previously described (14Go). The water samples were collected in commercially washed (mineral free) high density polyethylene bottles that meet Environmental Protection Agency standards for water collection (I-Chem, Newcastle, Delaware) or acid-washed bottles from a class 100 clean room. Bottles were kept in a washed, sealed plastic bag prior to and after collection. Wearing powderless latex gloves, the interviewer took the water samples after running the tap for 2–3 minutes. Duplicate samples were drawn for 10 percent of the households (every 10th interview), and field blanks were collected on a quarterly basis by each interviewer. All bottles were labeled with identification numbers that did not reveal the case-control status of the study participants or whether the sample was a replicate from the same household (14Go).

Arsenic determinations from toenail clipping samples
Toenail clipping samples were analyzed for arsenic and other trace elements by instrumental neutron activation analysis at the University of Missouri Research Reactor, using a standard comparison approach as described previously (15Go, 16Go). Using this approach, samples are not subjected to chemical or thermal destruction, and thus mechanical or chemical (volatility) loss, separation yield, matrix modification, and external contamination are reduced substantially or eliminated entirely.

Prior to analysis, any nails that contain nail polish were cleaned with acetone. The samples then were placed in 20-ml polyethylene vials. Vials were filled three fourths full with deionized water and sonicated for 10 minutes to remove external surface contamination. The nails were collected by vacuum filtration and thoroughly rinsed with deionized water. Samples were carefully separated from any insoluble debris and then freeze-dried. The dried samples were weighed into 0.2-ml high-density polyethylene vials closed with a friction-fit cap and then heat sealed.

Matrix-matched quality control samples, having known arsenic content, and analytical blanks were analyzed with the samples and standards. Two standard reference materials (orchard leaves (NBS SRM 1573) and bovine liver (NBS SRM 1577); National Institute of Standards and Technology, Gaithersburg, Maryland) and an internally certified keratin powder were used as quality control materials. Eight samples, one arsenic standard, and one keratin quality control sample were loaded into a polyethylene sleeve that is 7.5 cm long and 1.0 cm wide and then centered in a ventilated high-density polyethylene rabbit. In this configuration, the samples and standards were irradiated for a total of 60 minutes at a thermal neutron flux of 8 x 1013 neutrons/cm2/second. Following a decay period of approximately 24 hours, the samples and standards were live-time counted for 2 hours at a sample-to-detector distance of approximately 10 cm using a high resolution gamma-ray spectrometer equipped with an automatic sample changer. The 559-keV (kilo-electron volt) gamma ray from the decay of As-76 (half life = 26.3 hours), corrected for physical decay, was used to quantify arsenic by standard comparison. Arsenic concentrations measured in quality control samples were in good agreement (within 1 standard deviation) with the certified values or accepted values (i.e., for keratin). The detection limit of this method is approximately 0.001 µg/g.

Water measurements
Drinking water samples were analyzed for arsenic concentration using a Finnigan MAT GmbH ELEMENT (Finnigan MAT GmbH, Bremen, Germany) high resolution inductively coupled mass spectrometer equipped with a MY hydride generator (Finnigan MAT GmbH) (17Go). The water samples were acidified to pH 1 with ultrapure HNO3 (Seastar Chemicals, Inc., Sidney, British Columbia, Canada) upon arrival at the laboratory. At least 24 hours prior to analysis the water samples were spiked with Suprapur H2O2(Merck KGaA, Darmstadt, Germany) to the 0.01 percent level. All sample preparations and analyses were carried out in a trace-metal clean high-efficiency particulate air-filtered environment. Analytical blanks and potential instrumental drifts were carefully monitored, and instrument standardization and reproducibility were performed with certified standard reference materials.

Arsenic has only one stable isotope (75 atomic mass units) and therefore has a potential isobaric interference with the ArCl+ species. To enhance the sensitivity of the assay, we used a hydride generation technique. The applied membrane gas liquid separator allows the complete chemical separation of any chlorine in the sample matrix. The minimum detection limits for arsenic with a high-resolution inductively coupled mass spectrometer of 0.01 µg/liter have been reported by others (18Go), and in our laboratory we have found that minimum detection limits of 0.0005 µg/liter were easily obtainable. For practical reasons, the working range of the method was limited to 0.01–100 µg/liter arsenic. The dynamic range of the method covers more than six orders of magnitude (ng/liter to mg/liter range). Hydride-generation inductively coupled optical emission spectroscopy and hydride-generation atomic absorption spectroscopy methods are only capable of detection limits in the 1- to 5-µg/liter range (18Go). The analytical uncertainty of hydride-generation high-resolution inductively coupled mass spectrometry analyses using the external calibration method is typically 3–5 percent.

Statistical methods
After initial inspections of histograms and normal probability plots, log-transformed measurements were applied to both water and toenail arsenic concentrations to achieve approximately normal distributions for use in analytical statistical analyses. For undetectable water arsenic levels, a value of one-half the high-resolution inductively coupled mass spectrometer detection limit was used (0.002 µg/liter). Geometric means and standard errors were calculated for overall toenail and water arsenic measurements. Factors influencing water arsenic concentrations were examined using multivariate regression analysis (19Go). Variables included in the regression model for water arsenic concentrations were water supply (public vs. private drilled or private shallow well), year of interview, use of a filter and type of residence (urban vs. rural), and season of the year collected. Effect estimates for water arsenic were reported as percent changes or as ratios of the least squared means in 1-µg/liter units. The relation between water and toenail arsenic was initially summarized by an overall unadjusted Pearson correlation between the log-transformed measures. Separate correlations between water and toenail arsenic were computed for equal to or above and below 1 µg/liter of water arsenic to permit comparison with earlier studies, including ours (13Go), in which the detection limit for water arsenic was 1 µg/liter or higher. The drinking water concentration of arsenic as a determinant of toenail arsenic was analyzed using a multivariate regression analysis adjusting for influential factors including age, sex, smoking status, season collected, year of interview, and intake (defined as glasses per day). Results were expressed as percent changes or as ratios of the least squared means of toenail arsenic concentration (µg/g). The effect estimates for toenail arsenic were reported separately for above and below 1 µg/liter of water arsenic concentrations, again, for the purpose of comparison. Modifiers of the water-toenail relation were analyzed by subgroups and included stratification by median age, water supply, smoking status, season, year of interview, at least 1 year of using current water supply (yes/no), and median selenium.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A total of 540 controls were interviewed, of which 524 (97.0 percent) had an analyzable toenail sample. We restricted our analysis to the 506 individuals (93.7 percent) whose samples were of adequate weight to minimize any laboratory error due to sample weight. We requested water samples from 220 participants and obtained a sample from 217 of these (98.6 percent). There were a total of 208 subjects on whom we had both toenail and water analyses (excluding those with low-weight nail samples). The characteristics of subjects on whom we collected water samples were similar to those on whom we collected toenail samples (table 1).


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TABLE 1. Selected characteristics of subjects on whom toenail and water arsenic were measured, New Hampshire, 1994–1997

 
Water concentrations ranged from 0.002 to 66.6 µg/liter (geometric mean, 0.29 µg/liter; standard error, 0.04 µg/liter). Private drilled or artesian wells had higher water concentrations overall than did public water systems or shallow wells or springs (table 2). There were no statistically significant differences in water arsenic concentrations according to season or year in which the sample was collected, whether a water filter was used or urban versus rural residence (table 2).


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TABLE 2. Determinants of water arsenic concentrations (µg/liter), New Hampshire, 1994–1997

 
Toenail arsenic concentrations ranged from <0.01 to 0.81 µg/g (geometric mean, 0.09 µg/g; standard error, 0.003 µg/g). Overall, the correlation (r) between water and nail arsenic was 0.46 (p < 0.001) (figure 1). The correlation was highest among those with water concentrations at or above 1 µg/liter, the detection limit in our pilot study (r = 0.65, p < 0.001) (figure 1). Based on our regression model, a 10-fold increase in water arsenic was associated with a doubling (115 percent increase) in toenail arsenic concentrations in those with water arsenic of 1 µg/liter or greater (table 3). For water arsenic below 1 µg/liter, the correlation between toenails and water concentrations was close to zero (r = 0.08, p = 0.31) (figure 1) and associated with only a 4 percent, statistically nonsignificant, increase in toenail concentrations for a 10-fold increase in water concentrations (table 3). Only six individuals reported that they did not regularly consume their tap water; each had concentrations equal to or less than 1 µg/liter of arsenic. Reported water consumption (glasses/day) was not a statistically significant predictor of nail concentrations after water concentrations were taken into account (table 3). Toenail arsenic was slightly higher in the summer months than in other seasons, but only among those with water arsenic concentrations less than 1 µg/liter (table 3). Other factors such as sex, smoking status, and year of interview did not appear to influence toenail arsenic concentrations (table 3). No appreciable differences were found among any of the subgroups stratified by median age, water supply, smoking status, season, year of interview, at least 1 year of using current water supply (yes/no), and median level of selenium.



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FIGURE 1. Correlation between water and toenail concentrations of arsenic (n = 208), New Hampshire, 1994–1997

 

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TABLE 3. Determinants of toenail arsenic concentrations (µg), New Hampshire, 1994–1997

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our study, based on New Hampshire residents randomly selected from drivers' license records and Medicare enrollment files, indicates that tap water concentrations of arsenic are an important contributor to toenail arsenic concentrations. This finding was confined to those whose water contains at least 1 µg/liter of arsenic, the detection limit of standard methods of analysis. As a pilot study, we tested 21 individuals whose drinking water was supplied to their home by a private well in New Hampshire or Vermont (13Go). Approximately half the sample was selected from regions known to have arsenic-containing well water. In this study, the overall correlation between log-transformed values for toenail and well-water arsenic was 0.67. Thus, the results of the present, larger study confirm our initial findings based on a relatively small number of households.

Studies in more highly exposed populations also have observed a relation between drinking water and toenail concentrations of arsenic. Recently, Chiou et al. (20Go) studied a population on the northeast coast of Taiwan in whom drinking water concentrations ranged from <1 to >3,000 µg/liter and also found that water arsenic concentrations were positively correlated with toenail arsenic concentrations. Studies from Mexico (21Go) and Alaska (22Go) also found higher nail concentrations of arsenic in those consuming water containing high levels of arsenic. In the Alaskan study (22Go), the computed correlation between the measures was weak. However, the population they studied was somewhat transient; therefore, the current water levels may have differed from what they consumed several months previously.

Toenail arsenic may also reflect other sources or routes of arsenic exposure. Among workers in a gold mining operation, Agahian et al. (23Go) found a strong correlation between air and fingernail samples (r = 0.89). Based on data from 969 health professionals, toenail arsenic concentrations correlated with food frequency questionnaire data (r = 0.33, p < 0.0001) using stepwise regression approaches, but not when using published food content information (24Go). Improved food content databases are currently under development and may help to clarify the contribution of diet to toenail concentrations of arsenic. With the use of available databases, the average adult in the United States is estimated to consume between 11 and 14 µg of inorganic arsenic per day (25Go, 26Go). In our data, there was some indication of a seasonal difference in nail concentrations among those with water arsenic concentrations below 1 µg/liter. Dietary factors are one possible explanation for this finding, but, unfortunately, we did not collect dietary information on our subjects. Tobacco may also contain trace amounts of arsenic from pesticide residuals; although we detected higher toenail concentrations among current smokers, the difference may have been due to chance. Thus, aside from misclassification, dietary intake and other sources probably explain the residual variation in toenail concentrations in individuals with minimal drinking water exposure and perhaps occupational exposure or tobacco use; however, further studies are needed.

Arsenic readily binds to sulfhydryl groups. Scleroproteins, such as keratin in hair and nail tissue, are rich in these sulfhdryls and, thereby, accumulate arsenic (9Go, 10Go). Toenails take several months to a year to grow out and, for this reason, measure for past exposure. In a reproducibility study conducted among female nurses tested 6 years apart, toenail arsenic was among the most highly correlated of the 16 elements studied (11Go). These data suggest that toenail arsenic may be an indicator of exposure over even longer periods, that is, that exposure may remain relatively constant within individuals. This could be especially true for populations such as ours in New Hampshire that are relatively stable (1990 US Census) and in whom remediable efforts have not occurred. A biologic marker such as toenails is not susceptible to differential misclassification, such as recall bias, that can occur in case-control studies. However, the slight decline in toenail arsenic concentrations with age suggests that age should be considered as a potentially confounding factor in any analysis of toenail arsenic and health risks. Collection of toenail clippings is relatively simple and painless, resulting in high participation rates (97 percent of those interviewed in our study). Most participants collected their own sample with the exception of diabetics who had their toenails clipped at their podiatrist's office. While diabetes did not pose a problem, our experience was limited to the few diabetics chosen as controls for our case-control study. Very rarely (in only two subjects) were specimens unobtainable because of poor nail growth or fungal infection. A major advantage to measuring toenail samples for arsenic is that they are an integrated measure of all routes of exposure, including drinking water, occupation, and diet.

Another important feature of nail tissue is that it is generally considered less susceptible to external contamination than is hair. Agahian et al. (23Go) estimated that 98 percent of external arsenic was removed by washing fingernail samples exposed to particulate arsenic. We had similar results in our own laboratory using a high-specific-activity radiotracer. After exposure of toenail samples for 15 hours to even the highest concentrations of water arsenic we encountered, less than 1 percent of the arsenic detected in the lowest concentration toenails was due to absorption (data not shown). The association between dietary intake and toenail arsenic concentrations further argues that the source of arsenic is probably from ingestion rather than from exogenous sources. Genetic factors (e.g., GSTM1, GSTT1) were related to ratios of the urinary metabolites but were not associated with toenail concentrations in a study from Taiwan (20Go). These data suggest that toenail concentrations are a direct measure of exposure and are not influenced by individual differences in arsenic metabolism. In the specific regions we targeted for our pilot investigation (13Go), some people had stopped drinking their well water. When we removed these individuals from the analysis, the overall correlation improved, again suggesting that arsenic ingestion rather than external contamination is correlated with nail concentrations. However, among the larger set of controls randomly sampled for the current study, only six individuals did not usually drink tap water, and each had concentrations equal to or less than 1 µg/liter of arsenic. Therefore, we could not adequately assess the influence of ingestion on toenail measures.

Biologic tissues other than nails also contain arsenic, such as blood or urine, but concentrations in these tissues reflect relatively recent exposure. Arsenic is removed from the blood within a few hours and excreted through the kidneys and urine within a few days (1Go, 25Go). Urinary levels comprise both inorganic and organic forms of arsenic such as arsenbetaine ("fish" arsenic). Therefore, to assess inorganic arsenic exposure from urine requires separation of the specific forms. The correlation between drinking water and total urinary arsenic, inorganic arsenic, monomethylarsenic, and dimethylarsenic was relatively low (between 0.2 and 0.3) in a population exposed to drinking water concentrations of arsenic around 15 µg/liter (26Go). Thus, blood and urine measurements are unlikely to serve as markers of chronic low-level arsenic exposure.

Direct measurement of drinking water or other environmental samples is an alternative approach to estimating individual exposure, particularly if other sources of exposure (i.e., diet or job activities) are minimal. Yet, these measures are also subject to misclassification. For example, to assess drinking water intake of arsenic precisely requires measurements of water samples over a participant's lifetime combined with accurate (and nondifferential) recall of the amount of water consumed. The extent to which water concentrations vary over time is unclear. Moreover, little is known about the impact of misclassification regarding the recalled average intake of tap water on estimates of cumulative drinking water arsenic exposure. Misclassification of water intake may, in part, explain its lack of correlation with toenail arsenic concentrations. However, with careful precaution against contamination, water measurements themselves were highly accurate according to our findings. The intraclass correlation coefficient for our replicate samples was 0.98 with a detection limit of 0.01 µg/liter (14Go).

Arsenic levels in US water systems average between 1 and 2 µg/liter in most regions (1Go). Elevated levels of arsenic have been detected in water supplies in states including California (27Go), Oregon (6Go), and Alaska (22Go). In New Hampshire, detectable levels of arsenic are found throughout the state, with a few areas with particularly high levels (i.e., greater than 50 µg/liter) (28Go). Public water systems presumably have levels below the current Environmental Protection Agency maximum contaminant level for arsenic; however, private wells (serving fewer than 15 households or 25 individuals) are commonly used in rural areas and are not regulated. In our study, private systems serve roughly 40 percent of the households.

In our study, private drilled or artesian wells had higher arsenic concentrations than did public water supplies or shallow wells or springs. The relative absence of arsenic in surficial systems suggests that the source of arsenic may be natural occurring (i.e., from bedrock) rather than from anthropogenic sources (i.e., pesticide residual). In a geologic and spatial analysis, Peters et al. (28Go) found that arsenic concentrations correlate with pegmatite dikes within the granitic bedrock.

The US Environmental Protection Agency is considering whether to classify arsenic as a group A human carcinogen (with a zero maximum contaminant level goal) and whether to lower the drinking water standard (29Go). Part of the uncertainty about where to set the standard is that risk assessment models have relied on low-dose extrapolation of risk in highly exposed populations in Taiwan and other countries and very few epidemiologic data exist for the United States. Ecologic (correlational) studies in the United States have not found adverse heath effects to be correlated with drinking water concentrations (6GoGo–8Go). However, the results of these studies are difficult to evaluate because of the wide variability in arsenic concentrations within each region used as the unit of the analysis. Development of a reliable biologic marker of arsenic exposure would greatly improve our ability to assess the health effects of arsenic, particularly at low levels of exposure. In conclusion, our study along with others points to nail arsenic concentration as a quantitative estimate of internal (total) dose of arsenic exposure.


    ACKNOWLEDGMENTS
 
Funding for the study was provided by National Institutes of Health grants NIEHS ES-07373, NCI CA57494, and NCI CA61108.

The authors thank Mark Carey for his statistical assistance and Drs. Thérèse Stukel and E. Robert Greenberg for their contributions on the skin cancer case-control study.


    NOTES
 
Reprint requests to Dr. Margaret R. Karagas, Dartmouth Medical School, Section of Biostatistics and Epidemiology, 7927 Rubin 462M-3, One Medical Center Drive, Lebanon, NH 03756-001 (e-mail: margaret.karagas{at}dartmouth.edu).


    REFERENCES
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 RESULTS
 DISCUSSION
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Received for publication August 2, 1999. Accepted for publication December 27, 1999.