INTRODUCTION

Perchlorate (ClO4−) is a highly oxidized anion and is a widespread contaminant that when ingested can potentially cause adverse health effects in humans. Once in the body, perchlorate can competitively block iodide from entering the thyroid, potentially affecting further production of thyroid hormones. Deficiency of thyroid hormones has severe health effects especially for fetuses, infants, and young children. Because the thyroid hormones are crucial for neurodevelopment in the early stages of life, their deficiency can cause irreversible impairment in the nervous system, particularly in the brain.1, 2, 3, 4, 5

Perchlorate has been detected in surface water and groundwater across the United States6, 7, 8, 9, 10, 11, 12, 13, 14, 15 as well as in a wide range of food products including milk, infant formulas, fruits, and vegetables.16, 17, 18, 19, 20, 21, 22 The primary routes of perchlorate exposure to humans appear to be through oral intake of food and drinking water.22, 23, 24, 25, 26 Perchlorate has been detected in human US breast milk17, 27, 28, 29 and in all urine samples tested as part of the National Health and Nutrition Examination Surveys between 2001 and 2008,30, 31 indicating widespread human exposure across the United States. Urinary perchlorate is an indicator of exposure since perchlorate is not metabolized and exits the body primarily through urine.32 Children are exposed to significantly higher doses of perchlorate compared with adults, likely because of higher rates of consumption of foods that tend to contain higher perchlorate levels.22, 33, 34

Perchlorate originates from different natural and manufactured sources that can be distinguished by their relative isotopic signatures based on the stable chlorine isotope ratios reported in the conventional delta (δ) notation and on 36Cl/Cl ratios. Perchlorate that forms in the atmosphere deposits onto the surface of Earth, but it accumulates mainly in dry regions such as the Atacama Desert in Chile and the Death Valley in the United States.35, 36, 37, 38, 39, 40, 41 Two main types of isotopically distinct natural perchlorate have been reported to date: one type having generally heavier δ37Cl values (−4‰ to +6‰) combined with relatively high 36Cl/Cl ratios (3100 × 10−15 to 66,500 × 10−15) has been found at locations across the United States; and the second type characterized by anomalously low δ37Cl values (−14.3‰ to −11.8‰) and relatively low 36Cl/Cl ratios (22 × 10−15 to 590 × 10−15) has been found only in the Atacama Desert of northern Chile.10, 15, 42, 43 Atacama perchlorate is associated with nitrate-rich caliche deposits and is by far the largest known reservoir of natural perchlorate on Earth.8, 35, 44, 45 Substantial quantities of Atacama perchlorate were introduced into the environment across the United States during the first half of the 20th century due to the widespread application of imported Chilean nitrate fertilizer.36, 46, 47, 48 Synthetic perchlorate is primarily used as an oxidant in a variety of solid rocket propellants and explosives as well as in consumer products including fireworks, matches, air bags, chlorine bleach, safety flares, and chlorate herbicides.9, 49, 50, 51 The isotopic composition of synthetic perchlorate is distinctly different from both types of natural perchlorate in its relatively consistent δ37Cl value (+0.6±1.0‰) and its low 36Cl/Cl ratio (1 × 10−15 to 40 × 10−15).10, 43, 52

Recent studies evaluated dietary sources of perchlorate in the United States by estimating overall perchlorate contribution to the food chain from all potential sources46 or by using quantities of perchlorate in different food products, drinking water, and urine.22, 23 However, none of these studies can differentiate between indigenous natural perchlorate, natural Atacama perchlorate, and synthetic perchlorate as the primary source of perchlorate exposure. The objective of this study was to identify the exposure source of perchlorate by measuring the chlorine isotopic composition (δ37Cl, 36Cl/Cl) of perchlorate extracted from urine. This novel approach can distinguish between all potential sources of perchlorate, and identify the primary source of perchlorate exposure. We hypothesized that the chlorine isotopic composition of urinary perchlorate would resemble that of groundwater, surface water, and soil in the geographical region of the population.

METHODS

Sample Collection

Perchlorate was extracted from composite urine samples for isotopic analysis by using 100 cm3 clear PVC columns filled with Purolite A530E bifunctional anion exchange resin. This resin is exceptionally selective for perchlorate,53, 54, 55 and similar columns have previously been used to extract perchlorate from surface water and groundwater.11, 12, 13, 15, 42, 56 Detailed description of sampling column design and isotopic analytical procedures are given by Hatzinger et al.57 The first perchlorate sample was extracted from a total volume of 40-l urine pooled from ~133 anonymously collected urines from residents of the Atlanta metro area over a 2-week collection period. The second sample was extracted from a total volume of 1.05-l urine pooled from 49 clinical residual urine samples collected from adult residents of Taltal, Chile (Atacama region). Human specimen use was in accordance with human subjects review (University of California, Berkeley). After sampling was completed, the resin columns were shipped to the University of Illinois at Chicago in a cooler filled with ice packs, and the sample columns were kept refrigerated until analysis. The amounts of perchlorate retained on each column was estimated by analyzing perchlorate concentrations in urine samples before and after the Purolite columns by using an ion chromatography tandem mass spectrometry (IC-MS/MS) at CDC.58 Aliquots were run in triplicates for each composite sample.

Extraction of perchlorate from the resin columns was performed using procedures described by Hatzinger et al.57 Briefly, the resin was ultrasonically cleaned in deionized water, and then it was flushed with 4-M HCl to remove common anions including organic acids. Subsequently, perchlorate was eluted with 1-M FeCl3–4-M HCl,55 and iron was removed by passing the diluted eluant through a cation-exchange resin, then the iron-free solution was evaporated to ~2 ml volume. The final purification of perchlorate was performed by chromatography, then perchlorate was precipitated and recrystallized as tetra-n-pentylammonium perchlorate (TPAClO4).59 The overall sample processing blank for perchlorate was 5 μg, which was <5% of the perchlorate recovered for isotopic analysis.60

Stable Cl Isotope Analysis (δ37Cl) of Perchlorate by SIMS

The stable chlorine isotope ratio (37Cl/35Cl) in perchlorate is normally analyzed by isotope ratio mass spectrometry (IRMS).10, 11, 42 However, we used secondary ion mass spectrometry (SIMS) for stable chlorine isotope ratio measurements because of the small sample size. All analyses were performed on TPAClO4. Samples were mounted by pressing into indium metal in a 2.5-cm diameter sample holder and coating the surface with 50 nm of vapor-deposited gold. SIMS analyses were performed at the Center for Microanalysis of California Institute of Technology on an IMS 7 f-GEO (CAMECA, Gennevilliers, France). Analyses were performed with a 10-keV Cs+ primary ion beam. The ion beam was focused to a diameter of ~20 μm with 5–6 nA of beam current. All isotope ratio analyses were performed by pre-sputtering of a 100 × 100 μm raster area for 120 s, and then scanning a 75 × 75 μm raster during data collection. 34SH contribution to 35Cl results was negligible at a mass-resolving power (M/ΔM) of 1200 defined at the full width at 10% of the maximum peak height; this was also confirmed under a resolution of 5000. An energy bandwidth of 45 eV was set for all SIMS measurements, and secondary ion accelerating voltage was −9 keV for all isotopic analyses. Chlorine isotopes 35Cl and 37Cl were measured by Faraday cups with data collection times of 1 and 2 s per cycle, respectively, for a total of 20 cycles. Data are normalized to measurements of KClO4 isotopic reference materials USGS37 and USGS38,11 which were converted to TPAClO4 prior to measurement. Stable chlorine isotope ratios are reported in the conventional delta (δ) notation, as follows:

Where SMOC is the isotopic reference standard mean ocean chloride.61 Analytical precision corresponding to 1σ for δ37Cl values determined from replicate measurements of reference materials is ±1.0‰.

36Cl Abundance Analysis by Accelerator Mass Spectrometry

After the analysis of the stable chlorine isotope ratios by SIMS, the remaining samples were scraped from the indium metal sample holders and diluted with USGS37 standard in TPAClO4 form and homogenized by dissolving in 2-ml acetone. Sample J-1183 (Atlanta, USA) was diluted ~3.8 times, whereas sample J-1184 (Taltal, Chile) was diluted ~3.1 times, in order to get a larger sample size to facilitate loading of AgCl targets for accelerator mass spectrometry (AMS) measurements. Then, the diluted perchlorate samples were converted from TPAClO4 to KClO4 by using 0.5-mol/l KOH in absolute ethanol solution, and the KClO4 was sealed under vacuum into 20-cm lengths of 9-mm outer diameter borosilicate glass tubing. Afterwards, KClO4 was heated for 30 min at 600 °C in order to decompose KClO4 according to the following reaction: KClO4→KCl+2 O2(g). Subsequently, the chloride from KCl was purified by a standard ion chromatography method developed by the Purdue Rare Isotope Measurement (PRIME) Laboratory for 36Cl analyses, and then the chloride was precipitated as AgCl, which was used as a target material for the measurements of radioactive 36Cl isotopic abundances by AMS. Samples for the 36Cl abundances were analyzed at the PRIME Laboratory at Purdue University. The 36Cl results were corrected for the instrumental background and are reported in units of 36Cl/Cl × 10−15 along with errors based on counting statistics.

Comparative Isotopic Patterns

Chlorine-36 has previously been shown to be an effective tracer of the origin of perchlorate in various environmental matrices.15, 43 Previous work indicates unique clustering of 36Cl/Cl ratios vs δ37Cl values for perchlorate extracted from synthetic perchlorate, natural perchlorate from Atacama (Chile), and natural perchlorate from the western USA.42, 43 The chlorine isotope pattern for synthetic perchlorate was defined based on analysis of laboratory grade perchlorate salts. The chlorine isotope pattern for natural perchlorate indigenous of the western USA was extracted from groundwater and soil samples collected from Texas, New Mexico, California, and Oregon. The chlorine isotope pattern for natural perchlorate from the Atacama Desert was characterized based on bulk soil and groundwater samples collected in that region.42, 43 Concentration ellipses were computed using the “dataEllipse” function in the CAR (Companion to Applied Regression)62 package of R, an open source software environment for statistical computing and graphics,63 based on a bivariate normal distribution fit to the 36Cl/Cl ratios and δ37Cl values of perchlorate reference materials. The concentration ellipses define the confidence curves within which 95% of the data is expected to lie, assuming a bivariate normality.

RESULTS

Two perchlorate samples were extracted from composite urine samples collected from geographically distinct populations located in Atlanta, USA and Taltal, Chile regions for chlorine isotopic analyses of perchlorate. A total of ~202 μg of urinary perchlorate was recovered from the composite urine sample collected from the sample population of the Atlanta metro area, and ~155 μg of urinary perchlorate was recovered from the composite urine sample collected from the residents of Taltal, Chile (Table 1). The amounts of perchlorate retained on each column and the final yields of purified perchlorate were independently determined, and the estimates agreed well for both perchlorate samples.

Table 1 Stable and radioactive chlorine isotope ratios of perchlorate extracted from urine from Atlanta, USA and Taltal, Chile regions.
Full size table

The urinary perchlorate samples were analyzed for the stable (δ37Cl) and radioactive (36Cl/Cl) chlorine isotopic signatures in order to identify the primary exposure sources of perchlorate. The results demonstrate that the urinary perchlorate sample from the Atlanta area had a positive δ37Cl value of +4.1±1.0‰ and a relatively high 36Cl/Cl ratio of 1 811 (±136) × 10−15 (Table 1). In contrast, the perchlorate sample extracted from the composite urine sample collected from multiple residents of Taltal, Chile had significantly lower stable chlorine isotopic signature (δ37Cl value) of −11.0±1.0‰ and lower radioactive chlorine isotopic abundance (36Cl/Cl ratio) of 254 (±40) × 10−15 compared to the urinary perchlorate from the Atlanta region.

DISCUSSION

The chlorine isotopic compositions of perchlorate samples collected from the three known principal environmental sources of perchlorate in the United States and composite urine samples from Atlanta, USA and Taltal, Chile define three distinct, non-overlapping clusters in a diagram of 36Cl/Cl as a function of δ37Cl (Figure 1). The isotopic composition of indigenous natural perchlorate from the western USA appears to be rather consistent over a wide portion of North America in terms of δ37Cl values and 36Cl/Cl ratios. Similar isotopic signatures were observed in perchlorate from Texas, New Mexico, California, Oregon, and throughout the Great Lakes.15, 42, 43, 64 Indigenous natural perchlorate samples collected from the western USA and from the Great Lakes have the highest 36Cl/Cl ratios (3100 × 10−15 to 66,500 × 10−15) yet reported for perchlorate, and a range of δ37Cl values from −4‰ to +6‰. The Atacama Desert samples have much lower 36Cl/Cl ratios (ranging from 22 × 10−15 to 590 × 10−15) and δ37Cl values (−14.3‰ to −11.8‰) compared with those of indigenous natural perchlorate from the US. Synthetic perchlorate samples have δ37Cl values in the range 0.2‰ to 1.6‰ and 36Cl/Cl ratios of 1 × 10−15 to 40 × 10−15, which is consistent with a predominantly marine source of Cl in the brine used as for electrolytic production of perchlorate.43, 65

Figure 1
figure 1

Diagram comparing 36Cl/Cl ratios vs δ37Cl values for perchlorate extracted from urine from residents of the Atlanta, USA metro area and Taltal, Chile (Atacama region), in comparison with synthetic perchlorate, and natural perchlorate from Atacama (Chile) and the western USA.42, 43, 64 36Cl/Cl errors are smaller than data points. The concentration ellipses define the confidence curves within which 95% of the data is expected to lie, assuming bivariate normality.

Full size image

The urinary perchlorate sample from the Atlanta region more closely aligns with indigenous natural perchlorate extracted from groundwater and soil samples from the United States42, 43 in terms of its δ37Cl value and 36Cl/Cl ratio, indicating indigenous natural perchlorate as the primary source of exposure in the sampled Atlanta residents (Figure 1). Any addition of synthetic or Atacama perchlorate would reduce the 36Cl abundance in the urinary perchlorate sample from the Atlanta region, while also shifting the δ37Cl result toward lower values on Figure 1 since both synthetic and Atacama perchlorates have lower δ37Cl values than indigenous natural perchlorate from the United States. Overall, the shift in the δ37Cl value appears to be minimal in the Atlanta sample, but the 36Cl abundance is somewhat lower, compared with indigenous United States perchlorate. These features indicate negligible Atacama perchlorate contribution since it would produce a much larger shift in the δ37Cl value. However, a minor synthetic perchlorate contribution to a dominantly indigenous natural source could possibly explain both the δ37Cl value and the 36Cl/Cl ratio of the Atlanta urinary perchlorate sample.

The δ37Cl value and 36Cl/Cl ratio determined for perchlorate from the urine of residents from Taltal, Chile (Atacama region) are similar to perchlorate samples collected from the Atacama region, indicating that natural Atacama perchlorate is the main source of exposure in that region (Figure 1). Perchlorate contributions from synthetic or young atmospherically produced perchlorate appear to be relatively minor, because any synthetic perchlorate contribution would increase the δ37Cl value and decrease the 36Cl abundance, whereas addition of significant quantities of young atmospheric perchlorate would increase the 36Cl abundance while preserving the δ37Cl value if we assume that the chlorine sources for atmospheric perchlorate formation in a given region have relatively constant δ37Cl value over time.

Indigenous natural perchlorate samples having anomalously high 36Cl/Cl ratios may have been affected by the presence of the bomb-produced 36Cl pulse from the 1952–1962 thermonuclear tests.15, 43 However, 36Cl/Cl ratios >8000 × 10−15 were also measured in perchlorate extracted from southwest US groundwater having 14C ages of >10 ka.43 Even the lowest 36Cl/Cl ratio of ~3100 × 10−15 in indigenous perchlorate from pre-bomb groundwater is much higher than the highest pre-bomb 36Cl/Cl ratio of ~1200 × 10−15 in meteoric chloride near the Earth’s surface across the US.66, 67 The stratosphere is the only known location that has sufficiently high 36Cl isotopic abundances68 to account for the observed 36Cl/Cl ratios in indigenous perchlorate samples from the western US as well as in the urinary perchlorate sample from the Atlanta region, which strongly indicates that indigenous natural perchlorate is the dominant source of urinary perchlorate from Atlanta, USA.

The 36Cl/Cl ratios in natural Atacama perchlorate samples are significantly lower compared to those in the indigenous natural perchlorate from the western US, which might be explained chronologically since Atacama perchlorate is much older than the western US perchlorate.69,70 The low 36Cl/Cl ratios in synthetic perchlorate are consistent with chloride sources from ancient marine halite deposits.43 Thus, the 36Cl signature along with the stable chlorine isotope of perchlorate is a valuable tool, which can be used for tracing the source and origin of perchlorates in the environment. Although not analyzed in this study, stable oxygen isotopes (δ18O, Δ17O) of perchlorate offer additional tools for identifying perchlorate sources and origin.10, 11, 12, 13, 15, 42, 56, 71

In summary, our results demonstrate the use of chlorine isotope ratios of perchlorate extracted from pooled human urine samples for identifying the primary sources of perchlorate exposure in two geographically distinct sample populations. This novel method appears to be a robust approach, which can be used for differentiating between indigenous natural perchlorate, Atacama perchlorate and synthetic perchlorate as the primary source of perchlorate exposure, because the perchlorate molecule is stable in the environment, as well as in vivo,32 and can apparently retain its initial isotopic composition for centuries to millennia in groundwater and surface water systems.57, 72 The isotopic identification of perchlorate exposure sources can aid in targeting efforts to reduce exposure and improve public health.

Disclaimer

The findings and conclusions in this study are those of the authors and do not necessarily represent the official views or positions of the US Centers for Disease Control and Prevention. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the US Department of Health and Human Services, or the US Centers for Disease Control and Prevention.