Trace Element Distribution and Arsenic Speciation in Toenails as Affected by External Contamination and Evaluation of a Cleaning Protocol

Trace element concentrations in toenail clippings have increasingly been used to measure trace element exposure in epidemeological research. Conventional methods such as inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography ICP-MS (HPLC-ICP-MS) are commonly used to measure trace elements and their speciation in toenails. However, the impact of the removal of external contamination on trace element quantification has not been thoroughly studied. In this work, the microdistribution of trace elements (As, Ca, Co, Cu, Fe, K, Mn, Ni, Rb, S, Sr, Ti, and Zn) in dirty and washed toenails and the speciation of As in situ in toenails were investigated using synchrotron X-ray fluorescence microscopy (XFM) and laterally resolved X-ray absorption near edge spectroscopy (XANES). XFM showed different distribution patterns for each trace element, consistent with their binding properties and nail structure. External (terrestrial) contamination was identified and distinguished from the endogenous accumulation of trace elements in toenails—contaminated areas were characterized by the co-occurrence of Co, Fe, and Mn with elements such as Ti and Rb (i.e., indicators of terrestrial contamination). The XANES spectra showed the presence of one As species in washed toenails, corresponding to As bound to sulfhydryl groups. In dirty specimens, a mixed speciation was found in localized areas, containing AsIII–S species and AsV species. ArsenicV is thought to be associated with surface contamination and exogenous As. These findings provide new insights into the speciation of arsenic in toenails, the microdistribution of trace elements, and the effectiveness of a cleaning protocol in removing external contamination.


Table of contents
Table S1.As concentrations (mg/kg) in washed toenail samples of the 13 selected participants.
(p 2)         The retention time shift of iAs compared to its usual retention time (see below) is possibly due to heavy matrix effects.Instead, the presence of an additional peak (labelled U?) could be iAs as thioarsenate or arsenite eluting after the main iAs peak due to the heavy matrix in this particular sample.As/Ge (cps) Time (min)

Figure S1 .Figure S2 .Figure S3 .Figure S4 .Figure S6 .Figure S8 .Figure S9 .Figure S11 .Figure S14 .
Figure S1.Visual explanation of the sample preparation.(p 3) Figure S2.XFM maps of the measured trace elements in a thin section of the washed nail of participant #8.(p 4) Figure S3.Example of As binding types.(p 5) Figure S4.XFM maps of the measured trace elements in a thin section of the dirty nail of participant #8.(p 6) Figure S5.a) Whole toenails from participant #8.b) Optical microscope image of two thin sections.c) RGB images for samples 8b3 and 8a2, showing Co (red), S (green), and Fe (blue).(p 7) Figure S6.Traverse section with a visible distinction between endogenous and exogenous incorporation of Co, Mn, and Fe.(p 8) Figure S7.XFM maps of the measured trace elements in a thin section of the nails of participant #24 (dirty, left versus washed, right).(p 9) Figure S8.XFM maps of the measured trace elements in a thin section of the washed nail of participant #15.(p 10) Figure S9.XFM maps for multiple transversal thin sections of participant #8.(pp 11-13) Figure S10.Longitudinal thin sections.(p 14) Figure S11.As K-edge XANES spectra of the four standards used for analysis.(p 15) Figure S12.XANES analysis-participants #29 and #8 (washed samples).(p 16) Figure S13.XANES analysis-participant #8 (dirty sample).(p 17) Figure S14.HPLC-ICPMS chromatogram showing the As content in participant #8.(p 18) Figure S15.HPLC-ICPMS chromatogram showing the As content in participant #29.(p 18) References (p 18)

Figure S5 .
Figure S5.a) Whole toenails from participant #8 (washed, left panel versus dirty, right panel), with their ventral layers facing up.The thin sections were obtained by cutting the nail transversally, to obtain 150μm-thick sections that included all histological layers: dorsal (external arc), intermediate, and ventral (inner arc).b) Optical microscope image of two thin sections (washed, left panel versus dirty, right panel).A morphology change can be seen by the ventral layer, where the nail matrix is degraded and less uniform.In the dirty specimen (right panel), darker areas are observed at the edge of the ventral layer.For orientation, corresponding regions in a) and b) are highlighted in pink.c) RGB images for samples 8b3 and 8a2, showing Co (red), S (green) and Fe (blue).The dark green regions are areas of low S, coinciding with the areas of degraded keratin.The magentacoloured regions have no S and originate from the overlap of Co (red) with Fe (blue); they correspond to the darker regions observed in a) and b), likely indicating deposition of external contamination onto the nail.Less contamination is present on the washed sample (left), if compared to the dirty specimen (right).

Figure S6 .
Figure S6.The cyan traverse section was drawn in an area with a visible distinction between endogenous and exogenous incorporation of Co, Mn, and Fe.The high levels of these metals observed in proximity of the ventral layer correlate with high concentrations of K, Ti, and Rb, whereas the concentration peaks found in the dorsal layer are not associated with any of the elements indicative of external contamination.

Figure S7 .Figure S8 .
Figure S7.XFM maps of the measured trace elements in a thin section of the nails of participant #24 (dirty, left versus washed, right).In contrast to the sample in FigureS8, these XFM maps are indicative of the effectiveness of the cleaning protocol in removing loosely attached exogenous contamination.Prior to the washing steps, the two toenails were similar, with high levels of external material deposited onto the ventral side.The dark areas in the dirty sample (left), highly enriched in Fe, do not persist in the washed sample (right) after the cleaning steps.

Figure S9 .Figure S10 .Figure S11 .
Figure S9.XFM maps for multiple transversal thin sections of participant #8 (dirty first three from the left, and washed last three from the left).Trace element order: As, Ca, Co, Cu, and Fe.

Figure S13 .
Figure S13.(A) Nail region of participant #8 selected for XANES (dirty sample).(B) Energy association scatter plots corresponding to energies near the white line peaks of the As III -S bond in the nail specimen and of the As V standard.(C) Localisation of different populations of energy correlations.(D) Extracted XANES for the five different populations; the vertical lines correspond to the white line peaks of As III (GS)3, As III , DMA, and As V standards.

Figure S14 .
Figure S14.HPLC-ICPMS chromatogram showing the As content in participant #8.Eluting order: DMA (0.422 mg/kg), MMA (0.707 mg/kg), inorganic As, and unknown (8.910 mg/kg [iAs and unknown combined]).The retention time shift of iAs compared to its usual retention time (see below) is possibly due to heavy matrix effects.Instead, the presence of an additional peak (labelled U?) could be iAs as thioarsenate or arsenite eluting after the main iAs peak due to the heavy matrix in this particular sample.

Table S1 .
As concentrations (mg/kg) in washed toenail samples of the 13 selected participants.