WATER HARDNESS METALS AND HUMAN HAIR 385 spectroscopy (ATR-FTIR) method (5). Four measurements were averaged for each hair swatch. The swatches were then sorted into groups of three, which were balanced for cysteic acid. The lightness value (L*) was assessed to describe the degree of lightening imparted by the bleaching treatment. Eight measurements per hair swatch were made by a Konica Minolta CM3600D spectrophotometer under a D65 illuminant and 10° ob- server (Konica Minolta Opto, Tokyo, Japan). Table I summarizes the characterization of the hair substrates for this work. HAIR SAMPLE TREATMENT To test the effect of water hardness on metal uptake, virgin, slightly damaged, and highly damaged hair was subjected to six wash cycles in 2, 9, or 16 grain per gallon (gpg) water. These water hardness levels represent the categories of soft, moderately hard, and very hard water as identifi ed by the U.S. Geological Survey. The unit originates from the con- version of calcium and magnesium to an equivalent weight of calcium carbonate. A grain is a mass unit that is equal to 64.8 mg of material, and the concentration of water hard- ness is expressed as this quantity in a gallon (3.78 l) of water. Hence, 1 gpg is equal to 17.11 ppm CaCO3. The properties of the test water are summarized in Table II. Each wash cycle consisted of two thirty-second lathers with a commercial clarifying shampoo, thirty-second rinses before and after these two lathers, and fan-drying. The fl ow rate and temperature of the rinse water were 1.06 gallons per minute and 37°C, respectively. To test the effect of water pH on metal uptake, virgin and slightly damaged hair swatches were subjected to ten cycles of treatment with synthetic water hardness solutions contain- ing 1.2 mM calcium sulfate dihydrate (EMD Chemicals), 0.8 mM calcium chloride dihy- drate (EMD Chemicals), and 1.2 mM magnesium sulfate ( J.T. Baker Chemical) in buffers of pH 7 (5 mM bis-Tris [bis(2-hydroxyethyl) amino)] tris(hydroxymethyl)methane Organics Inc.) pH 8 (5 mM Tris tris(hydroxymethyl) aminomethane BDH Chemicals) or pH 9 (5 mM ethanolamine BDH Chemicals). The selected pH values represent a consumer-relevant pH range (14), and the resulting hardness of the solutions was 17 gpg. One treatment cycle consisted of one hour of soaking at a 1:100 hair/liquor ratio Table I Characterization of Hair Substrates Hair condition Bleaching treatment L* FTIR cysteic acid units Virgin n/a 22 25 Slightly damaged 15 min 31 56 Highly damaged 1 hour × 3 cycles 49 155 Table II Characterization of Treatment Water Water hardness (gpg) Ca (ppm) Mg (ppm) pH Alkalinity (as ppm HCO3) 2 11 4 8.4 80 9 40 13 8.4 175 16 71 23 8.5 285

JOURNAL OF COSMETIC SCIENCE 386 (with stirring) and overnight drying in a fume hood. Hair swatches that were soaked in buffer solutions containing no hardness ions served as the appropriate controls. Metal uptake was calculated by subtracting the average elemental content of the control groups from the respective treated groups. HAIR AND WATER ANALYSIS The metal content of hair and water samples was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) with an Optima 5300 DV optical emission spec- trometer (PerkinElmer Life and Analytical Sciences, Shelton, CT). For hair analyses, 200–250 mg of samples were digested overnight with 2 ml of high-purity concentrated nitric acid (70% v/v, Aristar® Plus BDH Chemicals). This mixture also contained 150 μ1 of 100 ppm yttrium internal standard (Inorganic Ventures, Christianburg, VA). The sam- ples were then heated to 75°–80°C for one hour, cooled to room temperature, and diluted to 15 ml with deionized water. Three samples from each hair swatch of the treatment groups were analyzed. For water analyses, 10-ml water samples were acidifi ed with 50 μl of trace metal analysis grade 50% v/v nitric acid. Three samples from each water treat- ment group were analyzed. The alkalinity of the water samples was determined using the Palintest Photometer 8000 and reagent tablets (Palintest Ltd., Erlanger, KY). STATISTICAL ANALYSIS The calcium and magnesium content of hair are reported as the mean plus/minus the standard deviation of three hair samples analyzed in triplicate. Treatment (water hard- ness, damage level, water pH) effects were assessed by univariate analysis of variance (ANOVA) and Tukey-Kramer HSD pairwise comparisons ( JMP 7.0.2 SAS Institute Inc., Cary, NC). Multiple linear regression was conducted to further examine the infl u- ence of water hardness and hair condition on water hardness metal uptake by hair, and this relationship was characterized by the partial coeffi cient of determination (r2) of each independent variable. Statistical signifi cance was established at p 0.05 for all analyses. RESULTS AND DISCUSSION The data highlighted the condition of the hair as the key driver for water hardness metal uptake. This was evidenced by the notable differences in metal content between the hair types within each water hardness group (p 0.001) and supports the idea that the bind- ing capacity of the hair is determined by the amount of anionic groups present within the fi ber (Figure 1). Upon treatment with alkaline hydrogen peroxide products, peptide and disulfi de bonds inside the hair and 18-methyleicosanoic acid (18-MEA) on the hair’s surface are cleaved. This exposes anionic carboxylate and sulfonate (of cysteic acid) groups (15–17), which render the hair an ideal cation exchange resin. It should be noted that the calcium and magnesium levels of the highly damaged hair are comparable to the levels found in the hair of consumers who regularly use oxidative colorant products (5). Selectivity for calcium over magnesium was exhibited by the hair substrates. The treated hair contained seven to nine times more calcium than magnesium, with the virgin hair lying at the lower end of this range and both levels of damaged hair equally lying at the