DILUTIONS Refer to Table 2

Sample dried in a wall oven over night at 60 °C. Sample transferred into clean/dry 15ml centrifuge tubes.

Sample made up to constant dilution using DDW (minimum volume = 4 ml). 0.5 ml conc. nitric acid added to the tube and

then tube is then covered in cling film.

Heated in a water bath at 70 °C for 1 hour or until digested.

Sample weighed into clean acid washed digestion tubes.

Filtered using 0.45 pm syringe filter into clean 15ml centrifuge tubes.

Digested material cooled and transferred to clean 25 ml Sterilins. Tube rinsed with DDW into the Sterilin. 5 to 10 ml acetone added to sample. Washed for 10

minutes in an ultrasonic bath.

5 to 10 ml DDW added to sample. Washed for 10 minutes in an ultrasonic bath (repeated 3 times).

5 to 10 ml acetone added to sample. Washed for 10 minutes in an ultrasonic bath.

Sample cut into small pieces using clean/dry stainless steel scissors. The scissors are washed with distilled deionised water (DDW) between samples.

2.2.3 Acid digestion

Each washed and dried sample was weighed into a clean weighing boat and then transferred into an acid-washed polypropylene digestion tube. A 0.5 ml aliquot of concentrated (conc.) Aristar® nitric acid (Fisher Scientific Ltd, Leicestershire, UK) was added to each tube, which were then covered with cling film™. The tubes were placed in a water bath and the samples were heated on a hot plate at 70 ± 1 °C for 1 hour or until all the sample was digested. The samples were then removed from the heat and allowed to cool before dilution. Each sample was diluted using distilled deionised water as the diluent. The dilution factor was weight dependent allowing for a minimum volume of 4 ml for analysis (section 2.2.4). Following dilution the samples were filtered using 0.45 pm membrane filters (Millex, Millipore, UK) before analysis by ICP-MS.

2.2.4 Diiution factor seiection

Due to the large variation in sample (hair/nail) mass provided by each study participant it was not possible to consistently use a low dilution factor (e.g. x1 0 0 dilution), in order

to get a minimum of 4 ml digested sample volume. Tests were therefore conducted to evaluate the impact of the dilution factor on the final calculation of elemental concentrations. A set of 14 dilution factors were selected across the range of 100 to 1000 times dilution (Table 2.1). Pooled control fingernail and hair samples were washed, digested and diluted according to the corresponding dilution factor (in duplicate). The samples were then analysed by ICP-MS.

Table 2.1: Hair, fingernail and toenail dilution factors and sample mass (SM) ranges for the preparation of a minimum of 4 ml digested sample volume for analysis by ICP-MS.

Dilution Factor Sample Mass (SM) Range (g)

100 0.040 < SM 150 0.027 < SM < 0.040 200 0.020 < SM < 0.027 250 0.016 < S M < 0.020 300 0.013 < S M < 0.016 350 0.011 < S M < 0.013 400 0.010 < S M < 0.011 450 0.0089 <SM <0.010 500 0.0080 < SM < 0.0089 600 0.0067 < SM < 0.0080 700 0.0057 < SM < 0.0067 800 0.0050 < SM < 0.0057 900 0.0044 < SM < 0.0050 1000 0.0040 < SM < 0.0044

Only a slight variation in arsenic concentration was observed across the dilution factor range for either hair (range: 0.009 - 0.06 mg/kg) or fingernail (range: 0.13 - 0.40 mg/kg) samples. Similar patterns were observed for other elements of interest (e.g. Mn,

Fe, V). However, a negative correlation was observed for selenium levels in both hair and fingernail samples with a decreasing selenium concentration as the dilution factor increased.

Due to the minimal variation in elemental concentrations with increasing dilution factor a dilution factor range of 1 0 0 to 1 0 0 0 times dilution has been applied in this study.

However, consideration must be taken when evaluating hair and nail data. Small differences in concentration (e.g. A < 0.2 mg/kg for fingernails) may be attributable to the dilution factor. Particular consideration needs to be made when evaluating selenium data due to the negative trend observed.

2.3 Sample Preparation: Urine

Human urine was collected by trained personnel from study volunteers in Eduardo Castex (La Pampa) as part of the ‘type-2 diabetes’ study; as stated in the ethical approval documentation (EC/2010/124/FHMS) (Appendix A1). Mid-stream urine samples were collected using clean 25 ml Sterilin® bottles (Fisher Scientific Ltd, Leicestershire, UK). Each sample was identifiable by a unique code which corresponded to a questionnaire relating to the volunteer (Appendix A2 - A3). Samples were transported in a polystyrene, ice-packed, cool box back to the UK, along with the required ethical approval documentation (Appendix A2 - AT). On arrival at the University of Surrey, all samples were filtered using a 0.45 pm syringe filter (Millex, Millipore, Bedford, USA), separated into aliquots and stored in a freezer at -20 °C. Sample aliquots (~ 5 ml) were defrosted as required to room temperature (~ 20 °C) to prevent continual re-freezing of the samples. Samples were diluted (x2) using distilled deionised water (DDW) prior to total elemental analysis by ICP-MS.

2.4 Sample Preparation: Whole Blood

Whole blood samples were collected by trained personnel from study volunteers in Eduardo Castex (La Pampa) as part of the ‘type-2 diabetes’ study; as stated in the ethical approval documentation (EC/2010/124/FHMS) (Appendix A1). The method used for sample collection is defined in section 2.4.1 and the preparation procedure is outlined in section 2.4.2.

2.4.1 Sample collection

Whole blood samples were collected by the collaborators in Eduardo Castex as part of routine health analysis. A 1 ml sub-sample was taken and sent to the University of Surrey for the purpose of this study. Each sample was identifiable by a unique code which corresponded to a questionnaire relating to the volunteer (Appendix A2 - A3). On arrival at the University of Surrey, all samples were stored in a freezer at -20 °C. Samples were defrosted as required to room temperature (~ 20 °C) prior to digestion.

Sample diluted to 5 ml using DDW

Filtered using 0.45 pm syringe filter into clean 15 ml centrifuge tubes.

Sample left at room temperature (-2 0 °C) for 1 2 hours before gradually

heating up to 70 ± 1 °C in a water bath for 1 hour.

0.5 ml whole blood transferred into a clean, weighed polypropylene digestion tube and the sample weight recorded ( - 0.5 g).

1 ml conc. Aristar® nitric acid added to each tube. Tubes covered with cling

Whole blood sample defrosted over night and allowed to reach room temperature ( - 20 °C).

Digested sample left to cool before adding 0.5 ml conc. hydrogen peroxide. Sample gradually heated up to 50°C in a water bath for 1

hour.

Fig. 2.2: Whole blood sample preparation (digestion) procedure.

2.4.1 Acid digestion

A 0.5 ml sample of whole blood was added to a clean, dry polypropylene digestion tube