## 2 Materials and Methods
### Sample Preparation  
The determination of mercury in the hair of prehistoric animals and museum exhibits is associated with a number of difficulties: obtaining a representative sample, choosing an appropriate method given the small amount of sample, and reliable analytical determination of Hg concentration in it. Loss of volatile Hg and sample contamination are possible during long-term storage and transportation of the sample. Samples of studied fossil animals' hair were stored in museum in glass cases or wooden boxes in conditions excluding their mercury contamination during storage and therefore, they are suitable for analysis.  
The amount of prehistoric animals' hair is very limited, and a single procedure of sample preparation must be developed not only for Hg, but also for a wide range of other trace elements. Here we aimed to determine the total concentrations of mercury in the hair of prehistoric animals both endogenous and exogenous in origin reflecting the intake from food and water, as well as from the air.  
Methylmercury easily incorporated into hairs as it grows and its concentration in the hair is proportional to the blood concentration. The high affinity of hair for metals is mainly due to the presence of cysteine or sulfhydryl (SH) groups [39]. Elemental mercury may also bind to the hydrophobic core of the melanin polymer in the hair structure [40]. The IAEA recommends hair washing procedure using acetone and deionized water [10]; it is not suitable for Hg because the fat and keratin structures of the hair are destroyed by acetone, which leads to the loss of endogenous mercury. Washing the hair with HCl solution can leach methyl mercury from hair samples [41]. We used a chemically inert detergent (\"SYNERGETIC Baby\", fragrance and color free) which removes only surface grease and dust from hair samples without disturbing their structure. All reagents were tested for Hg content and purified if necessary. Nitric and hydrochloric acids were purified using a Savillex DST-1000 distillation system (Savillex, Eden Prairie, MN, USA). Ultrapure water (MQ-water) was obtained using a Simplicity UV water purification system (Millipore SAS, Molsheim, France). All stages of sample preparation were carried out in a \"clean room\" equipped with outdoor air handlers that use progressively finer filters including high-efficiency particulate air (HEPA) filter and charcoal mercury filter, which remove particulate and elemental mercury from the incoming air.  
The washing procedure for hair comprises the following steps:  
1. Washing the samples in a solution of chemically inert detergent (30 min);
2. Three-fold soaking in MQ-water (during the day);
3. Air-drying in a clean room;
4. Cutting hair into small pieces of less than 0.3 cm (scissors pretreated with detergent and ultrapure water);
5. Careful homogenization of the obtained samples by mixing to provide a representative sub-sampling;
6. Storage in a double-sealed zipper-locked plastic bag, in a clean room.  
Microwave system MARS-5 (Thermo Fisher Scientific, Waltham, MA, USA) was used for digestion of hair samples by the program previously optimized for Hg analysis in biological objects (Table 2) [42].  
We tested acid and acid-peroxide digestion and demonstrated applicability of acid-peroxide digestion (2 mL HNO\({}_{3}\) + 1 mL H\({}_{2}\)O\({}_{2}\)) for Hg analysis. The latter method was used for the analysis of samples.  
After microwave digestion the samples were cooled to 25 \({}^{\circ}\)C, the pressure was brought to \(<\)50 psi, and sample volume was adjusted to 10-12 mL with MQ water. The procedure of sample preparation (washing, cutting, and digestion) was developed and tested by using hair samples of modern yak living in the Barnaul Zoo and Certified Reference Material of human hair (CRM, Hair NSC DC 73347, China). The developed sample-preparation procedure is suitable for both for Hg and multi-element analysis of prehistorical animals' hair and blood.