Materials and Reagents

Paper bags
10 mesh nylon standard inspection sieve (pore size 2 mm) (Shangyu Huafeng Hardware Instrument)
50 ml plastic centrifuge tube (SARSTEDT, catalog number: 62.559.001 )
National certified reference materials: GBW10010 (rice), GBW10014 (cabbage) and GBW10015 (spinach; All materials mentioned above were provided by the Institute of Geophysical and Geochemical Exploration, Chinese Academy of Geological Sciences). Lichen material IAEA-336 (International Atomic Energy Agency)
Lichens: Xanthoria mandschurica and Xanthoparmelia mexicana, collected from Taihang Mountains, Hebei Province, China
H₂O₂, > 30% (w/v) (Beijing Institute of Chemical Reagents, catalog number: 160215 )
¹⁰³Rh: 1.00 mg/ml (GSB-G62037-90, National Analysis Center for Iron & Steel, Beijing, China)
HCl, 35-37% (w/v) (Beijing Institute of Chemical Reagents, catalog number: 160718 )
Standard stocking solutions: 1.00 mg/ml Al (GBW[E]086219), Ba (GBW[E]080243), Ca (GBW[E]080261), Cd (GBW08612), Ce (GSB04-1775), Co (GBW08613), Cr (G130008614), Cs (GSB04-1724), Cu (GSB04-1725), Fe (GBW08616), K (GBW[E]080125), La (GBW08651), Mg (GBW[E]080126), Mn (GSB04-1736), Mo (GBW[E]080218), Na (GBW[E]080127), Ni (GBW08618), P (GBW[E]080186), Pb (GBW08619), Rb (GSB04-2836), S (GBW[E]080266), Sb (GBW[E]080545), Sc (GBW[E]3141), Sm (GSB64-1778), Sr (GSB04-1754), Tb (GSB04-1781), Th (GBW[E]080174), Ti (GBW3041), Tl (GSBG62070-90), V (GBW[E]080243), and Zn (GBW[E]080607) (National Research Center for standard materials, Beijing, China)
Standard intermediate solutions (see Recipes; Table 8)
Standard working solutions (see Recipes; Table 8)

Equipment

Inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies, model: Agilent 7700X ICP-MS )
Inductively coupled plasma optical emission spectrometer (ICP-AES) (Varian, model: Varian Vista MPX )
Microwave digestion system (equipped with Teflon digestion vessels) (CEM Corporation, Matthews, model: MARS X-press )
Grinding mill equipped with Tungsten Carbide jars (Retsch, model: Retsch MM400 )
Stereo microscope (Motic, model: Motic SMZ-140 )
Muffle furnace (Yiheng, model: SX2-4-10TP )
Oven (50-120 °C)
Tweezers
50 ml volumetric flask
30 ml nickel crucible (Jiangsu Plaza Premium Electric Instrument, catalog number: 30 ml crucible )
Deionized water: resistivity ≥ 18.0 MΩ cm (Aquapro International, model: Aquaplore3 )

Procedure

Sample preparation
1. Sample preparation for microwave digestion – ICP-MS
2. Sample preparation for microwave digestion – ICP-MS
Measurement of element concentrations in lichens
1. ICP-MS analysis
2. ICP-AES analysis
Evaluation of the optimized methods
1. Linear range and detection limit of the optimized methods
  Dilute the standard working solution for each element by 8% HNO₃ using serial dilution (see Recipes). Detection limits were determined by 8 technical replicates of blank solutions.
  The linear regression equation, linear correlation coefficient, linear range and detection limit of 22 elements (Cd [linear ranges, 0-0.1μg/ml; detection limits, 0.00007 μg/ml], Ce [0-1; 0.00002], Co [0-0.1; 0.0001], Cr [0-1; 0.00017], Cs [0-0.1; 0.00006], Cu [0-1; 0.00038], K [0-100; 0.0052], La [0-0.1; 0.00003], Mo [0-0.1; 0.00093], Na [0-30; 0.0051], Ni [0-1; 0.00007], Pb [0-1; 0.00031], Rb [0-30; 0.00003], Sb [0-0.1; 0.00008], Sc [0-0.1; 0.00009], Sm [0-0.1; 0.00004], Sr [0-30; 0.00011], Tb [0-0.1; 0.00004], Th [0-0.1; 0.00004], Tl [0-0.1; 0.00008], V [0-1; 0.000052], and Zn [0-5; 0.00095]) analyzed by ICP-MS can be seen in Table 4, of 9 elements (Al [Linear ranges, 0-100; detection limits, 0.0054], Ba [0-5; 0.0004], Ca [0-100; 0.0053], Fe [0-100; 0.004], Mg [0-30; 0.0049], Mn [0-5; 0.00014], P [0-30; 0.013], S [0-30; 0.0053], and Ti [0-30; 0.0006]) analyzed by ICP-AES can be seen in Table 7. The results show that both methods had low detection limits and wide linear ranges.
2. Accuracy and precision of the methods
  Prepare and decompose the certified reference materials (IAEA-336, GBW10014 and GBW10015) followed steps A1 and A2. The sample is analyzed with 8 technical replicates using ICP-AES/MS under optimal operating conditions (Tables 2 and 5). A comparison of the measured values with certified values is shown in Tables S1 and S2 for ICP-MS and ICP-AES analysis, respectively. The results show that both methods have a good accuracy (relative error < 10%) and precision (relative standard deviation ranging from 1.56% to 9.55%).

Data analysis

Accuracy and precision of the protocol for each element are obtained using Equation 1 and 2 (based on 8 technical replicates), respectively.

In both equations, C_j denotes the mean of certified reference material values based on n technical replicates; C_s denotes certified values of the certified reference material; C_i denotes the measured value of the certified reference material.

Notes

Lichen samples should be carefully cleaned to remove the attached particles on the surface of the lichen thalli to avoid inaccuracy in the determination of the element concentration.
Sample preparation is essential in this protocol. The classic preparation methods for plant samples are dry ashing, wet digestion, and microwave digestion. The wet digestion method is less suitable for our samples, because of the cumbersome, time-consuming operations, high risk of contamination, and high blank values leading to low precision. Although all elements can be analyzed by using both microwave digestion- ICP-MS and dry ashing-alkali fusion digestion- ICP-AES, we propose that it is better to analyze 22 elements (Cd, Ce, Co, Cr, Cs, Cu, K, La, Mo, Na, Ni, Pb, Rb, Sb, Sc, Sm, Sr, Tb, Th, Tl, V and Zn) using ICP-MS and 9 elements (Al, Ba, Ca, Fe, Mg, Mn, P, S and Ti) using ICP-AES. Microwave digestion with the HNO₃-H₂O₂ system is effective for most refractory elements, and the closed vessel helps in preventing losses of the volatile elements. Although the dry ashing method can lead to a lower result due to the loss of some volatile elements at high temperature, it can be used to prepare samples for analyzing 9 elements (Al, Ba, Ca, Fe, Mg, Mn, P, S and Ti) using ICP-AES analysis.
Because of the high content of metals in lichens, the lichen samples should be soaked in HNO₃ for 4 h, and then be digested by adding H₂O₂. If the sample is difficult to be completely digested, the sample size should be reduced and the soaking duration should be prolonged to overnight.
This protocol is suitable for lichens collected from North China, particularly Inner Mongolia, Shanxi, Hebei and Henan Provinces which are characterized by heavy air pollution.

Recipes

The standard intermediate solutions and standard working solutions for the measured elements are listed in Table 8.

Table 8. The standard intermediate solutions and standard working solutions for the measured elements

Acknowledgments

This work was supported by the National Natural Science Foundation of China under grant No. 31000239 and the Natural Science Foundation of Hebei Province under grant Nos. C2014201032 and C2010000268. The procedure was previously employed in Liu et al. (2016a; 2016b and 2016c) and Zhao et al. (2016).

References

Liu, H. J., Fang, S. B., Liu, S. W., Zhao, L. C., Guo, X. P., Jiang, Y. J., Hu, J. S., Liu, X. D., Xia, Y., Wang, Y. D. and Wu, Q. F. (2016a). Lichen elemental composition distinguishes anthropogenic emissions from dust storm input and differs among species: evidence from Xilinhot, Inner Mongolia, China. Sci Rep 6: 34694.
Liu, H. J., Liu, S. W., Wang, L., Liu, X. D., Zhao, L. C. and Wu, Q. F. (2016b). Effects of species and substrate preference on element concentration of six lichens in Taihang Mountains, Hebei, China. Mycosystema 35(10): 1258-1267.
Liu, H. J., Zhao, L. C., Fang, S. B., Liu, S. W., Hu, J. S., Wang, L., Liu, X. D. and Wu, Q. F. (2016c). Use of the lichen Xanthoria mandschurica in monitoring atmospheric elemental deposition in the Taihang Mountains, Hebei, China. Sci Rep 6: 23456.
Zhao, L. C., Jiang, Y. J., Guo, X. P., Li, X., Wang, Y. D., Guo, X. B., Lu, F. and Liu, H. J. (2016). Optimization of ICP-AES and ICP-MS techniques for the determination of major, minor and micro elements in lichens. Spectrosc Spect Anal 36(10): 3320-3325.