Determination of Elemental Concentrations in Lichens Using ICP-AES/MS

Materials and Reagents

1. Paper bags
   
2. 10 mesh nylon standard inspection sieve (pore size 2 mm) (Shangyu Huafeng Hardware Instrument)
   
3. 50 ml plastic centrifuge tube (SARSTEDT, catalog number: 62.559.001 )
   
4. 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)
   
5. Lichens: Xanthoria mandschurica and Xanthoparmelia mexicana, collected from Taihang Mountains, Hebei Province, China
   
6. H₂O₂, > 30% (w/v) (Beijing Institute of Chemical Reagents, catalog number: 160215 )
   
7. ¹⁰³Rh: 1.00 mg/ml (GSB-G62037-90, National Analysis Center for Iron & Steel, Beijing, China)
   
8. HCl, 35-37% (w/v) (Beijing Institute of Chemical Reagents, catalog number: 160718 )
   
9. 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)
   
10. Standard intermediate solutions (see Recipes; Table 8)
    
11. Standard working solutions (see Recipes; Table 8)
    

Equipment

1. Inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies, model: Agilent 7700X ICP-MS )
   
2. Inductively coupled plasma optical emission spectrometer (ICP-AES) (Varian, model: Varian Vista MPX )
   
3. Microwave digestion system (equipped with Teflon digestion vessels) (CEM Corporation, Matthews, model: MARS X-press )
   
4. Grinding mill equipped with Tungsten Carbide jars (Retsch, model: Retsch MM400 )
   
5. Stereo microscope (Motic, model: Motic SMZ-140 )
   
6. Muffle furnace (Yiheng, model: SX2-4-10TP )
   
7. Oven (50-120 °C)
   
8. Tweezers
   
9. 50 ml volumetric flask
   
10. 30 ml nickel crucible (Jiangsu Plaza Premium Electric Instrument, catalog number: 30 ml crucible )
    
11. Deionized water: resistivity ≥ 18.0 MΩ cm (Aquapro International, model: Aquaplore3 )
    

Procedure

1. Sample preparation
   1. Sample preparation for microwave digestion – ICP-MS
   1. The lichen samples (> 200 g per sample, fresh weight) are air-dried at room temperature in paper bags for at least 5 days. Carefully remove soil particles and other debris on lichen thalli under a stereo microscope using clean tweezers.
      
   2. The cleaned samples are oven-dried at 70 °C for 72 h to constant weight (determined by weighting each sample at an interval of 8-12 h). Cool down the samples to room temperature and ground each sample to powder in a grinding mill, then pass each sample through a 10 mesh nylon sieve.
      
   3. Transfer an accurately weighted portion of each sample (0.3000 g) to a Teflon digestion vessel. Add 4.0 ml HNO₃ to the vessel and incubate for 4 h at room temperature. Then add 2.0 ml H₂O₂, and transfer the digestion vessel to a microwave digestion system (MARS-X), applying the following temperature control procedure (Table 1).
      
      Table 1. Temperature control procedure for microwave digestion
      
      
      
   4. After digestion, when the pressure inside the vessel lowered to normal pressure and the vessel is cooled down to room temperature, open the vessel and wash the sample into a 50 ml volumetric flask by using deionized water (20-40 ml). Dilute the sample to 50 ml with deionized water for ICP-MS testing.
      
   5. Prepare blank samples and certified reference material samples (IAEA-336, GBW10010, GBW10014 and GBW10015) according to the above mentioned procedures.
      
   2. Sample preparation for microwave digestion – ICP-MS
   1. Transfer an accurately weighted portion of each sample (1.0000 g) of the lichen (obtained in step A1b) to a nickel crucible and then all samples are heated to 500 °C until they turn grayish white (and therefore are fully ashed).
      
   2. Add 1.5 g of NaOH to decompose each sample at 700 °C for 10 min. Cool down all samples to room temperature and wash each sample into a 50 ml plastic centrifuge tube using hot deionized water (70-80 °C, 20-30 ml). To ensure complete transfer of each sample, also wash the nickel crucible according to the method mentioned above. Dilute the solution in the tube to 50 ml and shake it well before ICP-AES testing.
      
   3. Prepare blank samples and certified reference material samples (IAEA-336, GBW10010, GBW10014 and GBW10015) according to the procedures mentioned above.
   
   
2. Measurement of element concentrations in lichens
   1. ICP-MS analysis
   1. Set the optimal operating conditions for ICP-MS (Table 2).
      
      Table 2. Optimal operating conditions of ICP-MS
      
      
      
   2. Measure twenty-two elements (Cd, Ce, Co, Cr, Cs, Cu, K, La, Mo, Na, Ni, Pb, Rb, Sb, Sc, Sm, Sr, Tb, Th, Tl, V and Zn) in samples obtained in step A1d using ICP-MS. Select the isotopes of the 22 elements for analysis to remove isotopic interference (Table 3).
      
      Table 3. Isotope of 22 elements for ICP-MS analysis
      
      
      
   3. Add 5 μg/L of the internal standard ¹⁰³Rh into each sample to remove physical interference in the ICP-MS analysis.
      
   4. The linear regression equation and correlation coefficient are determined using standard working solution series (see Recipes; Table 8), and are given by the machine (Table 4). The concentration (μg/ml) for each element is also given by the machine.
      
      Table 4. Parameters of the standard curves and detection limits for elements measured by ICP-MS
      
      
      
   2. ICP-AES analysis
   1. Set the optimal operating conditions for ICP-AES (Table 5).
      
   2. Measure nine elements (Al, Ba, Ca, Fe, Mg, Mn, P, S and Ti) in the samples obtained in step A2c using ICP-AES. The analytical spectral lines for these elements are listed in Table 6.
      
   3. The linear regression equation and correlation coefficient are determined using standard working solution series (see Recipes; Table 8), and are given by the machine (Table 7). The concentration (μg/ml) for each element is also given by the machine.
      
      Table 5. Optimal operating conditions of ICP-AES
      
      
      Table 6. Analytical spectral lines of the elements
      
      
      Table 7. Parameters of the standard curves and detection limits for elements measured by ICP-AES
      
      
   
   
3. 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

1. 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.
   
2. 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.
   
3. 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.
   
4. 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

1. 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

1. 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.
   
2. 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.
   
3. 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.
   
4. 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.