水消化法操作外，也另以X 射線螢光光譜儀(X-ray Fluorescence Spectrometer，XRF)
來測量土壤的全量銅濃度。XRF 的操作具有方便、快速、安全及機動性高的特性，
並且對供測樣本不具破壞性而可重複使用，與王水消化法的比較在(表 5)。藉由調 查使用這兩種測量土壤銅全量的結果，期望能增加未來試驗的效率及選擇性。
第一節、前人研究 單一萃取法
重金屬全量並不等於生物可利用量(Chojnacka et al., 2005; Ernst, 1996; Kidd et al., 2007)。且重金屬在土壤的不同型態決定了它們的移動性和生物可利用性(Meers et al., 2007) 。 過 去 常 以 連 續 萃 取 法 (sequential extraction) 或 單 一 萃 取 法 (single extraction)來進行土壤重金屬型態分析(Lo and Yang, 1998; Rao et al., 2008; Zhang et al., 2010; Zhao et al., 2018)。單一萃取法相對於連續萃取法的優勢為，土壤樣本可 同時進行測量和自動化，並節省化學藥品，相對簡單執行和解釋，具有成本效益，
並且通常具有很高的重複性，與連續萃取法相比，降低了將實驗性錯誤從一個步驟
表5. 王水消化與 XRF 之優缺點比較
Table 5. Comparison of the advantages and disadvantages of AR and XRF
Aqua Regia digestion(AR) X-ray fluorescence(XRF)
Advantages Low detection limit with ICP-OES, MS measurement
1. Non-destructive, sample reuse 2. Time-saving, Easy-to-use
Disadvantages
1. Samples need to be preprocessed 2. Time-consuming and laborious
1. Relatively high detection limit 2. Element interference effect ICP-OES : Inductively coupled plasma optical emission spectrometry
ICP-MS : Inductively coupled plasma mass spectrometry
帶到另一個步驟的可能性，另外單一萃取法試驗也不需要有劇毒的酸劑，如氟化氫 和過氯酸等(Li et al., 2017)。以不同單一萃取劑評估土壤重金屬之生物有效性已有 許多案例(表 6)，其中本試驗所使用之 0.1 M HCl、0.05 M EDTA、0.1 M NaNO3及 0.01 M CaCl2皆曾被使用於研究銅的生物有效性濃度。根據萃取機制的不同，又可 將化學萃取劑分類為交換性試劑、酸性試劑與有機螯合劑(Peijnenburg et al., 2007)。
(一)交換性試劑 量皆是最少的(Novozamsky et al., 1993)。
(二)酸性試劑 2009; Sutherland, 2002; Taylor et al., 1993)。酸性試劑在性質上模擬了植物根系有機 酸分泌所造成的微酸性環境(Wang et al., 2009)。根據研究結果表示，0.1 M HCl 的 重金屬萃取能力會受到土壤或重金屬性質的不同而有所差異，植物所吸收之重金 屬含量亦隨著植物及重金屬種類而有所不同(歐，2000)。另外也發現，雖然生物可
表6. 以不同萃取劑評估土壤重金屬之生物有效性案例
Table 6. Cases of assessing the bioavailability of heavy metals in soils with different soil extractants
Extraction solution Elements Reference
0.1 M NaNO3 Cu, Zn, Cd, Ni, Pb (Gupta and Aten, 1993) 0.01 M CaCl2 Cu, Zn, Y, La, Al, Cd (Takeda et al., 2006) 0.1 M HCl Cu, Zn, Cd, Cr, Ni, Pb (Romkens et al., 2009) 0.05 M EDTA Cu, Zn, Cd (Guo et al., 2006)
利用型態之含量和0.1 M HCl 土壤重金屬萃取量呈正相關性，但 0.1 M HCl 土壤重
金屬萃取量幾乎超過生物可利用型態含量，因此以0.1 M HCl 萃取量判定生物有效
性會是高估的(黃，2003)。
(三)有機螯合劑
Ethylenediaminetetraacetic acid(EDTA) 、 Diethylenetriaminepentaacetic acid (DTPA)、Hydroxyethyl ethylenediamine triacetic acid (HEDTA)、Nitrilotriacetic acid (NTA)、Ethylene glycol tetraacetic acid (EGTA)及檸檬酸等皆為有機螯合劑，含有二 個以上的配位基，能與土壤溶液中重金屬離子產生錯合作用形成穩定的複合物
（complex），而達到分離效果(李，2003)。0.1 M EDTA 被廣泛的用來測定土壤中的 重金屬濃度(Feng et al., 2005; Guo et al., 2006; Peijnenburg et al., 2007; Rauret et al., 2000)。有機螯合劑在性質上模擬了植物根系有機酸分泌物對金屬元素吸附的部分 (Wang et al., 2009)。評估土壤的生物有效性的研究中，EDTA 及 DTPA 被廣泛的應 用(Brun et al., 2001; Chaignon et al., 2003; Hammer and Keller, 2002; Haq et al., 1980;
Zhang et al., 2010)。並且發現到土壤重金屬的生物有效性與 EDTA、DTPA 及土壤 pH 值相關聯。酸性土壤以 EDTA 萃取之重金屬濃度，其與生物有效性有高的相關
析時間，約3-5 分鐘。XRF 機型為 Olympus Innov-X DELTA-D50，以金作為陽極靶 材，檢測器規格為≧30 mm2 SDD, Silicon Drift Detector，光管功率 50 KV，處理器 規格為530 MHz CPU, 128M RAM, 500 MHz Dual core DSP。 (promax syringe filter, 25 mm 0.45 μm pes, DIKMA)過濾萃取液，以感應耦合電漿原 子發射光譜儀(ICP - OES, Perkin Elmer OPTIMA 5300 DV)測定萃取液銅濃度。
(二)HCl 0.1 M 萃取法
本試驗的土壤須再過0.149 mm 標準篩網。試劑配置是以分注器注入 9.81 mL HCl(hydrochloric acid 32%, Emsure for analysis, Merck, Taiwan)，加入去離子水定量 至1 L。取 1 g 供試樣本置於 15 mL 離心管，加入 10 mL 0.1 M HCl 萃取液充分混 勻，再以水平震盪器200 rpm 下 25oC 震盪 1 小時，震盪後的樣本以 5390 xg 離心 10 分後，上清液以針筒濾膜(promax syringe filter, 25 mm 0.45 μm pes, DIKMA)過濾 萃取液，以感應耦合電漿原子發射光譜儀(ICP - OES, Perkin Elmer OPTIMA 5300
小時，震盪後的樣本以 5390 xg 離心 10 分後，上清液以針筒濾膜(promax syringe
模型的建立是以臺灣大學雲端共享軟體SPSS Statistics 17.0 製作，將各項生物 有效性因子(盆栽試驗為植體銅全量 mg·kg-1、土壤銅全量mg·kg-1與四種生物有效 性萃取液mg·kg-1；現地試驗則又包括黏粒%、pH、陽離子交換能力 cmol·kg-1及有 機質%)代入 SPSS 以複迴歸分析(multiple regression analysis)。由於各項目的單位不 同，因此先將數據統一換算成Log 值，除了 pH 原本即為氫離子的 Log 值不換算。
換能力及粒徑大小皆與植物銅的生物有效性相關(Bravin et al., 2009; Chaignon et al., 2002; Wang et al., 2015; Wu et al., 2010)。本章的目的為建立植體銅濃度預估模型，
將在調查供試土壤的基本理化性質後，用以後續模型的建立。
Jang, 2010; Wei and Yang, 2010)。
XRF 及王水消化後測得的土壤銅全量在比較後發現，除了 44 號樣本有顯著的
表7. 現地試驗土壤基本物理化學性質及不同單一化學萃取方法萃取重金屬銅之濃度
Table 7. The basic physical and chemical properties of the soil and the concentration of heavy metal copper extracted by different single chemical extraction methods
Cu
mg·kg-1 cmol·
kg-1 %
Sample
name Aqua
regiaz XRFy Ricex CaCl2w NaNO3v HClu EDTAt CECs pHr OMq Sandp Silto Clayn
1 19.3 19.7 3.408 0.067 0.04 7.979 6.368 11.2 6.1 3.31 32 48.6 19.4
2 21.48 19.4 2.431 0.004 0.027 8.957 58.9 11.9 5.37 4.67 23.6 54 22.4
3 22.11 20.9 3.751 0.017 0.026 8.045 9.586 13.4 6.25 5.18 25.6 58 16.4
4 22.78 22.3 3.512 0.026 0.038 10.28 10 15.4 5.61 4.87 21 60 19
5 23.26 22.1 3.567 0.031 0.059 8.61 8.954 13 6.3 4.46 37.6 46 16.4
6 23.38 24.3 3.883 0.025 0.025 9.025 10.28 14 4.79 5.16 23.3 58.3 18.4
7 23.53 21.7 3.518 0.022 0.03 8.743 10.5 15.7 6 4.98 31.6 47.6 20.8
8 23.89 24.3 4.067 0.014 0.065 9.029 9.727 11.4 6.03 5.03 35 51.6 13.4
9 24.16 23.2 3.393 0.017 0.032 8.328 9.94 13 6.38 4.24 23.6 54 22.4
10 24.27 22.3 3.976 0.021 0.029 8.399 10.01 14.9 6.25 4.07 29.6 50.6 19.8
z soil aqua regia digestion analysis. y soil x-ray fluorescence spectrometer analysis. x brown rice grains copper content analysis. w 0.01 M CaCl2 extraction. v NaNO3 extraction. u 0.1 M HCl extraction. t 0.05 M EDTA extraction. s cation exchange capacity. r pH value. q organic matter. p sand. o silt. n clay
表7. 現地試驗土壤基本物理化學性質及不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 7. The basic physical and chemical properties of the soil and the concentration of heavy metal copper extracted by different single chemical extraction methods
Cu
mg·kg-1 cmol·
kg-1 %
Sample name
Aqua
regiaz XRFy Ricex CaCl2w NaNO3v HClu EDTAt CECs pHr OMq Sandp Silto Clayn
11 24.29 24 3.475 0.019 0.033 8.882 10.49 15.1 6.21 4.77 27.6 58.6 13.8
12 24.6 20.47 1.635 0.052 0.016 9.025 9.585 15.8 5.61 4.69 27.6 52.6 19.8
13 24.71 24.8 5.223 0.026 0.041 9.374 10.91 13.3 6.07 4.26 33.6 46 20.4
14 26.89 25.1 2.695 0.037 0.06 11.48 11.62 14.5 6.35 5.22 39.6 48.6 11.8
15 27.85 22.1 3.337 0.021 0.028 12.11 11.82 14.5 6 5.9 37.6 50 12.4
16 28.9 25.8 2.586 0.018 0.035 10.36 12.52 12.4 6.34 5.58 25.6 58 16.4
17 31.27 30.8 2.55 0.007 0.066 12.81 13.79 12.5 6.14 4.26 23.6 56.6 19.8
18 44.29 39 2.757 0.056 0.2 24.83 21.33 10.5 6.98 4.09 27.6 56 16.4
19 46.87 41.8 1.773 0.045 0.145 26.93 22.18 11.1 6.56 3.71 33.6 50 16.4
20 48.67 34.9 2.502 0.326 0.496 24.76 19.17 9.6 6.62 6.23 33.6 54 12.4
z soil aqua regia digestion analysis. y soil x-ray fluorescence spectrometer analysis. x brown rice grains copper content analysis. w 0.01 M CaCl2 extraction. v NaNO3 extraction. u 0.1 M HCl extraction. t 0.05 M EDTA extraction. s cation exchange capacity. r pH value. q organic matter. p sand. o silt. n clay
表7. 現地試驗土壤基本物理化學性質及不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 7. The basic physical and chemical properties of the soil and the concentration of heavy metal copper extracted by different single chemical extraction methods
Cu
mg·kg-1 cmol·
kg-1 %
Sample
name Aqua
regiaz XRFy Ricex CaCl2w NaNO3v HClu EDTAt CECs pHr OMq Sandp Silto Clayn
21 51.28 44.9 3.989 0.067 0.294 27.09 27.84 15.4 6.61 5.83 39.6 48 12.4
22 52.67 51.1 5.945 0.103 0.294 26.8 28.84 11.8 6.14 6.5 53 40 7
23 57.68 53.2 3.924 0.011 0.098 28.98 27.36 13.7 6.76 3.93 21 58 21
24 63.78 61 3.671 0.026 0.139 36.03 31.92 13.2 6.27 4.3 19.6 56 24.4
25 69.44 66.8 5.997 0.058 0.216 43.16 39.39 13.7 6.51 5.93 43 49.2 7.8
26 69.46 61.5 5.37 0.108 0.226 40.86 40.07 13.1 6.48 5.92 51.6 38.6 9.8
27 75.45 69.4 7.227 0.065 0.178 38.74 40.26 8.7 7.18 2.75 21.6 56.6 21.8
28 76.35 70.1 7.36 0.047 0.171 41.79 36.69 13.4 6.53 4.33 21 52 27
29 79.02 72.6 3.084 0.039 0.174 49.94 44.86 10.6 6.28 4.11 29 52.6 18.4
30 82.01 77.4 3.451 0.146 0.369 46.89 45.88 12.3 6.3 5.08 27 54.6 18.4
z soil aqua regia digestion analysis. y soil x-ray fluorescence spectrometer analysis. x brown rice grains copper content analysis. w 0.01 M CaCl2 extraction. v NaNO3 extraction. u 0.1 M HCl extraction. t 0.05 M EDTA extraction. s cation exchange capacity. r pH value. q organic matter. p sand. o silt. n clay
表7. 現地試驗土壤基本物理化學性質及不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 7. The basic physical and chemical properties of the soil and the concentration of heavy metal copper extracted by different single chemical extraction methods
Cu
mg·kg-1 cmol·
kg-1 %
Sample name
Aqua
regiaz XRFy Ricex CaCl2w NaNO3v HClu EDTAt CECs pHr OMq Sandp Silto Clayn
31 82.26 74 4.932 0.322 0.574 46.38 45.86 15.7 6.56 6.42 43 46.6 10.4
32 83.27 78.3 3.422 0.062 0.175 48.93 46.39 12.4 5.91 4.39 27.6 56.6 15.8
33 85.16 72.1 5.349 0.071 0.169 49.46 55.55 12.5 6.41 4.69 25.6 52 22.4
34 88.89 84.2 3.735 0.349 0.588 50.47 47.39 12.2 5.77 4.8 29.6 54.6 15.8
35 92.26 124.8 4.53 0.405 0.672 56.59 54.57 13.3 7.06 4.21 37.6 44.6 17.8
36 94.35 76.7 4.406 0.063 0.328 57.61 61.67 14.2 7.12 4.74 33 52.6 14.4
37 99.22 85.6 4.493 0.14 0.266 58.65 51.48 10.5 6.04 3.84 33.6 42 24.4
38 102.2 89.8 5.043 0.069 0.24 50.97 50.47 15.2 6.62 4.2 23 50.6 26.4
39 105.1 90.5 5.062 0.053 0.174 59.62 63.71 17.9 6.66 4.91 29.6 50 20.4
40 108.1 97.1 2.8 0.067 0.166 66.8 58.12 12 6.17 4.58 33 46.6 20.4
z soil aqua regia digestion analysis. y soil x-ray fluorescence spectrometer analysis. x brown rice grains copper content analysis. w 0.01 M CaCl2 extraction. v NaNO3 extraction. u 0.1 M HCl extraction. t 0.05 M EDTA extraction. s cation exchange capacity. r pH value. q organic matter. p sand. o silt. n clay
表7. 現地試驗土壤基本物理化學性質及不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 7. The basic physical and chemical properties of the soil and the concentration of heavy metal copper extracted by different single chemical extraction methods
Cu
mg·kg-1 cmol·
kg-1 %
Sample name
Aqua
regiaz XRFy Ricex CaCl2w NaNO3v HClu EDTAt CECs pHr OMq Sandp Silto Clayn
41 124.4 97.8 3.694 0.195 0.509 80.03 73.91 16.1 6.89 4.73 37.6 46.6 15.8
42 130.6 113.9 5.055 0.043 0.202 74.93 77.47 12.7 6.04 5.46 39.6 46.6 13.8
43 177.4 151 5.65 0.191 0.46 101.4 95.8 14.4 6.53 5.6 41 44.6 14.4
44 180.8 85.5 4.284 0.254 0.5 54.02 52.5 12 6.06 5.55 29.6 56.6 13.8
45 181.3 152 8.69 0.088 0.284 108.1 86.14 11.2 6.18 3.75 27.6 54.6 17.8
46 245.5 218 4.857 0.138 0.424 125.91 125.9 14.8 6.52 5.28 27.6 54 18.4
47 320.3 268 3.671 0.144 0.375 178.7 182.7 11.8 6.27 4.65 29 54.6 16.4
48 341.6 290 4.128 0.201 0.543 238.2 172.7 11.8 6.89 3.8 37 45.2 17.8
49 446.8 392 5.627 0.621 0.971 335.1 264.6 12.5 6.9 4.33 43 40.6 16.4
50 691.1 585 7.074 0.568 1.515 498.8 419.8 14.5 7.18 4.33 33.6 48 18.4
z soil aqua regia digestion analysis. y soil x-ray fluorescence spectrometer analysis. x brown rice grains copper content analysis. w 0.01 M CaCl2 extraction. v NaNO3 extraction. u 0.1 M HCl extraction. t 0.05 M EDTA extraction. s cation exchange capacity. r pH value. q organic matter. p sand. o silt. n clay
圖19. 使用王水消化和 XRF 分析土壤銅濃度之迴歸分析
Fig. 19. Regression analysis of soil copper concentration using aqua regia digestion and XRF analysis
稻米銅濃度範圍介於1.63-8.69 mg·kg-1，平均銅濃度為4.21 mg·kg-1，相對於土壤的 (Peijnenburg et al., 2007)。單一萃取法就是其中一個用來研究土壤重金屬生物有效 性的方法。本試驗利用四種化學萃取劑，萃取劑的種類包括中性鹽類、酸劑及螯合
(Brun et al., 2001)。另外也可以發現，盆土可萃取的銅濃度與作物種類無關，意即
圖20. 稻米與土壤銅濃度的迴歸分析
Fig. 20. Regression analysis of copper concentration in rice and soil
表8. 蕹菜盆土以不同單一化學萃取方法萃取銅之濃度
Table. 8. Concentration of copper by different single chemical extraction methods for potting soil grown with water spinach
Water spinach
表8. 蕹菜盆土以不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table. 8. Concentration of copper by different single chemical extraction methods for potting soil grown with water spinach
Water spinach
表9. 白莧盆土以不同單一化學萃取方法萃取重金屬銅之濃度
Table 9. Concentration of copper by different single chemical extraction methods for potting soil grown with Amaranth
Amaranth
表9. 白莧盆土以不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 9. Concentration of copper by different single chemical extraction methods for potting soil grown with Amaranth
Amaranth
表10. 小白菜盆土以不同單一化學萃取方法萃取重金屬銅之濃度
Table 10. Concentration of copper by different single chemical extraction methods for potting soil grown with Pakchoi
Pakchoi
表10. 小白菜盆土以不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 10. Concentration of copper by different single chemical extraction methods for potting soil grown with Pakchoi
Pakchoi
ND : Not detectable
表11. 茼蒿盆土以不同單一化學萃取方法萃取重金屬銅之濃度
Table 11. Concentration of copper by different single chemical extraction methods for potting soil grown with Garland chrysanthemum
表11. 茼蒿盆土以不同單一化學萃取方法萃取重金屬銅之濃度(續)
Table 11. Concentration of copper by different single chemical extraction methods for potting soil grown with Garland chrysanthemum
Garland chrysanthemum
ND : Not detectable
* : Insufficient sample
四種作物在相同的銅濃度土壤，以一樣的萃取劑萃取，其結果會是相似的。
表12. 蕹菜銅濃度預估模型之植體銅濃度與各項自變數相關性
Table 12. Correlation between plant copper concentration and various independent variables in the water spinach copper concentration prediction model
Pearson Correlation (mg·kg-1) Soil Cu
concn.
Cu concn.
extracted by CaCl2
Cu concn.
extracted by NaNO3
Cu concn.
extracted by EDTA
Cu concn.
extracted by HCl Plant Cu
concn. 0.685 0.793 0.808 0.704 0.633
Significancex *** *** *** *** ***
x *** indicate significant p ≤ 0.001.
表13. 蕹菜銅濃度預估模型
Table 13. Prediction model of water spinach copper concentration
Multiple regression analysis R2 *
y = 0.325 a + 1.888 0.3360
y = 0.245 N + 2.812 0.5660
y = 1.537 N – 1.228 C + 2.805 0.6569
y = 0.495 N – 0.352 H + 3.735 0.7227
y = 0.305 N – 1.123 H + 0.978 E + 3.29 0.8144 y = 0.278 a – 1.268 C + 1.468 N + 1.058 E – 1.204 H + 2.377 0.8281 y = 1.256 N – 1.177 H + 1.225 E – 1.045 C + 2.801 0.8319 y : Log vaule of plant copper concentration (μg·kg-1)
a : Log vaule of soil copper concentration (μg·kg-1)
C : Log vaule of copper concentration extracted by CaCl2 (μg·kg-1) N : Log vaule of copper concentration extracted by NaNO3 (μg·kg-1) E : Log vaule of copper concentration extracted by EDTA (μg·kg-1) H : Log vaule of copper concentration extracted by HCl (μg·kg-1)
* : regression analysis of the estimated and actual values of plant copper concentration
圖 21. 蕹菜銅濃度實際值與預測值的關係(預測值以土壤銅全量為方程式的變量所 建構之模型所計算)
Fig. 21. The relationship between the actual and predicted copper concentration of water spinach. The predicted values are calculated by the multiple regression model, including soil copper concentration (mg·kg-1) as a variable in the equation.
圖 22. 蕹菜銅濃度實際值與預測值的關係(預測值以 NaNO3、CaCl2、HCl、EDTA 萃取的土壤銅濃度為方程式的變量所建構之模型所計算)
Fig. 22. The relationship between the actual and predicted copper concentration of water spinach. The predicted values are calculated by the multiple regression model, including soil copper concentration extracted by NaNO3, HCl, EDTA and CaCl2 (mg·kg-1) as a variable in the equation
圖23. 蕹菜銅濃度實際值與預測值的關係(預測值以土壤銅全量和 NaNO3、CaCl2、 HCl、EDTA 萃取的土壤銅濃度為方程式的變量所建構之模型所計算)
Fig. 23. The relationship between the actual and predicted copper concentration of water spinach. The predicted values are calculated by the multiple regression model, including soil copper concentration (mg·kg-1), copper concentration extracted by NaNO3, HCl, EDTA and CaCl2 (mg·kg-1) as a variable in the equation
圖 24. 蕹菜銅濃度實際值與預測值的關係(預測值 NaNO3、HCl、EDTA 萃取的土 壤銅濃度為方程式的變量所建構之模型所計算)
圖 24. 蕹菜銅濃度實際值與預測值的關係(預測值 NaNO3、HCl、EDTA 萃取的土 壤銅濃度為方程式的變量所建構之模型所計算)