⁶⁷Zn and ¹¹¹Cd labelled green manure to determine the fate and dynamics of zinc and cadmium in soil–fertilizer–crop systems

Figures & data

Table 1. Evaluation of the certified reference material (CRM) measured by combined ICP-MS and isotope mass balance calculations.

Sample type	ID	Certified Zn	Certified Cd	Measured Zn	Measured Cd	Zn recovery	Cd recovery
		mg (kg dry weight)^−1				Fraction recovered (%)
White cabbage	BCR-679	79.7	1.66	84.5	1.68	106	101
Hay powder	IAEA-V-10	24	0.050^a	23.1	0.050	96	99
Wheat flour	NIST 1567b	11.6	0.025	10.3	0.034	89	134

^a Certified value ranges from 0.03 to 0.05 mg (kg dry weight)⁻¹ for Cd.

Figure 1. Comparison of analytical methods used to analyse cadmium (Cd) and zinc (Zn) concentrations in wheat samples that contain enriched stable isotopes (⁶⁷Zn, ¹¹¹Cd). ‘ICP-MS + calculations’ refers to inductively coupled plasma mass spectroscopy combined with isotope mass balance calculations. ‘OES’ refers to inductively coupled plasma optical emission spectroscopy.

Figure 2. Comparison of analytical methods used to analyse Zn (A, B) and Cd (B, C) concentrations in wheat samples that contain enriched stable isotopes (⁶⁷Zn, ¹¹¹Cd). ‘ICP-MS + calc.’ refers to inductively coupled plasma mass spectroscopy combined with isotope mass balance calculations. ‘ICP-MS no calc.’ refers to ICP-MS results with no isotope mass balance correction to correct for isotope ratios at non-natural abundance. Regression line was only plotted when the trend between x and y was significant.

Table 2. Selected soil properties.

Parameter	Unit	Value
Clay	(%)	9^a
Silt	(%)	28^a
Sand	(%)	63^a
Total Zn	(mg kg^−1)	56.8^b
Total Cd	(mg kg^−1)	0.19^b
Zn/Cd ratio (total)	(-)	295
DTPA Zn	(mg kg^−1)	0.19^c
DTPA Cd	(mg kg^−1)	0.009^c
Zn/Cd ratio (DTPA)	(-)	21
Ctot	(g kg^−1)	73.7^b
N	(g kg^−1)	3.1^b
S	(g kg^−1)	0.4^b
C/N	(-)	23.7
Ptot	(g kg^−1)	0.5^a
Cinorg	(g kg^−1)	34.9^d
Corg	(g kg^−1)	38.8^b
pH (CaCl2)	(-)	7.7
WHC	(mL kg^−1)	529^e

^a Determined by WSL in July 1998.

^b Total digestion using hydrofloric and nitric acid.

^c Diethylenetriaminepentaacetic acid (DTPA) extractable Zn and Cd pool was extracted according to Lindsay and Norvell [39].

^d Determined by subtracting Corg (550°C, muffle oven) from total C (1050°C, varia El Cube, Elementar, Germany).

^e Water holding capacity.

Table 3. Wheat harvest data.

Soil Zn status	Green manure (g kg^−1 soil)	Dry weight
		Root			Shoot			Straw			Grain
		(g kg^−1 soil)			(g kg^−1 soil)			(g kg^−1 soil)			(g kg^−1 soil)
		Mean	±SD^a		Mean	±SD^a		Mean	±SD^a		Mean	±SD^a
Low soil-available Zn	0	0.18	0.12	b	2.50	0.22	c	1.28	0.17	b	1.23	0.17	b
	1	0.32	0.14	b	3.90	0.48	b	2.08	0.36	b	1.83	0.24	ab
	4	0.99	0.47	a	5.23	0.61	a	2.88	0.46	a	2.35	0.26	a
Mineral Zn fertilized	0	0.16	0.15	b	2.53	0.39	c	1.45	0.34	b	1.08	0.46	b
	1	0.20	0.11	b	2.80	0.75	bc	1.58	0.40	b	1.23	0.43	b
	4	0.31	0.05	b	3.90	0.32	b	2.25	0.24	a	1.65	0.21	ab
		ANOVA^b			ANOVA^b			ANOVA^b			ANOVA^b
p-values	GM	<0.05			<0.05			<0.05			<0.05
	Zn	<0.05			<0.05			<0.05			<0.05
	Zn*GM	>0.05			<0.05			>0.05			>0.05

^aSD = standard deviation of the mean of n = 4 replicates. Letter denote statistical differences within a column determined with post-hoc tests for ANOVA (Tukey HSD)

^bANOVA = two-way analyses of variance. P-values denote significant effects of the factors GM (green manure), Zn (mineral Zn fertilized) and Zn*GM for the combined factors (p-values: * < 0.05, ** < 0.01, *** < 0.001).

Table 4. Zinc (Zn) and cadmium (Cd) concentrations and masses in wheat samples.

(a)	Soil Zn status	Green manure	Zinc concentration									(b)	Soil Zn status	Green manure	Zinc mass
		(g kg^−1 soil)	Shoot			Straw			Grain				(g kg^−1 soil)	(g kg^−1 soil)	Shoot			Straw			Grain
			(mg kg^−1 dry weight)												(µg kg−^1 soil)
			Mean	±SD^a		Mean	±SD^a		Mean	±SD^a					Mean	±SD^a		Mean	±SD^a		Mean	±SD^a
	Low soil available Zn	0	26.3	4.12	c	22.6	7.57	b	29.7	1.74	b		Low soil available Zn	0	66.1	14.9	d	29.8	13.7	c	36.3	4.74	c
		1	25.3	1.41	c	21.8	3.96	b	29.0	4.89	b			1	98.1	7.48	d	44.8	8.56	bc	53.3	12.7	bc
		4	23.6	0.88	c	20.6	3.65	b	27.0	2.16	b			4	124	17.8	c	60.1	20.0	b	63.7	10.3	b
	Mineral Zn fertilized	0	96.1	10.1	a	81.6	19.0	a	113	8.94	a		Mineral Zn fertilized	0	242	35.7	b	122	49.9	a	120	45.5	b
		1	98.0	29.4	ab	87.7	44.0	a	110	8.57	a			1	259	34.5	a	127	34.3	a	132	41.8	a
		4	76.3	5.11	b	60.2	10.5	a	99.4	8.91	a			4	297	22.4	a	135	18.8	a	163	8.99	a
			Kruskal-Wallis^c			Kruskal-Wallis^c			Kruskal-Wallis^c						Kruskal-Wallis^c			Kruskal-Wallis^c			Kruskal-Wallis^c
	p-values	GM	>0.05			>0.05			>0.05				p-values	GM	>0.05			>0.05			>0.05
		Zn	<0.05			<0.05			<0.05					Zn	<0.05			<0.05			<0.05
		Zn*GM	<0.05			<0.05			<0.05					Zn*GM	<0.05			<0.05			<0.05
(c)	Soil Zn status	Green manure	Cadmium concentration									(d)	Soil Zn status	Green manure	Cadmium mass
		(g kg^−1 soil)	Shoot			Straw			Grain				(g kg^−1 soil)	Shoot			Straw			Grain
			(µg kg^−1 dry weight)											(ng kg^−1 soil)
			Mean	±SD^a		Mean	±SD^a		Mean	±SD^a				Mean	±SD^a		Mean	±SD^a		Mean	±SD^a
	Low soil available Zn	0	596	91.6	a	721	178	a	465	45.1	ab	low soil available Zn	0	1494	295	b	920	259	b	574	128	c
		1	483	37.2	b	529	67.7	a	427	35.7	b		1	1870	105	b	1089	161	b	782	131	b
		4	544	88.2	ab	582	146	a	494	18.9	a		4	2823	428	a	1664	443	a	1159	94.1	a
	Mineral Zn fertilized	0	187	27.3	c	198	37.3	b	169	21.5	c	mineral Zn fertilized	0	468	47.5	d	291	104	d	177	66.0	e
		1	203	67.4	c	225	102	b	167	14.6	c		1	533	66.5	d	329	72.1	d	204	70.6	e
		4	191	13.3	c	198	14.3	b	183	20.2	c		4	743	29.6	c	444	23.0	c	299	28.8	d
			Kruskal-Wallis^c			Kruskal-Wallis^c			Kruskal-Wallis^c					Kruskal-Wallis^c			Kruskal-Wallis^c			Kruskal-Wallis^c
	p-values	GM	>0.05			>0.05			>0.05			p-values	GM	>0.05			>0.05			>0.05
		Zn	<0.05			<0.05			<0.05				Zn	<0.05			<0.05			<0.05
		Zn*GM	<0.05			<0.05			<0.05				Zn*GM	<0.05			<0.05			<0.05

^aSD = standard deviation of the mean of n = 4 replicates. Letter denote statistical differences within a column determined with post-hoc tests for ANOVA (Tukey HSD) and Kruskal-Wallis (conover test).

^bANOVA = two-way analyses of variance. P-values denote significant effects of the factors GM (green manure), Zn (mineral Zn fertilzed) and Zn*GM for the combined factors.

^cKruskal-Wallis was applied when prerequisites for ANOVA were not fulfilled. Three Kruskal-Wallis tests were conducted: each one to test the effects of GM and Zn and one test for the combined effect of GM*Zn.

Figure 3. Fraction of Zn (A) and Cd (B) in wheat shoots either derived from the soil (brown) or the green manure (green) expressed in percentage. Error bars represent the standard deviation of the mean of n = 4 experimental replicates. Letters denote statistical differences of the mean between Zn or Cd that derived from the soil (brown) and from the green manure (green) determined by a Kruskal–Wallis test (Table S5).

Figure 4. The recovery of Zn (A) and Cd (B) in shoots of wheat that was applied with the green manure (GM). Error bars represent the standard deviation of the mean of n = 4 experimental replicates. Letters denote statistical differences of the mean determined by Kruskal–Wallis test (Table S6).

Figure 5. The transfer of Zn (A) and Cd (B) from soil (brown) and green manure (green) to shoots of wheat. Error bars represent the standard deviation of the mean of n = 4 experimental replicates. Horizontal lines (black) express the mean (straight line) and standard deviation (dashed line) of the control soil that has not received green manure. Letters denote statistical differences of the mean of Zn and Cd derived from the soil (brown) and green manure (green) determined by a Kruskal–Wallis test that includes the Zn and Cd derived from the soil in the control treatment (see details Table S7).

Figure 6. Schematic summary of the major effects of green manure addition on Zn and Cd dynamics in a soil–wheat system (alkaline soil with low Zn availability). Boxes are approximately proportional to the size of the Zn and Cd masses in the DTPA-extractable soil pools, green manure and wheat shoots. Size of the arrows symbolize Zn (blue) and Cd (red) transfer rates between the soil, fertilizer and plant compartments. Left: control treatment with no green manure added, Right: treatment with 4 g green manure per kg of soil. The direct transfer of Zn and Cd from the green manure to the wheat is small, but the transfer from the soil to the wheat significantly increases with green manure.

Lindsay WL, Norvell WA. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci Soc Am J. 1978;42:421–428. doi:10.2136/sssaj1978.03615995004200030009x.

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