Laboratory Procedure	LPFC-110
	August 1980

Determination of Lead and Cadmium in Foods by Atomic Absorption Spectrophotometry (1)

This content was archived on June 24, 2013.

HEALTH PROTECTION BRANCH LABORATORIES
Bureau of Chemical Safety
OTTAWA

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Definition: This method is applicable to the quantitative determination of lead and cadmium in foods. It can also be applied to copper.

Scope: This method has been evaluated for determination of lead and cadmium in biological materials and foods, including baby formulas. Using graphite-furnace atomization detection limits for 5 and 20 g samples are 8 and 2 ppb for lead, and 0.4 and 0.1 ppb for cadmium. See Tables 5-10 for recovery data for lead and cadmium and Table 10 for copper.

Principle: Samples of food are digested using, sequentially, HNO₃ and HCl. Metals released by the digestion are added to NH₄I solution, extracted into MIBK-TBP (80:20), then stripped into H₂O₂ (5%), HNO₃ (0.5%) and NH₄H₂PO₄ (0.1%). Samples are analyzed by injection into a carbon-rod atomizer of an atomic absorption spectrophotometer or by flame AAS.

Apparatus: 1. Atomic absorption spectrophotometer, Perkin-Elmer Model 403 or equivalent equipped with:

(a) P.E. HGA-400 graphite furnace with automatic stop-flow mode;
(b) simultaneous deuterium-arc background correction;
(c) fast-response (<0.5s Full Scale) strip-chart recorder;
(d) graphite tubes, not pyrolytically coated;
(e) lead and cadmium EDL lamps and power supply; or, hollow cathode lamps;
(f) air-acetylene burner.

2. Hot plate, Corning PC100 or equivalent.

3. Magnetic stirrer and stirring bars.

4. Oven, gravity convection, dust free, with stainless steel interior.

5. Plastic bottles and caps, preferably polypropylene or conventional polyethylene.

(a) various sizes. (Note 1)

6. pH-Meter; Corning Digital 112 or equivalent.

7. General laboratory glassware. (Note 2)

8. Safety equipment.

(a) goggles and rubber gloves.

Reagents: 1. Deionized water (H₂O) should be used wherever H₂O is specified or required.

2. Nitric acid, ca 70%, containing less than 1 ng/mL Pb and 0.1 ng/mL Cd.

3. Hydrochloric acid, 10 M, containing less than 1 ng/mL Pb and 0.1 ng/mL Cd.

4. Hydrogen peroxide (H₂O₂), 50%, reagent grade.

5. Ammonium hydroxide (NH₄OH), conc, reagent grade.

6. Chloroform (CHCl₃), reagent grade.

(a) preclean CHCl₃ by extracting 300 mL with 50 mL 1% HNO₃ for 3 min;
(b) store in acid-washed Pyrex bottle.

7. Diphenylthiocarbazone (dithizone), 0.1% chloroform solution.

(a) dissolve 0.10 g dithizone in 100 mL purified CHCl₃.

8. Ascorbic acid, 20% w/v.

(a) dissolve 20 g reagent grade L-ascorbic acid in ca 80 mL H₂O in a 100 mL volumetric flask;
(b) heat slightly to aid dissolution;
(c) cool;
(d) dilute to mark with H₂O.

9. Methyl isobutyl ketone (MIBK) and tri-n-butyl phosphate (TBP) solution, 4:1 v/v.

(a) to an acid-washed 2 litre borosilicate bottle, empty acid bottles are convenient, add 1200 mL MIBK and 300 mL TBP;
(b) add ca 200 mL 1% high-purity HNO₃;
(c) stir 15 min with magnetic bar;
(d) let phases separate and quickly insert 5 mL pipet into aqueous phase;
(e) draw off aqueous phase using suction from a water aspirator;
(f) add fresh portion of 1% HNO₃;
(g) stir 3 min;
(h) organic solution may be dispensed with a clean dry Pyrex repipet directly from the bottle containing 1% HNO₃.

10. Ammonium dihydrogen phosphate (NH₄H₂PO₄) solution, 10% w/v.

(a) dissolve 100 g of reagent grade NH₄H₂PO₄ in ca 400 mL H₂O;
(b) transfer to 500 mL separatory funnel;
(c) add 10 mL 0.1% dithizone solution and 30 mL CHCl₃;
(d) stopper;
(e) shake 1 min;
(f) drain CHCl₃ layer;
(g) add ca 40 mL CHCl₃;
(h) extract;
(i) drain CHCl₃;
(j) repeat until CHCl₃ layer is colourless;
(k) transfer aqueous phase to a round-bottom flask;
(l) remove CHCl₃ from solution at ambient temperature in vacuo for 1 hour;
(m) dilute to 1000 mL with H₂O;
(n) store in a clean polypropylene bottle;
(o) solution may be stored indefinitely.

11. Ammonium iodide (NH₄I) solution, 3M.

(a) dissolve 218 g of reagent grade NH₄I in 250 mL H₂O;
(b) transfer to separatory funnel;
(c) adjust pH to 8-9 with conc NH₃ solution;
(d) extract with dithizone and CHCl₃;
(e) remove excess CHCl₃ as in Reagent 10;
(f) dilute to 500 mL with H₂O.

12. Stripping solution, saturated with MIBK and TPB, containing 5% H₂O₂, 0.5% HNO₃ and 0.25% NH₄H₂PO₄.

(a) add the following to a 2000 mL volumetric flask in the sequence given:

(b) take to ca 2000 mL with H₂O;
(c) stopper;
(d) shake ca 1 min;
(e) let phases separate;
(f) take aqueous phase to 2000 mL with H₂O;
(g) stopper;
(h) mix well, ca 3 min;
(i) after separation of phases, remove excess organic phase using a clean pipet connected to a water aspirator;
(j) store in a clean Pyrex or polypropylene bottle.

13. Lead stock solution, 500 ppm. (Solution A).

(a) transfer 0.5000 g of lead to 1000 mL volumetric flask;
(b) add ca 20 mL of H₂O;
(c) add 20 mL of conc HNO₃;
(d) let stand until complete dissolution;
(e) cool;
(f) take to volume with H₂O;
(g) mix well.

14. Cadmium stock solution, 500 ppm (Solution B).

(a) transfer 0.5000 g of cadmium to 1000 mL volumetric flask.
(b), (c), (d), (e), (f) and (g) as in Reagent 13.

15. Cadmium stock solution, 50 pm in 1% HNO₃ (Solution C).

(a) transfer 10.0 mL of Solution B to 100 mL volumetric flask;
(b) add 1 mL of conc HNO₃;
(c) take to volume with H₂O;
(d) mix well.

16. Combined Pb-Cd stock solution, 10.0 ppm Pb and 1.00 ppm Cd. (Solution D)

(a) add 0.5 mL of conc HNO₃ and 2.00 mL of Solutions A and C to a 100 mL volumetric flask;
(b) take to volume with H₂O;
(c) mix well.

17. Combined Pb-Cd stock solution, 1.00 ppm Pb and 0.100 ppm Cd. (Solution E)

(a) add 0.5 mL of conc HNO₃ and 10 mL of Solution D to a 100 mL volumetric flask;
(b) take to volume with H₂O;
(c) mix will.

18. Working standards, graphite-furnace determination.

(a) into 8 well cleaned 100 mL volumetric flasks add volumes of Solution D according to Table 1;
(b) prepare every 6 months;
(c) take volume to 100 mL with stripping solution (Reagent 12).

Table 1. Standards for graphite furnace determinations.

Standard Number	mL added Solution D	Cd concn. (ng/mL)	Pb concn. (ng/mL)
1	0.00	0	0
2	1.00	1	10
3	2.00	2	20
4	3.00	3	30
5	4.00	4	40
6	5.00	5	50
7	6.00	6	60
8	7.00	7	70
9	8.00	8	80
10	9.00	9	90

19. Working standards, flame determination.

(a) into seven 100 mL volumetric flasks, pipet 0.5 mL conc HNO₃, 10 mL 50% H₂O₂, ca 30 mL H₂O, 2.5 mL 10% NH₄H₂PO₄, and standards according to Table 2.

Table 2. Standards for flame AAS

Standard No.	Volume (mL) Pb Std added	Volume (mL) Cd Std added	Conc (µg/mL) Pb Std added	Conc (µg/mL) Cd Std added	Final Pb conc (µg/mL)	Final Cd conc (µg/mL)
1	-	-	-	-	0.0	0.00
2	SD 1.00	-	10.0	1.00	0.1	0.01
3	SD 5.00	-	10.0	1.00	0.5	0.05
4	SD 10.00	-	10.0	1.00	1.0	0.10
5	SA 1.00	SC 1.00	500	50	5.0	0.50
6	SA 2.00	SC 2.00	500	50	10.0	1.00
7	SA 4.00	SC 4.00	500	50	20.0	2.00

SA = Solution A; SC = Solution C; SD = Solution D.

(b) to each flask add 3 mL MIBK-TPB solution;
(c) take to volume;
(d) stopper;
(e) mix well for 5 min;
(f) let stand 5 min;
(g) remove stopper;
(h) take aqueous phase to 100 mL mark;
(i) stopper;
(j) mix well for 5 min;
(k) let stand 30 min;
(l) suction off excess organic phase;
(m) store in clean 100 mL polypropylene bottles.

Procedure: A. Sample digestion

1. Label 12-20 250 mL Erlenmeyer flasks.

2. Weigh to ±0.5 g.

3. Homogenize samples.

4. Accurately weigh to ±0.01 g amount of sample into flasks according to Table 3.

Table 3. Sample weight (fresh foods)

Foods	Sample size (g)
1. Ready-to-use juices, milks, formulas, drinks.	20 g
2. Concentrated milks and formulas, leafy vegetables, fruits, canned vegetables, soups and fruits, potatoes. (Most foods containing >60% moisture).	10 g
3. Cheese, cream, fats, oils, meat, fish, dry goods, tomato paste, puddings, bread, mushrooms, carrots. (Foods containing <50% H2O.	5 g (limit fats and oil content to 3 g)

5. Dry foods overnight in oven at 120-140°C.

6. Carry a minimum of 3 blanks through entire procedure.

7. Add 15 mL conc HNO₃ to flasks and heat without spattering, at gentle to moderate temperature, to dryness.

8. 80-100% charring of the sample is acceptable.

9. Do not evaporate blanks to less than 2-3 mL.

10. Repeat steps 7-9.

11. Add 15 mL conc HNO₃.

12. Heat gently to a volume of ca 3 mL.

13. Charring must be avoided at this stage.

14. Remove from heat.

15. Add 40 mL of 10M HCl to flasks.

16. Increase temperature of hot plates to moderate heat.

17. Evaporate solutions to a final weight of 20±l g using the flask weights in step 2.

18. Remove flasks from hot plate.

19. Pipet 1 mL 20% ascorbic acid into each.

20. Place flasks on hot plate.

21. Heat at or near boiling until solutions begin to darken (light brown).

22. At this point remove from heat.

23. If darkening does not occur, after 10 min boiling, see Note 3.

B. Extraction

1. Premeasure 125 mL acid-washed separatory funnels to a volume of ca 60 mL and mark.

2. Pipette 4.00 mL of 3M NH₄I into each separatory funnel. This should be done accurately and reproducibly since the majority of the lead blank is due to this reagent.

3. Transfer warm digest solution carefully to separatory funnel.

4. Wash digestion flask 3 times with 10-15 mL portions of H₂O.

5. Transfer washings to separatory funnel.

6. Let solutions cool.

7. Bring volume in separatory funnels to 60 mL mark with H₂O.

8. Add 30 mL of MIBK-TBP solution to each separatory funnel.

9. Stopper.

10. Shake for 1 min.

11. Allow phases to separate.

12. Discard bulk of aqueous layer.

13. After 10 min drain residue of aqueous layer.

14. Pipette 20 mL of stripping solution into each separatory funnel.

15. Extract by inverting repeatedly for 3 min.

16. Rigorous shaking should be avoided.

17. Rinse funnel tips with H₂O.

18. Allow layers to separate.

19. Remove stopper.

20. Drain off a few mL of aqueous phase to rinse stop cock and funnel tip.

21. Collect remainder of aqueous phase in clean, dry 30 mL polypropylene bottles for analysis.

C. Determinations by Flame AAS

1. Set up AA spectrometer with simultaneous background correction and air-acetylene burner according to manufacturer's instructions.

2. Set up burner and gas controls according to manufacturer's specifications.

3. Analyse standards, samples and again standards.

4. Aspirate water between each sample to avoid cross contamination.

5. Plot a calibration curve and calculate blank and sample solution concentrations from the curve, allowing for the standard blank.

6. Calculate Pb and Cd concentrations (µg/g) in the original foods according to the formula:

_µg/g = (20 [sample concn (µg/g) - sample blank concn (µg/g)])/ sample wt(g)

7. If sample solutions contain less than 0.1 µg/mL Pb or less than 0.01 µg/mL Cd, analyze using graphite-furnace AAS.

8. Set up graphite furnace and recorder according to manufacturer's instructions.

9. Make peak-height measurements, operating in continuous mode.

10. Operating parameters are listed in Table 4.

Table 4. Operating Parameters

Graphite tubes:	Not pyrolytically coated
Aliquot volume:	10 µL maximum for HGA-2100, HGA-2200, HGA-400, HGA-76B, HGA-500. *20 µL maximum for HGA-2000. * 5 µL maximum for CRA-90.
Gas:	Argon (99.9%)
Mode:	Automatic stop-flow during atomization step
Drying temperature:	Adjust so that sample dries in 8-10 seconds
Charring temperature and time:	650°C, 20 s
Atomizing temperature:	2200°C for cadmium and 2400°C for lead
Atomizing time:	5 s
Precision expectancy:	< 3% RSD for any 5 replicates at absorbance values between 0.007 and 0.030

* For the HGA-2000 and CRA-90, calibration curves require that the maximum concentration of working standards be limited to ½ those noted Reagent 16.

11. Wash pipet tip of micropipet or autosampler in 50% high-purity HNO₃.

12. Rinse with H₂O.

13. Fire furnace several times to remove contamination.

14. Determine the lead and cadmium blanks of the working standards.

15. This should be done when the standards are first prepared and will not be necessary until fresh standards are prepared and is done by analyzing the standard solutions in the following sequence:

Sequence	Sample	Replicates
1	Standard 1	5
2	Standard 3	4
3	Standard 4	4
4	Standard 1	5

16. Average the peak heights for each of the four sequence steps.

17. Calculate the standard blanks according to the following formulas. Square brackets ( ) denote nominal standard concentration and PKH denotes mean peak height. The number following PKH denotes the sequence step.

i. y_l = (PKHl + PKH4)/2

ii. x₁ = y_{1[standard 3]}/(2 (PKH2-y1)) + y_{1[standard 4]}/(2 (PKH3-y1))

x₁ is the value (ng/mL) of the blank for the element determined and should be added to the nominal value of all the working standards for all subsequent calculations.

18. Determine the validity of working standards by analysing the standards in the following sequence.

19. This should be done

(a) when standards are first prepared;
(b) whenever standard contamination is suspected;
(c) every 2 months.

Sequence	Sample	Replicates
1	Standard 9	3
2	Standard 5	4
3	Standard 1	2
4	Standard 2	4
5	Standard 3	4
6	Standard 4	3
7	Standard 5	3
8	Standard 6	3
9	Standard 7	3
10	Standard 8	3
11	Standard 9	3
12	Standard 5	4

20. Average the peak heights for each sequence step.

21. Plot mean peak height vs corrected, for blank, standard concentrations.

22. Visually assess validity of standards.

23. Analyze standards and samples using graphite furnace AAS in the following sequence:

Sequence	Sample	Replicates
1	Dilution Solution	4
2	Standard 3	2
3	B1	3
4	B2	3
5	B3	3
6	Standard 3	3
7	Standard 5	3
8	Standard 7	3
9	Standard 9	3
10 - 19	S1-S10	2
20	Standard 2	3
21	Standard 4	3
22	Standard 6	3
23	Standard 8	3
24 - 33	S11-S20	2
34	Standard 3	3
35	Standard 5	3
36	Standard 7	3
37	Standard 9	3
38 - 47 etc.

B1-B3 refer to the sample blanks.

24. Calculate the mean peak height for each sequence number.

25. Calculate the mean sample blank (ng/mL) according to the formula:

SB=((PKH3+PKH4+PKH5)/6)x(2[standard 3]/(PKH2+PKH6) +[standard 5]/PKH7)

26. Calculate the sample solution concentrations (SSC) (ng/mL) directly from the calibration curve. For samples S1-S10 the calibration curve consists of the eight standards which block the samples. The standard concentration values are corrected for the standard blank (steps C14-C17).

27. Calculate the concentrations (µg/g) for the original samples according to the formula:

___µg/g = (SSC-SB)/(50W)

Note 1: Plastic Labware. Use only polypropylene or polyethylene, not linear (LPE), bottles and caps. No coloured plastics should be used. Plastics should be cleaned by soaking in 5% HNO₃, saturated with MIBK for 48 hr. Rinse thoroughly with H₂O and dry.

Note 2: All glassware should be rinsed with conc HNO₃ followed by deionized H₂O prior to use. Wash and rinse pipets by hand.

Note 3: If no darkening occurs after 10 minutes of heating, it means traces of HNO₃ are still present in the solution. If the addition of another 1.00 mL ascorbic acid does not cause darkening upon reheating, discard the sample. This problem generally can be avoided by careful observance of the final volume in step 7.

Reference: 1. A.D. McKenzie and R.D. Dabeka, Food Research Division, Bureau of Chemical Safety

Table 5. Lead in NBS SRM's

Material	Sample Size (g)	No. of Replicates	Found (µg/g±S)	Certified (µg/g±2S)
Pine Needles	0.5 1 2 5	3 2 3 4	9.9±0.2 9.7±0.5 9.61±0.04 10.0±0.2	10.8±0.5
Orchard Leaves	0.5 1 2 5	2 3 2 4	40±1 45±4 40.1±0.08 43±2	44±3
Tomato Leaves	0.5 1 2 5	3 2 3 3	5.12±0.09 5.3±0.1 5.6±0.2 5.4±0.1	6.3±0.3
Spinach Leaves	0.5 1 2 5	2 3 2 4	1.04±0.01 1.09±0.05 0.97±0.04 1.19±0.04	1.2±0.2
Bovine Liver	1 5	2 3	0.32±0.03 0.30±0.02	0.34±0.08

Table 6. Lead in Laboratory Reference Materials

Material	Sample Size (g)	No. of Replicates	Found (µg/g±S)	Reference Value (µg/g)
Skim milk Powder	10	11	0.038±0.002	0.042±0.004
Freeze-dried beef muscle	2 5	3 4	0.16±0.01 0.16±0.01	0.16±0.03
Wheat flour	2 10	2 4	0.014±0.003 0.010±0.001	0.012±0.004
Freeze-dried beef kidney	1 5	3 3	1.90±0.02 1.85±0.02	2.2±0.2

Table 7. Recovery of Lead from Milk Products

#	Material	Sample Size (g)	No. of Replicates	Pb Level Found (ng/g±S)	Spike Level ng/g	Recovery (%±S)
1	Cheddar Cheese	10 10 10	3 3 3	35±9	100 500	72±2 82±2
2	Margarine	10 10 10	3 3 3	<3	100 500	67±7 68±3
3	Homogenized whole milk	50 50 50	3 3 3	0.9±0.08	10 40	101±6 95±3
4	Evaporated milk	50 50 50	3 3 3	59±9	10 40	115±6 83±7
5	Concentrated liquid infant formula, milk base, Brand A	50 50 50	4 3 4	26±6	10 40	107±10 80±15
6	Ready-to-use infant liquid formula, milk base, Brand A	10 10	3 7	56±2	80	97±5
7	Concentrated liquid infant formula, milk base with added iron, Brand B	10 10	3 3	55±9	60	102±15
8	Ready-to-use infant liquid formula, milk base, Brand B	10 10	3 3	47±9	60	108±6
9	Concentrated liquid infant formula, soya base, Brand A	10 10	3 3	38±1	60	102±4
10	Concentrated liquid infant formula, milk base, Brand A	10 10	3 3	22±0.5	60	108±5
11	Ready-to-use liquid infant formula, milk base, Brand B	10 10	2 2	52±0.4	60	101±2
12	Ready-to-use liquid infant formula, milk base, Brand A	10 10	3 3	58±1	60	101±11
13	Concentrated liquid infant formula, milk base with added iron, Brand A	10 10	3 3	15±3	60	82±5
14	Concentrated liquid infant formula, soya base, Brand B	10 10	2 3	27±0.7	60	88±9
15	Concentrated liquid infant formula, milk base, Brand B	10 10	3 3	60±2	60	99±1
16	Concentrated liquid infant formula, soya base, Brand A	10 10 10	3 3 3	25±4	50 50	92±3 101±4

Table 8. Determination of Cadmium in NBS SRM's

Material	Sample Size (g)	No. of Replicates	Found (µg/g S)	Certified (Hot Certified) (µg/g)
Orchard leaves	0.5 1 2 5	3 1 3 3	0.10±0.01 0.123±0.004 0.113±0.004 0.119±0.002	0.11±0.02
Bovine liver	1 5	3 3	0.259±0.008 0.30±0.01	0.27±0.04
Pine needles	0.5 2 5	3 3 3	0.018±0.002 0.016±0.006 0.017±0.008	(<0.5)

Table 9. Recovery of Cadmium from Milk Products

#	Material	Sample Size (g)	No. of Replicates	Cd Level Found (ng/g±S)	Spike Level (ng/g)	Recovery (%±S)
1	Cheddar Cheese	10 10 10	3 3 3	2.0±0.6	10 50	140±17 81±1
2	Margarine	10 10 10	3 3 3	0.13±0.04	10 50	83±6 82±5
3	Homogenized Whole Milk	50 50 50	2 3 2	0.10±0.003	1.0 4.0	124±12 85±6
4	Evaporated Milk	50 50 50	3 2 2	0.632±0.12	1.0 4.0	90.0±0.7 85.0±0.2
5	Concentrated liquid infant formula, milk base, Brand A	50 50 50	3 4 5	1.54±0.18	1.0 4.0	91±12 94±3
6	Ready-to-use infant formula, milk base, Brand A	10 10	3 4	0.43±0.15	8.0	96±9
7	Ready-to-use liquid infant formula, milk base, Brand B	50 50 50	3 4 3	0.42±0.02	1.0 4.0	95±10 97±12
8	Concentrated infant formula, milk base with added iron, Brand B	10 10	2 1	1.16±0.06	6.0	81.7
9	Ready-to-use liquid infant formula, milk base, Brand B	10 10	2 2	0.65±0.01	6.0	104±14
10	Concentrated liquid infant formula, soya base, Brand A	10 10	3 2	9.7±2.1	6.0 6.0	97±9
11	Concentrated liquid infant formula, milk base, Brand A	10	2 3	0.81±0.01	6.0	73±4
12	Ready-to-use liquid infant formula, milk base, Brand B	10 10	3 3	0.50±0.01	6.0	99±9
13	Ready-to-use liquid infant formula, milk base, Brand A	10 10	1 3	0.71	6.0 6.0	104±16
14	Concentrated liquid infant formula, milk base with added iron, Brand A	10 10	3 2	0.72±0.16	6.0	105±6
15	Concentrated liquid infant formula, soya base, Brand B	10 10	2 3	2.81±0.10	6.0	93±2
16	Concentrated liquid infant formula, milk base, Brand B	10 10	2 3	0.92±0.11	6.0	85±2
17	Concentrated liquid infant formula, soya base, Brand A	10 10 10	4 4 3	12.6±0.4	10 50	106±11 103±9

Table 10. Flame AAS Determination of Lead, Cadmium and Copper in NBS SRM's

Material	Element	Sample Size (g)	No. of Replicates	Noted or Certified* Level (µg/g±2s)	Found (µg/g±s)
Pine needles	Pb*	0.5 1 2 5	3 2 3 4	10.8±0.5	10.1±0.5 11.6±0.4 10.1±0.3 10.0±0.4
	Cd	0.5 1 2 5	2 2 3 3	<0.5	0.22±0.03 0.27±0.04 0.20±0.02 0.193±0.007
	Cu*	0.5 1 2 5	2 3 3 4	3.0±0.3	2.2±0.3 2.7±0.6 2.68±0.06 2.7±0.1
Orchard leaves	Pb*	0.5 1 2 5	3 3 2 3	45±3	43±6 43±3 44±4 43±1
	Cd*	0.5 1 2 5	3 3 3 4	0.11±0.02	N.D. 0.11±0.01 0.13±0.02 0.090±0.001
	Cu*	0.5 1 2 5	3 3 3 4	12±1	11.6±0.8 11±1 12±1 11.7±0.4
Tomato leaves	Pb*	0.5 1 2 5	3 3 3 3	6.3±0.3	5.43±0.07 5.43±0.07 5.1±0.3 5.75±0.05
	Cd	0.5 1 2 5	3 3 3 4	3.0	2.69±0.06 2.53±0.05 2.61±0.07 2.60±0.07
	Cu*	0.5 1 2 5	3 3 3 4	11±1	10.5±0.3 9.1±0.1 9.94±0.06 9.5±0.3
Spinach	Pb*	0.5 1 2 5	3 3 3 4	1.2±0.2	2.9±0.3 0.5±0.1 1.6±0.2 1.24±0.07
	Cd	0.5 1 2 5	3 3 3 3	1.5	1.4±0.3 1.39±0.04 1.4±0.3 1.47±0.08
	Cu*	0.5 1 2 5	3 3 3 4	12±2	11.8±0.2 10.9±0.4 11.5±1.0 11.5±0.3