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1st - 5th July 2013
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Alliance of Cocoa
Producing Countries

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Proposed Methodology to Determine Source and Level of Lead Contamination in Cocoa

Background

One of the items examined at the Twenty-fourth Session of CODEX ALIMENTARIUS COMMISSION in Geneva, Switzerland in July n2001 was the Draft Revised Standard at Step 8 for Cocoa Butter (ALINORMO1/14, Appendix IV). The proposals were to reduced the levels of contaminants of lead (PB) from 0.5mg/kg to 0.1mg/kg for cocoa butter and 2mg/kg to 1mg/kg for cocoa mass and cocoa powders.

Earlier, in discussions at the Eighteenth Session of the CODEX COMMITTEE ON COCOA, in Fribourg, Switzerland, November 2002, Cocoa Producers were in full agreement as to the human health hazard of lead contaminants. However, some questions were raised, these were:

  1. in the absence of scientific data, on precise levels of lead that may result to exposure to risk, reduction in values may appear to be arbitrary;
  2. Varying levels of lead (that could be as high as 1 mg/kg Contamination) have been identified. Are these associated to specific geographical origin of the cocoa bean?
  3. While the possible source of lead that contributes to residue build-up in the bean have not been determined, a search for sources should not be only restricted to soil or chemical inputs used on the farm.

At the Meeting of the Scientific Research Committee of the Cocoa Producers' Alliance, in Accra, Ghana I March 2002, the matter of lead contaminants was added to the agenda and gave rise to debate. It was generally agreed that in order to attempt at obtaining a reduction in level in cocoa, the source that is the cause of this contaminant has to be first identified.

It was also agreed that just as much as soil or farm chemical inputs were pointed as a possible source, could also exist. Sources of contaminants could result from exposure/introductions after the farm-gate. Therefore, one has to examine the whole process during which cocoa beans are exposed, up to the manufacture of the final products. These could also include sources in warehouses at origin and at destination, in the holds of ships and in the actual grinding process, and if one considers the final product, other products that are added, also require investigation. The period factor - duration of exposure to contribute to elevate levels.

Possible origin of Pb contaminations

Therefore, the proposals of reducing levels of lead residues in cocoa butter, cocoa mass and cocoa powders would require that in the first instance, the source of lead that contributes to contamination, be determined. This would also encompass possible origins such as:

  1. juvenile geographic distribution - planting origin of cocoa in respect to soil (lead is ubiquitous in soil and rocks - average concentrations could be 16 ppm in the lithospere and 15 to 25 ppm in soil);
  2. introduced lead contamination - resulting from Pb containing pesticides in farm practices, example: lead arsenate or fertilizers that may have trace 'contaminants' of heavy metals;
  3. atmospheric deposition of anthropogenic Pb - released in burning of fossil fuels, example: in gasoline from internal combustion engines, with greater contamination risk to beans where the practice of cocoa drying along road sides is common;
  4. enclosed areas - such as warehouses, holds of ships were Pb-base paints are used;
  5. grinding/manufacturing process - may be experience wear and tear of machinery/part;
  6. mixtures/additions to the final product - sugar, milk, nuts fruit and others;
  7. packing/wrapping material - 'silver paper ' and others.

Methodology

Quantitation:
In literature *, numerous techniques are described for analysis of Pb. In the context of a determined protocol, to ensure standardization of single method it would be necessary to ensure that the basic equipment required is available in the various research institutes that would participate in the study. Alternatively, if different techniques are used then the precision between techniques would have to be determined using one specific sample, to then allow for a factor of concentration or use a lead standard (dissolve 0.100 g of 99.9% Pb metal in 1:1nitric (HN03), dilute to 1litre to give 100mg of Pb/litre)

Techniques that might be applied include X-ray fluorescence, emission spectrometry, photoelectron spectrometry, atomic absorption spectrometry colorimetry, nuclear activation, mass spectrometry and electrochemistry. Techniques may vary in sensitive or may have some limitations on certain samples. However, it is very possibly equipment for atomic absorption spectrometry would be available in analytic laboratories in different cocoa growing zones, in different countries thus allow for ample studies to be undertaken. Accepting that limitations also occur in atomic absorption overcome this by modifying the process.

Sampling:

  • On farms

    Soil
    Soil type: Representing soil in the major part of countries' production - possibly three or more types.
    Soil depths: starting at soil surface and two sampling depth up to 20 cm;
    One depth sample at C-horizon top
    Plant tissue: Roots
    Leaves
    Mature pods - husk and mucilage.

    Cocoa beans
    Before fermentation
    After fermentation
    After drying
    husk
    cotyledon

  • On-farm stores (at country of origin)

    Beans at monthly intervals

    Other areas of analysis - inputs on farms -
    Fertilizers - all types in use.
    Lime - all types in use.
    Insecticides - all types in use.
    Fungicides - all types in use.

  • After farm-gate to manufacture
    Warehouses in country of origin - beans, at monthly intervals and just before embark.

    Ship holds - beans, soon after disembark.

    Warehouses (in country of destination)
    - beans, at monthly intervals
    - husks, at least once
    - cotyledon, at least once

    After grinding
    - cocoa mass
    - cocoa butter
    - cocoa cake
    - cocoa powers

  • Final products
    - dry mixtures of cocoa and sugars
    - other components in the final product
    - wrapping ('silver paper', etc.)

Solutions, Equipment, Procedure (see Annex 1, Annex 2 and Annex 3)

Final observations

Except, for proposing the use of atomic absorption spectrometry and a comprehensive sampling procedure to cover possible determinants of lead contaminants, adjustments will have to be made in many aspects of the process. New grounds will be covered as the study will call for extractions over a wide range of sources, and this, in itself, may require development of new analytic methods.

Therefore, the proposal at this stage cannot be considered as a protocol. However, there is a need to have a pre-project meeting aimed at forming a specialized group to prepare acceptable standards that can be adhered too by all participates of the study. Therefore, funding will be need for a pre-project Formulation Meeting. After an agreement on the protocol has been reached, a project would be formulated together with necessary financial budgeting required for its implementation.


Annex 1

Solutions

  1. Nitric acid (HNO3), concentrated
  2. Nitric acid (HNO3), 1%, 1:100 (vol/vol) HNO3 /H2O
  3. Perchloric acid (HCIO4), concentrated.
  4. Hydrochloric acid (HCI), concentrated.
  5. Hydrochloric acid (HCI), 1:5 (vol/vol) HCI3 /H2O.
  6. Ammonium hydroxide (NH4OH), concentrated,
  7. Ammonium hydroxide (NH4OH), 1:3 (vol/vol) HH4OH/H2O.
  8. Citric acid buffer: Weigh out 200 g of citric acid, and dissolve in 500 ml of distilled water. Add concentrated NH4OH to pH 5.0 as measured by glass electrode potentiometry. Dilute to 1 liter.
  9. Chelating mixture: Make a solution that is 1% (wt/vol) in ammonium prryolidine dithiocarbanmate (APDC) and 1% (wt/vol) in diethylammonium diethyldithiocarbanmate (DDDC). Make fresh daily.
  10. Indicator: to 100 mg of bromophenol blue, add 1.5 ml of 0.1M sodium hydroxide (NaOH), and, make up to 100 ml.
  11. Aceylacetone (2,4-pentanedione).
  12. Chloroform (CHCI3), or trichloramethane.
  13. Methylisobutyketone (4-methyl 2-pentanone, or MIBK): Transfer 50 ml of citrate buffer into a large separatory funnel. Add 7.3 ml of cone hydrochloric acid (HCl) and 250 ml of distilled water. (The pH should be 3.0 to 3.2). Add 1 liter of MIBK. Shake for 30 sec. Allow to stand, and then discard the aqueous phase. Filter the water-saturated MIBK through acid-washed filter paper.
  14. Lead standard: Dissolve 0.100 g of 99% Pb metal in 1:1 acid (HNO3). Dilute to 1 liter to give 100 mg of Pb/liter.

Annex 2

Equipment

  1. Electric hotplate.
  2. Heating blocks: Aluminum blocks are machined from A1 stock into 11 cm diam cylindrical shapes. Four cavities 3.7 cm in diam are drilled into the top of the cylinder to accept the glass test tubes. Two blocks heights are required. The short block is 7.5 cm high, and the test tube cavities are drilled to a 13 cm depth. A thermometer well is drilled in the center to the same depth as the test tube cavities.
  3. Test tubes: Rimless test tubes 3.5 cm o.d and 20 cm in length are constructed form standard wall borosilicate glass.
  4. Funnels: Small funnels (3.5 cm rim by 6000 are inserted in the test tubes to condense acid vapors during digestion. It is advisable to cut away most of the stem of standard short-stem funnels. Larger funnels for filtering should be matched to the size of the filter paper.
  5. Filter paper, Whatman no. 41 or equivalent.

Annex 3

Procedure

  1. Weigh 2.0 g of sample (crushed to pass through a 2 mm sieve) into a 20 by 3.5 cm test tube.
  2. Add 10 ml of NHO3, and insert a funnel condenser. Heat in a low block at 80 to 90° C overnight.
  3. Transfer tube and funnel to a tall block, and heat at 125 to 130°C to dryness.
  4. Cool. Add 1 ml of HNO3 and 4 ml of HCO4.
  5. Heat at 200 to 210°C to dryness with funnel condenser in place in tall block.
  6. Cool. Add 4 ml of HCl and 50 ml of distilled water. Heat to 70°C for 1 hour.
  7. Quantitatively transfer tube contents through retentive. Acid-washed filter paper receiving the filter in a 100 ml volumetric flask. Rinse the tube and filter residue with 1% HNO3. Make up to volume with 1% HNO3
  8. Transfer 50.0 ml of the filtered, diluted digest to a 250 ml seperatory funnel.
  9. Add 10 ml of citric acid and 10 drops of indicator solution.
  10. If the mixture is blue, add 1:5 HCl just to fading blue. If it is yellow, add 1:3 NH4OH to first detectable blue color: then 1:5 HCl to the fading of blue. The pH should be 3.0 to 3.2.
  11. Add 10 ml of acetyl acetone. Mix.
  12. Add 10 ml of CHCl3. Shake for 1 min. Allow to settle. Tap off and discard the CHCl3 layer.
  13. Repeat steps 11 and 12 three times.
  14. Add 5 ml of chelating solution and 5 ml water-saturated MIBK.
  15. Shake for 30 sec. Let stand for 1 hour. Discard aqueous layer.
  16. Filter MIBK layer through acid-washed filter paper.
  17. Prepare standards by transferring into seperatory funnels, 0, 0.2, 0.4, 0.7 and 1.0 ml of a 100 ppm PB solution. To each funnel, add 2 ml of HCl and 50 ml of 1% HNO3. Threat according to the above protocol beginning at step 9.
  18. Aspirate samples and standards into a convenient air-acetylene flame of an atomic absorption spectrometer.
Source: Copyright © ASA-SSSA, 677s, Segoe Road, Madison, WI 53711, USA Sources of Soil Analysis, Part 2. Chemical and Microbiological Properties - Agronomy Monograph no. 9 2nd Edition).
 

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