Identification of Irradiated Foodstuffs: Results of a European Test Intercomparison

J. RAFFI,” J-J. BELLIARDO,‘J-P. AGNEL’ and P. VINCENT’

‘DPVE/SIV, CEN Cadarache, BP N”1, 13108 Saint-Paul-lez-Durance, France

‘BCR, CCE, 200 rue de la Loi, 1049 Brussels, Belgium

The results of an intercomparison, organized by the Community Bureau of Reference (Commission of the European Communities), on the use of Electron Spin Resonance spectroscopy for the identification of irradiated food are presented. A qualitative intercomparison was carried out using beef and trout bones, sardine scales, pistachio nut shells, dried grapes and papaya. Protocols are proposed for meat bones, fish bones (with some restrictions) and fruits such as dried grapes and papaya. The protocol for pistachio nuts and fruits such strawberries is more complicated and further research is needed prior the organization of future intercomparisons. A quantitative intercomparison on poultry bones was also organized. Laboratories were able to distinguish between chicken bones irradiated at 1 to 3 kGy or 7 to 10 kGy.

KEYWORDS: Electron Spin Resonance, European intercomparison, food, radiation.

INTRODUCTION

The irradiation of food by X- and gamma-rays and by electron beam has been introduced recently as a new technological process to reduce food losses and to improve the hygienic quality of foodstuffs. In 1980, a Joint Food and Agriculture (FAO)/International Atomic Energy Agency (IAEA)/World Health Organization (WHO) Expert Committee meeting (FAO/IAEA/WHO, 1981) concluded that “the irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard; hence, toxicological testing of foods so treated is no longer required.” As a consequence, the radiation treatment of a number of foods is now legally accepted in several countries, although it is still prohibited in others. To facilitate trade of irradiated foods regulatory authorities in all countries are interested in having simple and reliable methods to detect foods treated by irradiation (FAO/IAEA, 1989) and, consequently, to check on compliance with labelling regulations. Several methods for the detection of irradiated foods, including Electron Spin Resonance (ESR), have been discussed previously (Bog1 et al., 1988; Raffi and Belliardo, 1991) and these led to the first collaborative ESR trial (Desrosiers et al., 1990).

The Commission of the European Communities (CEC) is interested in the legislative aspects of food irradiation. Inside the CEC, the Community Bureau of Reference (BCR) has for its main objective the improvement of measurements when differences in results create problems of trade and make obstacles to the implementation of the Single European Market. Consequently, a BCR cooperative project was initiated to deal with the identification of irradiated food. After two meetings organized in Brussels (2 June and 3-4 October 1989) it was proposed to, 1) hold a general meeting in order to examine other potential new methods of detection of irradiated food (Rafft and Belliardo, 1991), 2) develop a concerted action in this field and, 3) carry out two large intercomparisons, at European level, on the use of Electron Spin Resonance (this document and Rafti, 1991) and Thermo-

407

408 ESR dosimetry and applications

luminescence to identify irradiated foodstuffs. These two techniques were judged to be the most promising methods at present.

ESR is a spectroscopic method to detect unpaired electrons typically derived from, ions from transition elements [which often lead to signals present in a reference sample, as the action of many enzymes involves such ions] and radicals, including those induced by irradiation. ESR can by used as an identification test (Dodd ef al., 1988; Rafft ef al., 1989) if the radicals are stable during commercial storage of the food. This only occurs in the solid and dry components of the food, where the reactivity of the radicals with each other is low. Moreover, the radiation-induced signals must be clearly distinguishable from those of a reference sample. During the meeting held in Brussels in October 1989, it was generally considered by the participants that the theoretical studies on bones and fruits were sufficiently advanced to enable a qualitative and a quantitative test to be carried out on these products.

Meat and Fish. Studying the ESR signals induced in bones is not new and details about the recording conditions are not dealt with here as these have been reported elsewhere (Desrosiers and Simic, 1988; Dodd et al.. 1988; Lea et al., 1988; Gray and Stevenson, 1989; Rafft et al., 1988, 1989; Stevenson and Gray, 1989a, b; Desrosiers cf al., 1990). In the unirradiated samples, there is a symmetric absorption with a g-factor - 2.0044 and a peak-to-peak linewidth (AH) of - 0.6 mT which is due to radicals in the marrow (Raffi er al., 1989); in irradiated samples, there is an asymmetric absorption characterized by

g1 - 2.0030 - 2.0033 and g1 - 1.9969 - 1.9975 which is due to hydroxyapatite. Since the signal height is linearly proportional to the dose (up to 14 kGy for frog legs) and quite stable in meat bones, it is possible to obtain an estimate of the dose by “standard addition” technique. The sample is re-irradiated several times and its ESR signal recorded after every reirradiation (Desrosiers et al., 1990). We chose chicken bones for the quantitative intercomparison, because poultry are likely to be irradiated to enhance the safety of the product by reduction of bacterial contamination. For the qualitative intercomparison, we chose beef and two fish species where the lifetime of their radiation-induced ESR signal is well known (Raffi ef al., 1989).

The radicals induced in the fruit pulp are not stable because the water content of fruit is generally high, but ESR can be used in dried fruit or in the dry components such as achenes, pips or stones (Dodd et al., 1988; Desrosiers and McLaughlin, 1989; Goodman ef al., 1989; Raff and Agnel, 1989a). Different responses are observed, depending on the fruit: in type 1 (papaya, dried grapes), and ESR multicomponent signal is radiation-induced, but the unirradiated fruit presents no ESR signal or a single line. In type 2 fruits (pistachio nut, berries), a triplet is induced (Rafft et uf., 1988). But a six-line signal due to Mn” and a central single line may also be present both in irradiated and non-irradiated sample.

During storage, the ESR signal may have different decay rates depending on the species. Thus we carried out kinetic experiments (Table 1) before fruit selection, i.e., papaya and dried grapes (type 1) and pistachio nuts (type 2).

ESR dosimetry and applications 409

Table 1. Kinetic study of the ESR signal of fruit products.

PRODUCT

onion

reference

(a)

B

irradiated

(a)

CC’

Maximum Observable Time (days)

1.5 kGy 8 kGy

- 60 0~) - 150

apricot 1 B 1 m, CC’ 1 < 17 1 - 90

dried grapes B m > 530 (b) > 692 (c)

pistachio nuts

french prunes

B CC’ - 388 > 687

B CC’ > 687

banana 1 B 1 m I > 631 I > 687

papaya

dates

B

B

m

m

> 631

> 690 (d)

> 687

> 687

coconuts I B I m, CC’ I I > 687

figs B CC’ < 50 - 685

(a) type of signal: no signal (o), signal central line (B), cellulose triplet (CC’) or multicomponent signal (m) (b) dose of only 0.5 kGy (c) same result for a dose of 4 kGy (d) irradiation in commercial conditions, i.e., 0.7-0.9 kGy

RESULTS AND DISCUSSION

This ESR intercomparison (Raffl, 1991) involved 22 European laboratories and was carried out as follows: preliminary study (November 1989-April 1990), mailing and recording of results (June-September 1990), first statistical analysis (October-November 1990), preliminary report (December 1990-January 1991), discussion and technical evaluation of results with participants (Cadarache, 19 February 1991), final statistical analysis and report (March-July 1991).

Six samples of each product were sent to each of the 21 laboratories involved in the qualitative test. The samples were irradiated at one of the six different doses (‘0’, 0.5, 1, 2, 4 and 7 kGy); the dose is not known to the participants. The 15 laboratories involved in the quantitative test received 6 samples of each of the three chosen doses (0.3, 3, 7 kGy). All the data are published in a BCR document (Raffi, 1991) and only the main results of the qualitative intercomparison are summarized here, in Table 2.

When the experimental protocol introduces numerous, but only small and independent errors, data are expected to follow a Gaussian distribution. The integral of such a distribution gives the probability of obtaining a result greater than or equal to a given value. Statistical tests presented below used 21 measurements per dose value, i.e., 21x6= 126 per foodstuff, and were run with the ‘VOYONS’ program (TX&y, 1985). Thus, a probability of 50% represents a 50% success rate for detection and the dose associated with this level is defined as ‘DoseSO’. Similarly the probability of 84.1% represents the success rate at ‘Dose84’ plus one standard deviation of the distribution. The ratio Dose84/Dose50 is a measurement of the spread on the distribution of the ‘difficulty ratio.’

410 ESR dosimetry and applications

Table 2. Results of the qualitative test (in percentage of correct answers).

Papaya 100 90.9 100 100 100 100 0.35 1.3

Pistachio 95.2 12.5 70.5 60 79.5 85.7 0.19 35

Qualitative test on meat bones. The results (Table 2) are very good. There was only one error in the 126 samples examined thus giving 95% correct identifications. The shape of spectra were unchanged after 220 days, even though a small loss with time occurred when bone was stored with flesh still attached. Moreover the preliminary work of Desrosiers et al. (1990) allows the extrapolation of these results to other sources of bone, e.g., pork, chicken and frog. Consequently, we can now propose a final protocol (Appendix IA).

Quantitative test on noultrv bones. Considering the results of each individual laboratory, there is no ambiguity in distinguishing between a dose of 3 kGy (upper limit of the dose range l-3 kGy) and a dose of 7 kGy (lower limit of the dose range 7-10 kGy). Consequently, each laboratory can at least deduce the technological purpose of the irradiation. However, despite the dosimetric corrections, there is a slight overlap between the results of the different laboratories, a fact which is not explained. But, taking the average of 8 measurements, there should be no problem, for a specialist laboratory, to ascertain the technological purpose of the irradiation; thus, we can propose a protocol for a quantitative test (Appendix IB).

Qualitative test on fish species. The results (Table 2) are less conclusive in the case of fish. But for fish, data fitting gives values of the minimum detectable doses (0.56 and 0.77 kGy respectively). Low dose irradiation of fish may be not too difficult to ascertain. The ESR signals induced in fish bones were stable for at least 2.5 months. It was clear that, for commercial products where bone may not be present, it may be necessary to record the spectra from scales or teeth (Raffi et al., 1989). The use of these additional components should also improve the reliability of the results. The final protocol (Appendix IA) must include a list of fish species for which information is available on the stability of the ‘characteristic’ ESR signal.

Qualitative test on dried draDes and nanava (fruits of ivne 1). The results for dried grapes (raisins) and papaya are conclusive (Table 2). The minimum detectable doses are low: 0.12 kGy for grapes and 0.35 for papaya, which guarantees a high success rate for irradiation detection. The papaya signal seems quite characteristic (very low Dose84/Dose50 ratio: 1.3). The multi-line spectra allows a high level of correct identifications, with a very simple protocol (Appendix II) but other fruits need to be studied before these results can be extrapolated to them.

Oualitative test on nistachio nuts (fruits of tvDe 2). The results for pistachio nuts are more complicated. They did not improve with increasing doses. Moreover, some of the laboratories had a high success rate while others achieved minimal success, which explains a minimum detectable dose for pistachio nut relatively low (0.19 kGy) with a large difficulty ratio (Dose84/Dose50 = 35). A detailed protocol would be necessary in this case to identify the characteristic signal which is masked by the steep sides of a large spectral line. Let us remember that pistachio nuts were chosen as a model for fresh fruit (berry achenes, Raft? and Agnel,

ESR dosimetry and applications 411

1989). The changes in spectral shape, that is two satellite lines, on irradiation are small and very dependent on instrument variables. Before the organization of an ESR intercomparison on fresh fruits, further research and training of participants may be necessary.

CONCLUSIONS

Generally, there was no evidence of a link between the results of each laboratory and their spectrometers (company, magnet size, etc) or, for the quantitative test, their irradiation facility (gamma cell or electron beam, dose rates, etc.). It should be noted that the surface state contamination of the ESR cavity may have slightly influenced the accuracy of the results especially for low doses. Finally it is concluded that:

a) the protocol for meat bones, fish bones, scales or teeth is definitive. But a list of fish species must be added, indicating the maximum lifetime of the ESR signal in relation with the irradiation dose and irradiation and storage temperatures;

b) an estimate of the dose range (1-3 or 7-10 kGy) can be reached by the post-irradiation method, in case of meat bones;

c) the protocol for fruit may be improved. A list of fruits must also be added, as above, indicating the type (1 or 2) of the ESR signal to search for;

d) further research must be carried out to improve the estimate of the dose delivered to bones and to determine the parameters influencing the ESR signal radiation-induced in fruit of type 2;

e) systematic studies of the ESR signal lifetime must be carried out on the different fish and fruit species and further research have to be done on other foodstuffs such as shell fish.

ACKNOWLEDGEMENTS

This work was sponsored by the Community Bureau of Reference of the Commission of the European Communities [Agreement 5348/1/5/340/90/4_BCR F(lO)]. We also thank all the participants [listed in Rafft, 19911 involved in sample preparation, recording of spectra and interpretation of results, and especially Dr. J.M. Thiery (DPVE, Cadarache, France) for the statistical analysis of test results, and Dr. M.H. Stevenson (Queen’s University, Belfast, UK), Dr. M. Kent (MAFF, Aberdeen, UK) for ESR discussions.

Appendix I: Protocol for ESR identification test of irradiated meat and fish.

A - QUALITATIVE TEST Samnling: take fragments of bones, scales or teeth; clean them (if possible, remove the marrow inside the bone) and dry them on a filter paper. Put into an ESR tube. Recordinp: with a 9 GHz spectrometer, set the medium field at 350 mT (3500 G) with a scan range of It 10 mT (100 G), a modulation frequency of 100 kHz, a modulation amplitude of 0.20 mT (2 G) and a microwave power of l-l.5 mW. j&& the unirradiated bones present no signal or a small symmetric absorption with a g-factor -2.044 and a peak-to-peak width of -0.6 mT (6 G); the irradiated bones present in addition an asymmetric signal characterized by g, -2.003 and gt - 1.997, and “two” peak-to-peak widths of - 0.35 and 1 mT (3.5 and 10 G).

B - OUANTITATIVE TEST The spectrum must be recorded after noting precisely the position of the bone in the tube. Then, give to the bone an additional dose of 1 kGy, and record the new ESR spectrum, the bone being placed in the same position in the tube. Repeat the experiment four times. Extrapolate the initial delivered dose.

C - APPLIED TO - All meat containing bones and teeth - fish bones, scales and teeth for which the lifetime is longer than the commercial lifetime of the foodstuff (list to be drawn with regard to irradiation and storage parameters)

412 ESR dosimetry and applications

Annendix II: 9. Samnling take about 100 mg of seeds, pips or fragments of stones or of dried fruits; if necessary, wipe them on filter paper and place them in a standard ESR tube. Close the tube. Recording: with a 9 GHz spectrometer, set the medium field at 349mT (3490 G) with a scan range of f5 mT (50 G), a modulation frequency of 100 kHz, a modulation amplitude around 0.1-0.2 mT (1-2 G) and a microwave power of 2.0 mW. Test: if there is a multi-component signal, the fruit (type 1) has been irradiated. If there is no signal or only a single line, you must record another spectrum, very carefully, saturating this central line and using a modulation amplitude of 0.4 to 1 mT (4-10 G). If there is a peak or a shoulder at 3 mT (30 G) on the left of the central line, the food has been irradiated (type 2); note that, generally, a weaker line may be observed on the right. If you do not see the left line, you have to vary the modulation amplitude in order to be sure not to miss it.

A list must be added indicating for each fruit, the type of ESR signal (1 or 2) and its lifetime with regard to the commercial lifetime of the foodstuff and its irradiation and storage parameters.

REFERENCES

Bog1 K., Regulla D. and Suess M. (1988) Health Impact Identification, and Dosimetry of Irradiated Foods, MMV Medizin Verlag, Munchen.

Desrosiers M. and McLaughlin W. (1989) Examination of gamma-irradiated fruits and vegetables by ESR spectroscopy. Radiat. Phys. Chem., 34, 895-898.

Desrosiers M., McLaughlin W., Sheahen L., Dodd N., Lea J., Evans J.C., Rowlands C., Raffi J. and Agnel J.-P. (1990) Co-trial on identification and estimates of gamma-ray and electron absorbed doses given to meat and bones. Int. J. Food Sci. Tech., 25, 682-69 1.

Desrosiers M. and Simic M. (1988) Post-irradiation dosimetry of meat by ESR spectroscopy of bones. J. Agric. Food Chem., 36, 601-603.

Dodd N., Lea J. and Swallow A.J. (1988) ESR detection of irradiated food. Nature, 334, 387. FAO/IAEA/WHO (1981) Wholesomeness of Irradiated Food, WHO, Geneva, No. 659. Goodman B.A., McPhail D.B. and Duthie D. (1989) ESR of some irradiated foodstuffs. J. Sci. Food

Agric., 47, 101-111. Gray R. and Stevenson M.H. (1989) Detection of irradiated deboned turkey meat using ESR

spectroscopy. Radiat. Phys. Chem., 34, 899-902. Lea J., Dodd N. and Swallow A.J. (1988) A method of testing for irradiation of poultry. Int. J. Food

Sci. Tech., 23, 625-632. Raffi J. (1991) Electron Spin Resonance Intercomparison Studies on Irradiated Foodstuffs.

Proceedings of a BCR Workshop, CEC Luxembourg, EUR No. 13630. Raffi J. and Agnel J.-P. (1989) ESR Identification of Irradiated Fruits. Radiat. Phys. Chem., 34,

891-894. Raffi J., Agnel J.-P., Buscarlet L. and Martin C. (1988) ESR Identification of Irradiated

Strawberries. J. Chem. Sot. Faraday Trans. I, 84, 3359-3362. Raffi J. and Belliardo J.-J. (1991) Potential New Methods of Detection of Irradiated Food.

Proceedings of a BCR Workshop, CEC, Luxembourg, EUR No. 1333 1. Raffi J., Evans J.C., Agnel J.-P., Rowlands C., Lesgards G. (1989) ESR Analysis of Irradiated Frog

Legs and Fishes. Appl. Radiat. Isot., 40, 1215-1218. ThiCry J.M. (1985) ‘VOYONS’, programme de simulations conversationnelles en Physico-chimie et en

Agronomie, in Logicielspour la Chimie, C6me G.-M, Ducloy J., Soulie E. and Thiery J.M. Eds., Sot Chimie (Paris) and Assoc. Nat. Logiciels (CNRS, Nancy), 156-157.