Radiat. Phys. Chem. Vol. 35, Nos 1-3, pp. 284--287, 1990 Int. J. Radiat. Appl. lnstrum., Part C Printed in Great Britain

0146-5724/90 $3.00+ 0.00 Pergamon Press plc

THE EFFECT OF IRRADIATION DOSE AND AGE OF BIRD ON THE ESR SIGNAL IN IRRADIATED CHICKEN DRUMSTICKS

Richard Gray I M Hilary Stevenson 1'3 and David J Kilpatrick 2'3

iFood and Agrlc~itural Chemistry Research Division, Biometrics Division,

Department of Agrlculture for Northern Ireland and

3The Queen's University of Belfast, Newforge Lane, Belfast BT9 5PX.

ABSTRACT

Groups of 20 broiler chickens of the same genetic strain and reared under identical conditions were slaughtered at either 4, 5, 6, 7 or 8 weeks of age. Fairs of drumsticks were removed from each bird and groups were either not irradiated or irradiated at 2.5, 5.0, 7.5 or i0.0 kGy using a cobalt 60 source. Bone samples were excised, fragmented, freeze dried and ground prior to the determination of free radical concentration using electron spin resonance (ESR) spectroscopy. Increasing irradiation dose gave a highly significant increase in free radical concentration whilst for each irradiation dose, bones from younger birds gave significantly lower concentrations compared to those for older birds. Crystallinity coefficient increased linearly with age of bird and this may account in part for the increased signal observed as the birds aged.

KEYWORDS

Irradiation; ESR; chicken; age; bone.

INTRODUCTION

Recent research has demonstrated that electron spin resonance (ESR) spectroscopy has potential for the qualitative detection of irradiated food containing bone (Doddet al., 1985; Desrosiers and Simic, 1988; Dodd et al., 1988; Lea et al., 1988; Stevenson and Gray, 1989 a, b). However, little work has been published which attempts to quantify the dose received by the food product. The magnitude of the signal produced has been shown to be linearly related to the irradiation dose (Stevenson and Gray, 1989 a, b) but in order to establish a quantitative relationship between ESR signal strength and irradiation dose all relevant secondary factors affecting this relationship must be investigated. For example, Gray and Stevenson (1989) found that post-lrradiation cooking of chicken carcasses reduced the free radical concentration of the excised bones by approximately 20%.

Another factor which has been shown to affect the ESR signal of irradiated bone is its crystallinlty (Ostrowskl and Dzledzic-Goclawska, (1976). According to these workers, the measurement of crystalllnlty evaluates the proportion of the crystalline fraction of tissue mineral to the total amount of mineral. The crystallinlty coefficient can be determined by calculating the ratio of the concentration of stable free radicals induced by a saturating dose of ionizing radiation to the ash content of the tissue (Ostrowski et al., 1974). As the bird matures, crystallinity of bone may change and this present investigation was designed to study the effect of age of bird on the ESR signal strength of irradiated chicken drumsticks (tibia).

MATERIALS AND METHODS

One hundred broiler chickens from the same genetic strain were reared under identical, commercial conditions. Day-old chickens were introduced to the experiment at weekly intervals so that at the time of slaughter, groups of 20 birds were available which were either 4, 5, 6, 7 or 8 weeks of age. The average weights at slaughter were 1.08, 1.55, 1.92, 2.37 and 2.59 kg respectively. For each age of bird, four samples were selected at random and irradiated at either 2.5, 5.0, 7.5 or I0.0 kGy (dose rate = 0.74 kGy/h) or used as non-irradiated controls. Fairs of drumsticks were removed from each carcass, wrapped in cling film and stored at 5OC for 36 h prior to irradiation. The samples were prepared for

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7th International Meeting on Radiation Processing 285

irradiation and dosimetry was measured according to the procedure described by Stevenson and Gray (1989 b). Cobalt 60 was used as the source of ionizing radiation (Gamma-Beam 650, Nordion International Inc., Kanata, Ontario, Canada) and the environmental temperature was maintained at 16°C during irradiation. After irradiation, the pairs of drumsticks were prepared fOE analysls as detailed by Stevenson and Gray (1989 b).

ESR spectroscopy w~.performed using a Bruker ESP 300 Spectrometer (Bruker, Karlsruhe, West Germany) with a TE "-4 double rectangular cavity. Operating conditions for the spectrometer together with the derivation and correction of the spectra produced followed the procedure set out by @tevenson and Gr_a~ (1989 b). The spectra were double integrated over the range 3.444 x 10-~T to 3.544 x IO-~T and the results are tabulated under 'Basic' (Table i). The relationship between the value of the ESR integral and the applied dose was determined statlstlcally and found to be llnear over the range 2.5 to 10 kGy and of the form:-

Corrected ESR integral = 0.2905 (Z 0.011) x (Actual Dose - Intended dose)

This relationship was then used to correct the basic ESR signal for differences between the intended dose and the actual dose received. These results are tabulated under 'Dose CorE' (Table 1). The ash, calcium and phosphorus concentrations of the bone samples were determined (Stevenson and Gray, 1989 b) and the integrals were corrected to a standard ash of 640g/kg, calcium of 240g/kg and phosphorus of 100g/kg and the results tabulated under 'Ash CorE', 'Ca CORE' and 'P CorE' respectively in Table i.

Crystalllnlty coefficients were determined for each of the bone samples by subjecting them to an irradiation dose of 80 kGy, measuring the ESR signal produced and calculating the ratio of the ESR integral to the corresponding ash content of the bone (Ostrowski et al., 1974). The results were subjected to both analysis of variance and regression analysis.

RESULTS

The effect of irradiation dose on the mean values of the ESR signal strengths for the irradiated chicken bones are given in Table i. In each case the effect was highly significant (P<0.001) and the response was linear.

Table I. Effect of irradiation dose on the observed strengths from irradiated chicken drumsticks. 20 observations)

and corrected ESR signal (Values are the mean of

Dose (kGy)

ESR SIGNAL

Basic Dose Ash Ca P COrE CorE CorE CorE

2.5 1.064 1.028 1.027 0.962 0.922

5.0 1.969 1.899 1.874 1.769 1.696

7.5 2.653 2.546 2.555 2.393 2.278

i0.0 3.265 3.203 3.229 3.108 2.847

SEM 0.0552 0.0571 0.0564 0.0673 0.0540

CV% II.0 11.8 11.6 14.6 12.5

Significance of effect

Overall *** *** *** *** ***

Linear *** *** *** *** ***

The mean results for the relative signal strength of bones from each of the five age groups together with the crystallinlty coefficients are presented in Table 2. There was a highly significant llnear Eelatlonshlp (P<O.O01) between the age of the bird and the ESR signal strength (Table 2, 'Basic'), the ESR slgnal increasing wlth increasing age of the bird.

286 RICHARD GRAY e t al.

Regression analysis was carried out to relate the ESR integral to both the age of the chickens and the applied irradiation dose. For each age, there was a linear relationship between ESR integral and irradiation dose over the range 2.5 to I0.0 kGy. Tests indicated that a common intercept could be assumed for each age but that the regression coefficient increased linearly with age. This gave the following equations with y = ESR integral and x = irradiation dose:

8 weeks: y = 0.3847 + 0.3296 x 7 weeks: y = 0.3847 + 0.3078 x 6 weeks: y = 0.3847 + 0.2859 x 5 weeks: y = 0.3847 + 0.2640 x 4 weeks: y = 0.3847 + 0.2428 x

SE (intercept) = 0.0735; SE (regression coefficient) = 0.0113

R 2 = 0.913; residual standard deviation = 0.267

These regression lines are shown in Fig. i.

The crystalllnity coefficient increased linearly with age (Table 2, P<O.OI). The ash, calcium and phosphorus concentrations of the dried bones were not significantly affected by age and the results are not presented.

DISCUSSION

The increase in free radical concentration with increasing irradiation dose confirms earlier findings (Stevenson and Gray, 1989 a, b). As with previous experiments (Gray and Stevenson, 1989; Stevenson and Gray, 1989 b) there was no indication that the corrections for ash, calcium or phosphorus content of the bones changed the relationship obtained.

Also, correction for calcium and phosphorus content of the bone increased the coefficient of variation of the results in comparison to that of the ash corrected values (Tables 1 and 2).

Table 2. Effect of age on the ESR signal strength in irradiated chicken drumsticks. (Values are the mean of 16 observations).

ESR SIGNAL

Age Basic Dose Ash Ca P Crystallinity (Weeks) Corr Corr Corr Corr coefficient

8 2.537 2.489 2.464 2.367 2.215 2.60

7 2.413 2.323 2.304 2.227 2.008 2.55

6 2.172 2.099 2.107 1.912 1.854 2.48

5 2.104 2.021 2.036 1.997 1.840 2.42

4 1.963 1.913 1.946 1.787 1.761 2.32

SEM 0.0617 0.0638 0.0630 0.0752 0.0604 0.080

CV% ii.0 11.8 11.6 14.6 12.5 13.0

Significance of effect

Overall *** *** *** *** *** NS

Linear *** *** *** *** *** **

The increase in ESR signal with increasing age (Table 2, Fig. I) over the range 2.5 to I0 kGy is not due to a change in mineral composition since this was constant over the age range investigated. Thus it appears that the bones investigated had reached their mature mineral composition by 4 weeks of age. Regression analysis of the results from this experiment indicated a common intercept which was significantly different from zero.

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co

D

X 7 weeks

V 6 weeks

5 weeks

O 4 weeks

2~5 5:0 7~5 Irradiation dose (kGy)

Fig. i. Relationship between corrected ESR Integral and Irradiation dose for different ages of bird.

id.o

However, further experimentation at doses between 0 and 2.5 kGy would confirm if this is in fact the case. The observed increase in crystallinity coefficient which is a measure of bone crystallinity and which has been shown to affect ESR signal strength (Ostrowskl et al.~ 1974), may account for at least part of the enhanced signal obtained as the bird aged.

CONCLUSIONS

Age of the bird is another factor which should be considered when using ESR spectroscopy to quantify the dose received by irradiated bone.

ACKNOWLEDGEMENT

The authors wish to thank the Ministry of Agriculture, Fisheries and Food for provision of funds to undertake the research and Mr W D Graham and Miss E M Stewart for technical assistance.

REFERENCES

Desrosiers, M.F. and M.G. Simic (1988). Post irradiation dosimetry of meat by electron spin resonance spectroscopy of bones. J. A~ric. Food Chem., 36, 601-603.

Dodd, N.J.F., A.J. Swallow and J.S. Lea (1985). Use of ESR to identify irradiated food. Radiat. Phys. Chem., 26, 451-453.

Dodd, N.J.F., A.J. Swallow and J.S. Lea (1988). ESR detection of irradiated food. Nature, 334, 387.

Gray, R. and M.H. Stevenson (1989). The effect of post-lrradlation cooking on the ESR signal in irradiated chicken drumsticks. Int. J. Food Sci. Tech., in press.

Lea, J.S., N.J.F. Dodd and A.J. Swallow (1988). A method of testing for irradiation of poultry. Int. J. Food Sci. Tech., 23, 625-633.

Ostrowski, K. and A. Dziedzic-Goclawska (1976). Electron spin resonance spectrometry in investigations on mineralised tissues. In: The biochemistry and physiology of bone. (G.H Bourne, ed.), Vol. IV, 2nd ed., pp. 303-327. Academic Press, New York, San Francisco, London.

Ostrowski, K., A. Dziedzic-Goclawska, W. Stachowicz and J. Michalik (1974). Accuracy, sensitivity and specificity of electron spin resonance analyses of mineral constituents of irradiated tissues. Ann. N.Y. Acad. Sci., 238, 186-201.

Stevenson, M.H. and R. Gray (1989 a). An investigation into the effect of sample preparation methods on the resulting ESR signal from irradiated chicken bone. J. Sci. Food A~ric., in press.

Stevenson, M.H. and R. Gray (1989 b). The effect of irradiation dose, storage glme and temperature on the ESR signal in irradiated chicken drumsticks. J. Sci. Food A~ric., in press.