## 4 Discussion
### Time Stability of the Radiation Induced Free Radicals Studied by EPR Spectroscopy  
It is known that EPR spectroscopy can be applied for the identification of previous radiation treated products from plant origin (herbs, spices, nuts, etc.). This depends on the life time of the free radicals induced after irradiation. The storage conditions of the samples, as the temperature, humidity and access to light, have an impact on the shelf life of the radicals. It was discovered that at normal storage conditions the \"cellulose-like\" EPR signal can be observed within a period of 60-80 days after irradiation. However, in the current study this period is longer. Figure 2a exhibits the decay of the central EPR peak with time for the peeled seed samples irradiated by different doses, whereas Figure 2b shows the decay of the shell seeds. It was observed that in the first days after irradiation the intensity increases, and then begins to slowly decrease. Usually when some mechanical treatments are carried out on the samples, it leads to additional creation of free radicals. Very often the radicals are similar to these induced upon irradiation. The horse chestnuts samples were ground before gamma treatment. Probably, the grinding process generates the same kind of free radicals as these occurring in irradiated samples. The natural oxidation of chestnut ingredients, triggered by the exposure of fresh surfaces to atmospheric oxygen, also leads to formation of radicals. So, probably the reason for the observed increase in the intensity of the EPR signals in the first days is just that these radicals, which are more unstable in comparison to the radiation induced radicals, decay for several days. The results show that the EPR signal intensities of irradiated by 1, 5, and 10 kGy peeled seeds decreased with 84%, 86%, and 82%, respectively, for a period of 250 days. For the same time period the intensity of the signal of horse chestnut shells decreased with 35%, 42%, and 37% for 1, 5, and 10 kGy irradiated samples, respectively. It is interesting to mention that in the samples from peeled seeds the intensity of the line recorded at irradiation with 5 kGy is higher in comparison those at 10 kGy. This could be explained by the saturation of the free radicals at higher doses due to recombination. On the other hand, on the whole, higher quantities of free radicals are recorded with shell seeds than peeled seeds. The evidence is the more intensive EPR signal arising from cellulose in the EPR spectrum. Also, in the shell samples, the saturation of the EPR signal at 10 kGy of irradiated samples is not observed. The different behavior of the EPR signal with various kinds of horse chestnut samples (peeled seeds and shell) is probably due to their different composition and water content. Figure 3 shows the decay over time of the two satellite peaks attributed to the cellulose radical formed upon irradiation. As it was mentioned before, these are the peaks used to prove that cellulose-containing foods were subjected to high energy ionizing radiation. After the irradiation, the signal intensity decays exponentially with time. For the samples irradiated by 1 kGy, the signal becomes difficult to detect 45 days after the irradiation in both case (peels seeds and shell seeds). For this time period the EPR signal intensity of the samples irradiated by 1 kGy decreased with 85% for peeled seeds and 73% for shell seeds. The samples irradiated by 5 and 10 kGy gamma rays show higher stability. Their intensity decreased with 82% and 71% for peeled seeds and 91% and 92% for shell seeds, respectively, but for the 250 days period. On the basis of the obtained results, it can be concluded, that for time periods longer than 250 days after irradiation, the identification of the radiation treatment of horse chestnut is very difficult because of the disappeared \"satellite-lines\" of the \"cellulose-liked\" EPR spectrum.