- Email: [email protected]
Identification of irradiated oysters by EPR measurements on shells
Radiation Measurements 46 (2011) 816e821
Contents lists available at ScienceDirect
Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Identification of irradiated oysters by EPR measurements on shells S. Della Monaca*, P. Fattibene, C. Boniglia, R. Gargiulo, E. Bortolin Istituto Superiore di Sanità, viale Regina Elena, 299 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 November 2010 Received in revised form 28 January 2011 Accepted 28 March 2011
In this paper the EPR spectra of the radicals induced in oyster shells after irradiation to (0.5e2) kGy ionizing radiation doses are analyzed. EPR spectra of irradiated shells showed the complex radical composition of biocarbonates, characterized by the presence of SO2, SO3 and CO2 radicals with different symmetries. In particular, the radiation-induced line at g ¼ 2.0038, due to the gx component of the orthorhombic SO3, was well distinguishable from the rest of the spectrum. The gx component of the orthorhombic SO3 was found to be intense and stable enough to allow the identification at least for the whole shelf life of the oyster. Furthermore, it is still well visible at low microwave powers for which the other signals are weak or nonvisible and has a linear dose response in the (0.5e2) kGy range. A possible procedure protocol for the identification of irradiated oysters, can be based on acquisitions of the spectrum at low microwave power values (tenths of milliWatt) and low modulation amplitude values (0.03e0.05 mT) and on the identification of the g ¼ 2.0038 signal as a proof of the ionizing radiation treatment performed on the sample. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Electron paramagnetic resonance Biocarbonates Shells Food irradiation
1. Introduction Oysters, as other shellfish, are known to be an important vehicle of Vibrio species (Vibrio parahaemolyticus and Vibrio vulnificus) and other foodborne pathogens (Su and Liu, 2007; Jacabi et al., 2003; U.S. FDA, 2005a). Ionizing radiation treatment with high doses (not exceeding 5.5 kGy) is one of the methods authorized by U.S. Food and Drug Administration to eliminate such bacterial pathogens (U.S. FDA, 2005b). To permit checks on compliance with existing regulation (Framework Directive 1999/2/EC; Directive 1999/3/EC), in EU the availability of reliable analytical methods for the detection of irradiated food is required. EPR is a method validated for the identification of irradiated food containing bones, cellulose or crystalline sugar. The objective of this work is to investigate the feasibility to use electron paramagnetic resonance as a method to detect irradiated oysters. This study was conducted because no validated protocol exists for the detection of irradiated oysters. Oyster shells are mainly composed by biocarbonates, whose EPR spectrum is well known from the literature. EPR spectra of irradiated biocarbonates show the complex radical composition characterized by the presence of at least five paramagnetic centers assigned to carbonate- and sulfate-derived radicals with different symmetries, well studied in dating literature (Ikeya, 1993; Callens
* Corresponding author. Tel.: þ39 06 4990 2614; fax: þ39 06 4990 2137. E-mail address: [email protected] (S. Della Monaca). 1350-4487/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2011.03.028
et al., 1997) and widely analyzed in the past by multi-frequency EPR approaches (Strzelczak et al., 2001; Callens et al., 2002). These signals have different properties of dose response and time stability. The presence of such a high number of EPR signals in field range of about 2 mT makes it difficult to select a single line as a dose indicator. In the past a draft protocol for EPR identification of irradiated oysters was suggested by Raffi et al. (1996). In that work EPR spectra were recorded using measurement parameters which overmodulated radiation-induced signals. The use of over-modulated spectra in EPR dosimetry is a consolidated method in relative measurements (e.g. when a calibration curve is used, as it is the case in dosimetry), provided that the signals contributing to the over-modulated line have the same dose response. This is not the case for biocarbonates, where the CO2 response is linear up to 250 Gy. In fact the dose response of the spectrum recorded as proposed by Raffi et al. (1996) was not linear in the 1e5 kGy dose range. In the present work a different approach is used; the experimental activity concerned mainly a systematical study of spectra recorded at different measurement parameters on both unirradiated and irradiated samples. The main goal was to find parameters allowing detection of a single species responding at doses of the order of the kGy. In particular, a radiation-induced line at g ¼ 2.0038, attributed to the gx component of the orthorhombic signal due to SO3, was found to be intense and enough stable to allow the identification at least for the whole shelf life of the oyster. Furthermore, it is well visible at low
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
microwave power values (tenths of milliWatt), for which the other signals are weak or non-visible. Scope of this paper is the characterization of the dose response and the time stability of this signal, as a preliminary step for a procedure aimed at the identification of irradiated oysters.
817
In order to investigate the EPR spectrum of unirradiated oyster shells, six samples non-irradiated in the laboratory were measured. EPR spectra of one sample recorded over a 12 mT field range, at a field modulation amplitude of 0.05 mT and various microwave power values are shown in Fig. 1. The spectra obtained from different samples were found to have the same shape.
As expected from the literature on EPR on carbonate-containing biological systems (Ikeya, 1993), two large Mn2þ lines are present, whose intensities grow as the microwave power increases until about 20 mW. Besides those larger signals, two smaller peaks are present, which may probably be identified with a superposition between the two satellite peaks of Mn2þ and defects induced by natural radiation (Seletchi and Duliu, 2007; Piligkos et al., 2007); an increase of peak intensities with microwave power until 50 mW is evident in this case. In Fig. 2 the spectrum of one sample irradiated at 1 kGy is shown compared with the spectrum of an unirradiated sample, recorded at 0.05 mT field modulation amplitude and microwave power of 3.2 mW, over a field range of 12 mT Mn2þ lines intensities do not grow with ionizing radiation. Intensities of the radiation-induced signals were found to be about two orders of magnitude more intense than the Mn2þ lines observed in the unirradiated sample spectra. In Figs. 3 and 4 spectra of a 1 kGy irradiated sample recorded at different values of the microwave power and different values of the field modulation amplitude are shown, respectively. Stable carbonate-derived radicals signals well known from the literature (Schramm and Rossi, 1996; Callens et al., 1997), such as the isotropic and the orthorhombic CO2, are well distinguishable in the spectrum. Moreover, sulfate-derived radical signals (such as orthorhombic and isotropic SO3 and isotropic SO2) are recognizable, which are coming from impurities contained in natural biocarbonates (Barabas et al., 1992; Schramm and Rossi, 1996; Seletchi and Duliu, 2007). Spectra recorded for all specimens investigated in the present study showed the same signal composition. The microwave study shown in Fig. 3a showed that radiation-induced line at g ¼ 2.0038, attributed to the gx component of the orthorhombic signal due to SO3, is well distinguishable at microwave power values of tenths of milliWatt, for which the other signals are weak or non-visible. This is also evident from Fig. 3b, where the behavior of the peakepeak intensity of the signal as a function of the square root of the microwave power is reported. The maximum intensity of the signal appears to be located at a microwave power of about 0.2e0.3 mW. The study reported in Fig. 4a, with spectra recorded at a microwave power of 0.3 mW and different values of the field modulation amplitude, showed that an over-modulation of the spectrum occurred for field modulation amplitude values above 0.05 mT. This was also confirmed by the behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the modulation amplitude, reported in Fig. 4b, showing
Fig. 1. Unirradiated sample. Spectra of an unirradiated sample recorded over a field range of 12 mT, with a field modulation amplitude of 0.05 mT and recorded at different values of the microwave power. Two large Mn2þ signals are present, whose intensities grow with the microwave power until about 20 mW. Two smaller peaks are present, which may probably be identified with a superposition between the two satellite peaks of Mn2þ and defects induced from natural radiation.
Fig. 2. Irradiated sample. Spectrum of a sample irradiated at 1 kGy compared with the spectrum of an unirradiated sample, recorded at 0.05 mT field modulation amplitude and microwave power of 3.2 mW, over a field range of 12 mT Mn2þ lines intensities do not grow with ionizing radiation and intensities of the radiation-induced signals are about two orders of magnitude more intense than the Mn2þ lines observed in the unirradiated sample spectrum.
2. Material and methods Twelve oysters, Crassostrea gigas, imported from France, were bought in an Italian supermarket. Shells were opened and flesh was removed from valves. Then, shells were washed and dried in oven for 12 h at 50 C for a preliminary water removal process. Shells were crushed by a chisel and fragments of about 50 mg, 6 mme10 mm length and 3 mme3.5 mm thickness were selected, choosing the ones presenting a bigger amount of carbonate (thus showing a whiter coloring) in order to avoid, within reason, organic component (the protein called “conchiolin”). The inner and the outer part of the shell have not been separated. To get powdered samples fragments were sieved and powdered samples were subjected to a further water removal process in vacuum oven at 50 C for 2 h before measurements. EPR measurements, in X band, were performed using a CW spectrometer Bruker Elexsys E500. For the positioning of the samples in the cavity quartz tubes were used, having internal diameters of 5 mm. Six oysters were kept unirradiated, six oysters were irradiated at doses of 0.5, 1, 1.5 and 2 kGy. During the irradiation and the transfer from the irradiation facility to ISS, oysters were kept at the controlled temperature of about 0 C. Irradiations and measurements were performed in air. g-ray irradiation was carried out at ISOF-CNR (Bologna) in a 60Co source Nordion Gammacell 220. 3. Results and discussion
818
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
Fig. 3. Spectra recorded at different microwave power values. In (a): spectra of a sample irradiated at 1 kGy recorded at different values of microwave power and field modulation amplitude of 0.03 mT. The radiation-induced line at g ¼ 2.0038, due to the gx component of the orthorhombic SO3, is found to be intense and well visible at low microwave power values (tenths of milliWatt), for which the other signals are weak or non-visible. In (b): behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the square root of the microwave power. The maximum intensity of the signal appears to be located at a microwave power of about 0.3 mW.
Fig. 4. Spectra recorded at different field modulation amplitude values. In (a): spectra of a sample irradiated at 1 kGy recorded at different values of the field modulation amplitude and a microwave power of 0.3 mW. Over-modulation of the signal occurs for values of the field modulation amplitude above 0.05 mT. In (b): behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the modulation amplitude. A saturation plateau starting at about 0.3 mT and a loss of linearity already at modulation amplitude of 0.02e0.05 mT are evident.
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
819
Fig. 5. Dose response. (a): spectra of samples irradiated at dose values of 0.5, 1, 1.5 and 2 kGy. The intensity of the signal located at g ¼ 2.0038 grows as the radiation dose increases. (b): the behavior of the peakepeak intensity of the g ¼ 2.0038 signal exhibits a linear increase of the signal intensity with radiation doses in the studied dose range. Spectra are recorded at 0.05 mT modulation amplitude and 0.3 mW microwave power.
a saturation plateau starting at about 0.3 mT and a loss of linearity already at modulation amplitude values of 0.02e0.05 mT. In Fig. 5 the dose response of the sample is analyzed. In particular, in Fig. 5a spectra of samples irradiated at dose values of 0.5, 1, 1.5 and 2 kGy are shown and it is evident that the intensity of the signal located at g ¼ 2.0038 grows as the radiation dose increases. In Fig. 5b the behavior of the peakepeak intensity of the g ¼ 2.0038 signal is shown, exhibiting a linear increase with radiation doses up to 2 kGy.
Fig. 6. Signal fading. Comparison between spectra of a sample irradiated at 1 kGy and measured immediately (solid line) and three months (dashed line) after the irradiation. The g ¼ 2.0038 signal due to the gx component of the orthorhombic SO3 is still well visible after three months (a loss of around 8% has occurred), a time range much longer than the shelf life of an oyster. Spectra are recorded at 0.03 mT modulation amplitude and 0.2 mW microwave power.
In Fig. 6 the spectrum of the shell of an oyster irradiated at 1 kGy and measured three months after the irradiation is shown. It is evident that the g ¼ 2.0038 signal due to the gx component of the orthorhombic SO3 radical is still visible after a time range which is much longer than the shelf life of an oyster. A loss of signal of about 8% is evident in the former case. Due to the simplicity of the spectrum recorded at low microwave power values, the time stability and the linear dose response, a possible procedure protocol for the identification of irradiated oysters, addressed to laboratories responsible for food safety controls, could be based on acquisitions of the spectrum at low microwave power values and on the identification of the g ¼ 2.0038 signal as an evidence of the ionizing radiation treatment performed on oysters. In the past a draft protocol for ESR identification of irradiated oysters was presented by Raffi et al. (1996). In that work EPR spectra were recorded using measurement parameters which highly deformed and over-modulated radiation-induced signals, using a field modulation amplitude of 0.45 mT and a microwave power of 12 mW (Fig. 7a). Such a proposal is valid in the case of a qualitative identification of an occurred irradiation of specimens. Nevertheless, when detection is performed with high levels of microwave power and modulation amplitude, the resulting signal is a convolution of CO2 and SO3 and measured intensity is a combination of the intensity of four signals. SO3 signals are linear with dose up to at least 5 kGy, whereas CO2 loses linearity around 250 Gy. Therefore, in cases where a quantitative estimation of the exposure level is required for a verification of the delivered dose declared by the producer, a dosimetric analysis on single signals could be more appropriate. Thus, an alternative procedure protocol for the identification of irradiated oysters is here proposed, which suggest acquisitions of
820
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
Fig. 7. Protocol proposals. (a) Spectrum recorded following the proposal by Raffi et al. (1996), with measurements parameters that highly over-modulate the radiation-induced spectrum (12 mW microwave power, 0.45 mT modulation amplitude). (b) Spectrum recorded according to the proposal of the present work, based on acquisitions of the spectrum at low microwave power (0.2 mW) and field modulation amplitude (0.03 mT) values and on the identification of the g ¼ 2.0038 line due to the orthorhombic SO3 as a proof of the ionizing radiation treatment performed on specimens.
the spectrum at low microwave power values and the identification of the g ¼ 2.0038 signal (attributed to the gx component of the orthorhombic SO3 signal) as a proof of the ionizing radiation treatment performed on oysters (Fig. 7b). 4. Conclusions The goal of the present work was to investigate the possibility of identifying irradiated oysters by EPR measurements on shells. The signal located at g ¼ 2.0038, attributed to the gx component of the orthorhombic SO3 radical was found to be very well distinguishable at low microwave power values (tenths of milliWatt) and stable in time for a time range much longer than the shelf life of an oyster. Moreover, it exhibited a linear dose response. For those reasons, a procedure protocol for the identification of irradiated oysters was proposed, which suggested acquisitions of the spectrum at low microwave power values (tenths of milliWatt) and the identification of the g ¼ 2.0038 signal as a proof of the ionizing radiation treatment performed on oysters.
References Barabas, M., Mudelsee, M., Walther, R., Mangini, A., 1992. Dose response and thermal behaviour of the ESR signal at g ¼ 2.0006 in carbonates. Quat. Sci. Rev. 11 (1e2), 173e179. Callens, F., Vanhaelewyn, G., Matthys, P., Boesman, E., 1997. EPR of carbonate derived radicals: applications in dosimetry, dating and detection of irradiated food. Appl. Magn. Reson. 14, 235e254. Callens, F., Vanhaelewyn, G., Matthys, P., 2002. Some recent multi-frequency electron paramagnetic resonance results on systems relevant for dosimetry and dating. Spectrochim. Acta Mol. Biomol. Spectros. 58, 1321e1328. Ikeya, 1993. New Applications of Electron Spin Resonance e Dating, Dosimetry and Microscopy. World Scientific. Jacabi, M., Gelli, D.S., Torre, J.C.M.D., Rodas, M.A.B., Franco, B.D.G.M., Destro, M.T., Landgraf, M., 2003. Inactivation by ionizing radiation of Salmonella enteritidis, Salmonella infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana). J. Food Prot. 66 (6), 1025e1029. Piligkos, S., Laursen, I., Morgenstjerne, A., Weihe, H., 2007. Sign and magnitude of spin Hamiltonian parameters for Mn2þ impurities in calcite. A multi- and lowfrequency EPR study. Mol. Phys. 105 (15e16), 2025e2030. Raffi, J., Hasbany, C., Lesgards, G., Ochin, D., 1996. ESR detection of irradiated seashells. Appl. Radiat. Isot. 47, 1633e1636. Schramm, D.U., Rossi, A.M., 1996. Electron spin resonance (ESR), electron nuclear double resonance (ENDOR) and general triple resonance of irradiated biocarbonates. Appl. Radiat. Isot. 47, 1443e1455.
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821 Seletchi, E.D., Duliu, O.G., 2007. Comparative study on ESR spectra of carbonates. Rom. J. Phys. 52, 657e666. Strzelczak, G., Vanhaelewyn, G., Stachowicz, W., Goovaerts, E., Callens, F., Michalik, J., 2001. Multifrequency EPR study of carbonate- and sulfate-derived radicals produced by radiation in shells and corallite. Radiat. Res. 155, 619e624. Su, Y.-C., Liu, C., 2007. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. 24, 549e558.
821
U.S. Food and Drug Administration (FDA), 2005a. Quantitative Risk Assessment on the Public Health Impact of Pathogenic Vibrio parahaemolyticus in Raw Oysters Available at. www.fda.gov/Food/ScienceResearch/ResearchAreas/RiskAssessment SafetyAssessment/ucm050421.htm. U.S. Food and Drug Administration (FDA), 2005b. Ionizing radiation for control of Vibrio species and other foodborne pathogens in fresh or frozen molluscan shellfish. Fed. Regist. 70 (157), 48057.
Contents lists available at ScienceDirect
Radiation Measurements journal homepage: www.elsevier.com/locate/radmeas
Identification of irradiated oysters by EPR measurements on shells S. Della Monaca*, P. Fattibene, C. Boniglia, R. Gargiulo, E. Bortolin Istituto Superiore di Sanità, viale Regina Elena, 299 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 November 2010 Received in revised form 28 January 2011 Accepted 28 March 2011
In this paper the EPR spectra of the radicals induced in oyster shells after irradiation to (0.5e2) kGy ionizing radiation doses are analyzed. EPR spectra of irradiated shells showed the complex radical composition of biocarbonates, characterized by the presence of SO2, SO3 and CO2 radicals with different symmetries. In particular, the radiation-induced line at g ¼ 2.0038, due to the gx component of the orthorhombic SO3, was well distinguishable from the rest of the spectrum. The gx component of the orthorhombic SO3 was found to be intense and stable enough to allow the identification at least for the whole shelf life of the oyster. Furthermore, it is still well visible at low microwave powers for which the other signals are weak or nonvisible and has a linear dose response in the (0.5e2) kGy range. A possible procedure protocol for the identification of irradiated oysters, can be based on acquisitions of the spectrum at low microwave power values (tenths of milliWatt) and low modulation amplitude values (0.03e0.05 mT) and on the identification of the g ¼ 2.0038 signal as a proof of the ionizing radiation treatment performed on the sample. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Electron paramagnetic resonance Biocarbonates Shells Food irradiation
1. Introduction Oysters, as other shellfish, are known to be an important vehicle of Vibrio species (Vibrio parahaemolyticus and Vibrio vulnificus) and other foodborne pathogens (Su and Liu, 2007; Jacabi et al., 2003; U.S. FDA, 2005a). Ionizing radiation treatment with high doses (not exceeding 5.5 kGy) is one of the methods authorized by U.S. Food and Drug Administration to eliminate such bacterial pathogens (U.S. FDA, 2005b). To permit checks on compliance with existing regulation (Framework Directive 1999/2/EC; Directive 1999/3/EC), in EU the availability of reliable analytical methods for the detection of irradiated food is required. EPR is a method validated for the identification of irradiated food containing bones, cellulose or crystalline sugar. The objective of this work is to investigate the feasibility to use electron paramagnetic resonance as a method to detect irradiated oysters. This study was conducted because no validated protocol exists for the detection of irradiated oysters. Oyster shells are mainly composed by biocarbonates, whose EPR spectrum is well known from the literature. EPR spectra of irradiated biocarbonates show the complex radical composition characterized by the presence of at least five paramagnetic centers assigned to carbonate- and sulfate-derived radicals with different symmetries, well studied in dating literature (Ikeya, 1993; Callens
* Corresponding author. Tel.: þ39 06 4990 2614; fax: þ39 06 4990 2137. E-mail address: [email protected] (S. Della Monaca). 1350-4487/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.radmeas.2011.03.028
et al., 1997) and widely analyzed in the past by multi-frequency EPR approaches (Strzelczak et al., 2001; Callens et al., 2002). These signals have different properties of dose response and time stability. The presence of such a high number of EPR signals in field range of about 2 mT makes it difficult to select a single line as a dose indicator. In the past a draft protocol for EPR identification of irradiated oysters was suggested by Raffi et al. (1996). In that work EPR spectra were recorded using measurement parameters which overmodulated radiation-induced signals. The use of over-modulated spectra in EPR dosimetry is a consolidated method in relative measurements (e.g. when a calibration curve is used, as it is the case in dosimetry), provided that the signals contributing to the over-modulated line have the same dose response. This is not the case for biocarbonates, where the CO2 response is linear up to 250 Gy. In fact the dose response of the spectrum recorded as proposed by Raffi et al. (1996) was not linear in the 1e5 kGy dose range. In the present work a different approach is used; the experimental activity concerned mainly a systematical study of spectra recorded at different measurement parameters on both unirradiated and irradiated samples. The main goal was to find parameters allowing detection of a single species responding at doses of the order of the kGy. In particular, a radiation-induced line at g ¼ 2.0038, attributed to the gx component of the orthorhombic signal due to SO3, was found to be intense and enough stable to allow the identification at least for the whole shelf life of the oyster. Furthermore, it is well visible at low
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
microwave power values (tenths of milliWatt), for which the other signals are weak or non-visible. Scope of this paper is the characterization of the dose response and the time stability of this signal, as a preliminary step for a procedure aimed at the identification of irradiated oysters.
817
In order to investigate the EPR spectrum of unirradiated oyster shells, six samples non-irradiated in the laboratory were measured. EPR spectra of one sample recorded over a 12 mT field range, at a field modulation amplitude of 0.05 mT and various microwave power values are shown in Fig. 1. The spectra obtained from different samples were found to have the same shape.
As expected from the literature on EPR on carbonate-containing biological systems (Ikeya, 1993), two large Mn2þ lines are present, whose intensities grow as the microwave power increases until about 20 mW. Besides those larger signals, two smaller peaks are present, which may probably be identified with a superposition between the two satellite peaks of Mn2þ and defects induced by natural radiation (Seletchi and Duliu, 2007; Piligkos et al., 2007); an increase of peak intensities with microwave power until 50 mW is evident in this case. In Fig. 2 the spectrum of one sample irradiated at 1 kGy is shown compared with the spectrum of an unirradiated sample, recorded at 0.05 mT field modulation amplitude and microwave power of 3.2 mW, over a field range of 12 mT Mn2þ lines intensities do not grow with ionizing radiation. Intensities of the radiation-induced signals were found to be about two orders of magnitude more intense than the Mn2þ lines observed in the unirradiated sample spectra. In Figs. 3 and 4 spectra of a 1 kGy irradiated sample recorded at different values of the microwave power and different values of the field modulation amplitude are shown, respectively. Stable carbonate-derived radicals signals well known from the literature (Schramm and Rossi, 1996; Callens et al., 1997), such as the isotropic and the orthorhombic CO2, are well distinguishable in the spectrum. Moreover, sulfate-derived radical signals (such as orthorhombic and isotropic SO3 and isotropic SO2) are recognizable, which are coming from impurities contained in natural biocarbonates (Barabas et al., 1992; Schramm and Rossi, 1996; Seletchi and Duliu, 2007). Spectra recorded for all specimens investigated in the present study showed the same signal composition. The microwave study shown in Fig. 3a showed that radiation-induced line at g ¼ 2.0038, attributed to the gx component of the orthorhombic signal due to SO3, is well distinguishable at microwave power values of tenths of milliWatt, for which the other signals are weak or non-visible. This is also evident from Fig. 3b, where the behavior of the peakepeak intensity of the signal as a function of the square root of the microwave power is reported. The maximum intensity of the signal appears to be located at a microwave power of about 0.2e0.3 mW. The study reported in Fig. 4a, with spectra recorded at a microwave power of 0.3 mW and different values of the field modulation amplitude, showed that an over-modulation of the spectrum occurred for field modulation amplitude values above 0.05 mT. This was also confirmed by the behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the modulation amplitude, reported in Fig. 4b, showing
Fig. 1. Unirradiated sample. Spectra of an unirradiated sample recorded over a field range of 12 mT, with a field modulation amplitude of 0.05 mT and recorded at different values of the microwave power. Two large Mn2þ signals are present, whose intensities grow with the microwave power until about 20 mW. Two smaller peaks are present, which may probably be identified with a superposition between the two satellite peaks of Mn2þ and defects induced from natural radiation.
Fig. 2. Irradiated sample. Spectrum of a sample irradiated at 1 kGy compared with the spectrum of an unirradiated sample, recorded at 0.05 mT field modulation amplitude and microwave power of 3.2 mW, over a field range of 12 mT Mn2þ lines intensities do not grow with ionizing radiation and intensities of the radiation-induced signals are about two orders of magnitude more intense than the Mn2þ lines observed in the unirradiated sample spectrum.
2. Material and methods Twelve oysters, Crassostrea gigas, imported from France, were bought in an Italian supermarket. Shells were opened and flesh was removed from valves. Then, shells were washed and dried in oven for 12 h at 50 C for a preliminary water removal process. Shells were crushed by a chisel and fragments of about 50 mg, 6 mme10 mm length and 3 mme3.5 mm thickness were selected, choosing the ones presenting a bigger amount of carbonate (thus showing a whiter coloring) in order to avoid, within reason, organic component (the protein called “conchiolin”). The inner and the outer part of the shell have not been separated. To get powdered samples fragments were sieved and powdered samples were subjected to a further water removal process in vacuum oven at 50 C for 2 h before measurements. EPR measurements, in X band, were performed using a CW spectrometer Bruker Elexsys E500. For the positioning of the samples in the cavity quartz tubes were used, having internal diameters of 5 mm. Six oysters were kept unirradiated, six oysters were irradiated at doses of 0.5, 1, 1.5 and 2 kGy. During the irradiation and the transfer from the irradiation facility to ISS, oysters were kept at the controlled temperature of about 0 C. Irradiations and measurements were performed in air. g-ray irradiation was carried out at ISOF-CNR (Bologna) in a 60Co source Nordion Gammacell 220. 3. Results and discussion
818
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
Fig. 3. Spectra recorded at different microwave power values. In (a): spectra of a sample irradiated at 1 kGy recorded at different values of microwave power and field modulation amplitude of 0.03 mT. The radiation-induced line at g ¼ 2.0038, due to the gx component of the orthorhombic SO3, is found to be intense and well visible at low microwave power values (tenths of milliWatt), for which the other signals are weak or non-visible. In (b): behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the square root of the microwave power. The maximum intensity of the signal appears to be located at a microwave power of about 0.3 mW.
Fig. 4. Spectra recorded at different field modulation amplitude values. In (a): spectra of a sample irradiated at 1 kGy recorded at different values of the field modulation amplitude and a microwave power of 0.3 mW. Over-modulation of the signal occurs for values of the field modulation amplitude above 0.05 mT. In (b): behavior of the peakepeak intensity of the g ¼ 2.0038 signal as a function of the modulation amplitude. A saturation plateau starting at about 0.3 mT and a loss of linearity already at modulation amplitude of 0.02e0.05 mT are evident.
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
819
Fig. 5. Dose response. (a): spectra of samples irradiated at dose values of 0.5, 1, 1.5 and 2 kGy. The intensity of the signal located at g ¼ 2.0038 grows as the radiation dose increases. (b): the behavior of the peakepeak intensity of the g ¼ 2.0038 signal exhibits a linear increase of the signal intensity with radiation doses in the studied dose range. Spectra are recorded at 0.05 mT modulation amplitude and 0.3 mW microwave power.
a saturation plateau starting at about 0.3 mT and a loss of linearity already at modulation amplitude values of 0.02e0.05 mT. In Fig. 5 the dose response of the sample is analyzed. In particular, in Fig. 5a spectra of samples irradiated at dose values of 0.5, 1, 1.5 and 2 kGy are shown and it is evident that the intensity of the signal located at g ¼ 2.0038 grows as the radiation dose increases. In Fig. 5b the behavior of the peakepeak intensity of the g ¼ 2.0038 signal is shown, exhibiting a linear increase with radiation doses up to 2 kGy.
Fig. 6. Signal fading. Comparison between spectra of a sample irradiated at 1 kGy and measured immediately (solid line) and three months (dashed line) after the irradiation. The g ¼ 2.0038 signal due to the gx component of the orthorhombic SO3 is still well visible after three months (a loss of around 8% has occurred), a time range much longer than the shelf life of an oyster. Spectra are recorded at 0.03 mT modulation amplitude and 0.2 mW microwave power.
In Fig. 6 the spectrum of the shell of an oyster irradiated at 1 kGy and measured three months after the irradiation is shown. It is evident that the g ¼ 2.0038 signal due to the gx component of the orthorhombic SO3 radical is still visible after a time range which is much longer than the shelf life of an oyster. A loss of signal of about 8% is evident in the former case. Due to the simplicity of the spectrum recorded at low microwave power values, the time stability and the linear dose response, a possible procedure protocol for the identification of irradiated oysters, addressed to laboratories responsible for food safety controls, could be based on acquisitions of the spectrum at low microwave power values and on the identification of the g ¼ 2.0038 signal as an evidence of the ionizing radiation treatment performed on oysters. In the past a draft protocol for ESR identification of irradiated oysters was presented by Raffi et al. (1996). In that work EPR spectra were recorded using measurement parameters which highly deformed and over-modulated radiation-induced signals, using a field modulation amplitude of 0.45 mT and a microwave power of 12 mW (Fig. 7a). Such a proposal is valid in the case of a qualitative identification of an occurred irradiation of specimens. Nevertheless, when detection is performed with high levels of microwave power and modulation amplitude, the resulting signal is a convolution of CO2 and SO3 and measured intensity is a combination of the intensity of four signals. SO3 signals are linear with dose up to at least 5 kGy, whereas CO2 loses linearity around 250 Gy. Therefore, in cases where a quantitative estimation of the exposure level is required for a verification of the delivered dose declared by the producer, a dosimetric analysis on single signals could be more appropriate. Thus, an alternative procedure protocol for the identification of irradiated oysters is here proposed, which suggest acquisitions of
820
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821
Fig. 7. Protocol proposals. (a) Spectrum recorded following the proposal by Raffi et al. (1996), with measurements parameters that highly over-modulate the radiation-induced spectrum (12 mW microwave power, 0.45 mT modulation amplitude). (b) Spectrum recorded according to the proposal of the present work, based on acquisitions of the spectrum at low microwave power (0.2 mW) and field modulation amplitude (0.03 mT) values and on the identification of the g ¼ 2.0038 line due to the orthorhombic SO3 as a proof of the ionizing radiation treatment performed on specimens.
the spectrum at low microwave power values and the identification of the g ¼ 2.0038 signal (attributed to the gx component of the orthorhombic SO3 signal) as a proof of the ionizing radiation treatment performed on oysters (Fig. 7b). 4. Conclusions The goal of the present work was to investigate the possibility of identifying irradiated oysters by EPR measurements on shells. The signal located at g ¼ 2.0038, attributed to the gx component of the orthorhombic SO3 radical was found to be very well distinguishable at low microwave power values (tenths of milliWatt) and stable in time for a time range much longer than the shelf life of an oyster. Moreover, it exhibited a linear dose response. For those reasons, a procedure protocol for the identification of irradiated oysters was proposed, which suggested acquisitions of the spectrum at low microwave power values (tenths of milliWatt) and the identification of the g ¼ 2.0038 signal as a proof of the ionizing radiation treatment performed on oysters.
References Barabas, M., Mudelsee, M., Walther, R., Mangini, A., 1992. Dose response and thermal behaviour of the ESR signal at g ¼ 2.0006 in carbonates. Quat. Sci. Rev. 11 (1e2), 173e179. Callens, F., Vanhaelewyn, G., Matthys, P., Boesman, E., 1997. EPR of carbonate derived radicals: applications in dosimetry, dating and detection of irradiated food. Appl. Magn. Reson. 14, 235e254. Callens, F., Vanhaelewyn, G., Matthys, P., 2002. Some recent multi-frequency electron paramagnetic resonance results on systems relevant for dosimetry and dating. Spectrochim. Acta Mol. Biomol. Spectros. 58, 1321e1328. Ikeya, 1993. New Applications of Electron Spin Resonance e Dating, Dosimetry and Microscopy. World Scientific. Jacabi, M., Gelli, D.S., Torre, J.C.M.D., Rodas, M.A.B., Franco, B.D.G.M., Destro, M.T., Landgraf, M., 2003. Inactivation by ionizing radiation of Salmonella enteritidis, Salmonella infantis, and Vibrio parahaemolyticus in oysters (Crassostrea brasiliana). J. Food Prot. 66 (6), 1025e1029. Piligkos, S., Laursen, I., Morgenstjerne, A., Weihe, H., 2007. Sign and magnitude of spin Hamiltonian parameters for Mn2þ impurities in calcite. A multi- and lowfrequency EPR study. Mol. Phys. 105 (15e16), 2025e2030. Raffi, J., Hasbany, C., Lesgards, G., Ochin, D., 1996. ESR detection of irradiated seashells. Appl. Radiat. Isot. 47, 1633e1636. Schramm, D.U., Rossi, A.M., 1996. Electron spin resonance (ESR), electron nuclear double resonance (ENDOR) and general triple resonance of irradiated biocarbonates. Appl. Radiat. Isot. 47, 1443e1455.
S. Della Monaca et al. / Radiation Measurements 46 (2011) 816e821 Seletchi, E.D., Duliu, O.G., 2007. Comparative study on ESR spectra of carbonates. Rom. J. Phys. 52, 657e666. Strzelczak, G., Vanhaelewyn, G., Stachowicz, W., Goovaerts, E., Callens, F., Michalik, J., 2001. Multifrequency EPR study of carbonate- and sulfate-derived radicals produced by radiation in shells and corallite. Radiat. Res. 155, 619e624. Su, Y.-C., Liu, C., 2007. Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol. 24, 549e558.
821
U.S. Food and Drug Administration (FDA), 2005a. Quantitative Risk Assessment on the Public Health Impact of Pathogenic Vibrio parahaemolyticus in Raw Oysters Available at. www.fda.gov/Food/ScienceResearch/ResearchAreas/RiskAssessment SafetyAssessment/ucm050421.htm. U.S. Food and Drug Administration (FDA), 2005b. Ionizing radiation for control of Vibrio species and other foodborne pathogens in fresh or frozen molluscan shellfish. Fed. Regist. 70 (157), 48057.