SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 983 FOOD COMPOSITION AND ADDITIVES
Photostimulated Luminescence Detection of Irradiated Shellfish: International Interlaboratory Trial DAVID C.W. SANDERSON, LORNA A. CARMICHAEL, and SAFFRON FISK Scottish Universities Research and Reactor Centre (SURRC), Scottish Enterprise Technology Park, Rankine Ave, East Kilbride, G75 OQF, UK Collaborators: P. Brown; Y. Burton; H. Nootenboom; S. Pinnioja; G.A. Schreiber; and U. Wagner
An interlaboratory trial was conducted to validate photostimulated luminescence (PSL) detection of irradiated shellfish. Five species of shellfish (Nephrops norvegicus, mussels, black tiger prawns, brown shrimps, and king scallops) were presented blind as nonirradiated and irradiated to 0.5 and 2.5 kGy. Precharacterization analysis of each product and treatment was performed on both whole (including shell) and intestinal samples. The results for whole samples (including shell) confirmed that the method was able to distinguish between nonirradiated and irradiated samples, regardless of dose. Intestinal data have identified that the method is dependent on the quantity and sensitivity of grits present within the intestinal tract, which can be assessed using calibration by normalization to 1 kGy. Five laboratories returned both initial screening and calibrated data and sample classification. All laboratories correctly identified all irradiated products using the screening criteria. There were no false positives. The results confirm the validity of the PSL method for shellfish, which has been adopted as a European standard method and by the Codex Alimentarius Commission. Calibration is required where only intestinal material is available. For whole samples with shell, screening alone is sufficient.
or routine detection of irradiated foods such as herbs, spices, fruits, vegetables, and shellfish (1–3), the standard validated methods have been based on thermoluminescence (TL). TL methods require a physical separation to extract minerals for calibrated analysis. Relatively lengthy laboratory preparation and the need for access to a calibrated source of ionizing radiation have limited the use of TL analysis of foods to a small number of laboratories. Shellfish commonly consist of an intimate mixture of inorganic and organic components, which are hard to separate.
F
Received February 27, 2003. Accepted by SG May 23, 2003. Corresponding author’s e-mail:
[email protected].
Bioinorganic food components, such as bones or shells, inhibit high temperature TL analysis (4). There is, therefore, scope for development of faster methods requiring less sample preparation for routine commercial or enforcement testing. Photostimulated luminescence (PSL) techniques combine simpler processing with nondestructive measurements. The development of PSL for detecting irradiated foods, where the energy to release trapped charge carriers is provided optically (5–8), has been aimed at resolving the practical limitations of silicate TL methods. In 1992 (9–13), an instrument was developed for high-sensitivity PSL measurements from food samples, using the highly radiation-specific UV-Vis luminescence signals which can be stimulated by using IR sources together with pulsed lock-in techniques for background suppression. Samples are measured in disposable Petri dishes and are undamaged by the measurement process; if necessary calibration can be performed by irradiation followed by repeat measurement (CalPSL). This allows the sensitivity of the sample to irradiation to be assessed. The viability of this technique for measuring unprepared whole samples was investigated using a set of herbs, spices, and seasonings (10, 14), presented both as nonirradiated and irradiated. Using intensity measurements, more than 90% of irradiated samples could be identified. There was a small overlap between high-sensitivity nonirradiated samples and low-sensitivity irradiated samples. This can be resolved with CalPSL. Luminescence intensity detected from the sample, as received, is used for preliminary classification into negative, intermediate, or positive bands (screening mode) using predefined thresholds (6, 14). Calibrated PSL measurements to distinguish between low- and high-sensitivity samples resolve the classification of intermediate screening results. Low-sensitivity samples may require further investigation by TL. Since 1992, more than 2000 samples (mostly herbs and spices) have been analyzed at Scottish Universities Research and Reactor Centre (SURRC) using PSL methods. A number of these samples have also been analyzed using the validated TL method. There has been no conflict between PSL and TL analyses performed on the same material. PSL investigations of shellfish have been undertaken using both research spectrometers (15) and the pulsed PSL instrument, where signals from intestinally trapped silicates are
984 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
Figure 1. Flow chart of decision-making process for classifying shellfish using PSL thresholds of 1000 and 4000.
stimulated not only through the membranes of dissected guts, but also in some cases through the whole body of the creature (9, 10, 14). The IR stimulation system is also capable of responding to signals from shell for at least several weeks after irradiation. Practical limits for application of this approach to shellfish have been investigated at SURRC (10) using blind analysis of commercial samples, previously analyzed by TL in laboratories in Finland and The Netherlands. Correct identification of approximately 84% of irradiated shellfish samples was obtained by screening; calibrated PSL measurements showed that the remaining 16% had low PSL sensitivity, presumably due to the lack of suitable minerals in the dissected intestinal material. The SURRC PSL instrument was also used informally to analyze samples of Vietnamese shrimp during the 1994 BGVV TL blind trial (16), producing results with a 100% correct detection rate. In 1996, a national surveillance exercise conducted in the United Kingdom on behalf of the Ministry of Agriculture, Fisheries and Food (MAFF; 17), included luminescence testing at SURRC. A combined PSL/TL approach with calibrated PSL measurements was used to select shellfish samples for confirmation by TL. The need to measure a large number of samples rapidly was met by screening paired aliquots of each submitted sample by PSL and calibrating for sensitivity. If the response to radiation was below a predetermined threshold, 6 portions of intestinal material were prepared for similar measurements in pairs, continuing until adequate sensitivity was obtained. If all 6 aliquots failed to
produce adequate sensitivity, the sample remained unclassified. TL analysis was performed on a random 10% selection of negative samples in addition to all intermediate or positive screening results. It was notable in this case that a significant proportion of samples tested failed to produce adequate PSL sensitivity. Many of these products were submitted without shell and/or any visible intestinal tract. PSL screening systems have now been installed in more than 15 laboratories in the United Kingdom and overseas. In 1995, when shellfish samples were being prepared for TL trials (18), there were 5 laboratories with PSL capabilities. These laboratories agreed to participate in the parallel PSL study. The same 5 species of shellfish that had been examined in the TL trial were distributed for examination by PSL. These were Nephrops norvegicus, mussels, brown shrimps, black tiger prawns, and king scallops. Samples were presented for blind analysis as nonirradiated and irradiated to 0.5 and 2.5 kGy. Several of the laboratories participating in the TL trial were also equipped to perform PSL measurements; 2 received further samples for this purpose. In addition, one laboratory, which does not have TL facilities, analyzed a set of samples with PSL; another laboratory with PSL only was unable to return any results. Participants were asked to classify their initial screening measurements on the basis of the highest reproducible PSL measurements from a replicated set, and to
Figure 2.
Precharacterization data for whole shellfish.
SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 985
re-irradiate the samples and perform calibrated measurements where possible. Experimental Protocol Instructions were sent to participants giving details of sample preparation, screening and calibrated measurements, assessment, and reporting of results. To help classify screening results as nonirradiated and irradiated, a decision-making chart was also enclosed (Figure 1). The protocol suggested that any sample with 2 or more positive results be identified as irradiated, any sample with 2 or more intermediate results be identified as requiring further investigation, and any sample with 2 or more negative results be identified as nonirradiated. For calibrated PSL results, participants were asked to identify samples with low sensitivity to irradiation and classify them as requiring further investigation.
Figure 3. Precharacterization data for intestinal samples.
Precharacterization Precharacterization testing was performed at SURRC on all of the products distributed as blind samples. This ensured that there was sufficient sensitivity for the irradiated material to be detected. For each product, 6 aliquots of whole samples and 6 aliquots of dissected intestines were measured for each of the 3 categories (nonirradiated and irradiated to 0.5 and 2.5 kGy). Choice of Samples and Predistribution Treatment The same 5 species of shellfish used for the TL trial (18) were used for the PSL trial: N. norvegicus, mussels, brown shrimps, black tiger prawns, and king scallops. The material was purchased from a Glasgow shellfish wholesaler; enough material was purchased to supply both trials. Each product was immediately bagged into 500 g aliquots; control samples were freezer-stored. Those samples randomly selected for irradiation doses to 0.5 and 2.5 kGy were repacked and frozen, ready for irradiation at SURRC. The 0.5 kGy samples received a mean dose of 0.49 ± 0.01 kGy and
Figure 4. Participants’ screening results for whole shellfish samples.
986 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
the 2.5 kGy samples received 2.50 ± 0.09 kGy (determined by using Harwell Amber [Oxfordshire, UK] perspex dosimeters). Participants received samples as whole shellfish, including shell material. For analysis of intestinal samples, participants were required to dissect some of these. Preparation of Blind Samples Sample preparation was very simple; samples were dispensed into 50 mm Petri dishes as whole shellfish (including shell) and as intestines. Participants were asked to analyze 6 portions of each sample in whole form and 6 portions of dissected intestines. All laboratories were asked to make screening measurements, followed by irradiation to 1 kGy, then a second measurement (CalPSL) was made after overnight chilled storage. PSL Measurement and Recording Participants were asked to conduct measurements for 60 s, with signals recorded every second and as a cumulative terminal count. Thresholds of 1000 (T1) and 4000 (T2) counts for 60 s were set to separate negative, intermediate, and positive
Figure 5. Participants’ screening results for intestinal samples.
categories (6, 14). Results were to be recorded as PSL (recording each separate count) and summary (terminal counts only) files on disk for both screening and calibrated measurements, leading to classification as nonirradiated, irradiated, or requiring further investigation. Results and Discussion Precharacterization Preccharacterization of the 5 product types and treatments provided a means of assessing signal response from a single laboratory. Materials presented for routine testing are likely to have undergone some fading; the signal from calcite shell is less stable than the signal from intestinal material. It is therefore worthwhile to investigate the photostimulated luminescence signals from both whole samples (including shell) and intestinal material. Figures 2 and 3 show the precharacterization data from the whole shellfish samples and the intestinal material, respectively. Thresholds T1 and T2, used for the decision-making process, and the instrumental preset of 256 counts, are shown as dotted vertical lines. Good separation was observed between the irradiated and nonirradiated whole shellfish samples, but there was no significant separation between the 2 doses. Less distinction between irradiated and nonirradiated samples was observed for the intestines, and there is again no separation between the doses. There is also much product-to-product variation than was observed for the whole samples. With intestinal tracts, it is very difficult to assess the min-
Figure 6. Participants’ calibrated versus screening results for all products of whole shellfish.
SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 987
Figure 7. Participants’ calibrated versus screening results for all products of intestinal samples.
eral content by direct observation, and poor mineral yield may be responsible for the poorer resolution between treatments. Calibration of the majority of these samples identifies them as having low sensitivity. Participants’ Results Results were returned from 5 laboratories in the form of initial and calibrated PSL counts for both whole and intestinal samples, and a qualitative decision for each sample. (a) Screening results.—Figures 4 and 5 display the participants’ initial screening results for whole samples and intestinal samples, respectively. For the whole samples, all 5 prod-
ucts show good separation between irradiated and nonirradiated samples. Intestines of the samples show the effect observed in the precharacterization study, where for some products the separation between irradiated and nonirradiated products is less well-defined. This is associated with low mineral yield and sensitivity. There is no clear separation between the 0.5 and 2.5 kGy doses for either whole or intestinal samples. This also corroborates the precharacterization data. No measurements from nonirradiated samples (either whole or intestinal) exceeded T1. (b) Calibrated results.—Participants’ calibrated results were similar to the observations from precharacterization measurements, where both whole and intestinal samples showed good separation between the calibrated nonirradiated and irradiated samples when plotted against screening results for the same samples (Figures 6 and 7). There are some outlying points for both whole and intestinal material. In the majority of cases, low sensitivity accounted for these, but the possibility of unusual mineralogy or contamination cannot be ruled out completely. Calibration of intestinal samples which had given screening results below the upper threshold T2 (negative or intermediate screening) successfully identified low-sensitivity samples (negative or intermediate calibrated results). Under the circumstances of the trial, participants were able to compare whole and intestinal results from the same sample, and they appear to have taken this into account when reaching their qualitative decisions. In routine analysis, however, this may not always be possible. If, therefore, only intestinal material is presented, calibration is necessary for assessing sensitivity and avoiding false negatives. Whole (unpeeled) shellfish should be preferred where they are available. (c) Qualitative results.—Participants’ qualitative results were compared with the true identity of each sample. Tables 1 and 2 summarize the results for screening (whole and intestinal samples). Participants stated whether a sample was irradiated or not, basing their interpretation on the guidelines provided in the protocol. The protocol suggested that any sample with 2 or more positive results be identified as irradiated, any sample with 2 or more intermediate results be identified as requiring further investigation, and any sample with 2 or more
Table 1. Qualitative screening results for whole shellfish samplesa No. of nonirradiated samples Product
No. sent
+
–
0.5 kGy +
All irradiated samples
2.5 kGy –
+
–
+
–
Total +
–
Nephrops
15
5
0
5
0
5
0
10
0
15
0
Mussels
15
5
0
5
0
5
0
10
0
15
0
Brown shrimps
15
5
0
5
0
5
0
10
0
15
0
Black tiger prawns 15
5
0
5
0
5
0
10
0
15
0
King scallops
15
5
0
5
0
5
0
10
0
15
0
Total
75
25
0
25
0
25
0
50
0
75
0
a
Five laboratories returned qualitative results from a total of 75 blind coded samples. + = Correct and – = incorrect.
988 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 Table 2. Qualitative screening results for intestinal shellfish samplesa No. of nonirradiated samples
0.5 kGy
All irradiated samples
2.5 kGy
Total
No. sent
+
–
+
–
+
–
+
–
+
–
Nephrops
15
5
0
5*
0
5*
0
10
0
15
0
Mussels
15
5
0
5
0
5
0
10
0
15
0
Brown shrimps
15
5
0
5*
0
5*
0
10
0
15
0
Black tiger prawns
15
5
0
5
0
5
0
10
0
15
0
King scallops
15
5
0
5*
0
5*
0
10
0
15
0
Total
75
25
0
25
0
25
0
50
0
75
0
Product
a
Five laboratories returned qualitative results from a total of 75 blind coded samples. + = Correct, – = incorrect, and * = requiring further investigation.
negative results be identified as nonirradiated. The 0.5 and 2.5 kGy treatments were correctly identified as irradiated for all cases, but distinction between doses had not been requested. The nonirradiated samples were also correctly identified. For a total of 75 blind samples for whole and intestinal samples, 100% correct identification was achieved. For intestinal qualitative screening results (Table 2), all samples were correctly identified, but for those identified by asterisks, participants commented that these would require further investigation because of low signals. It is clear from this that participants referred back to whole sample results when making their decisions. Participants observed that there was greater variation in signal intensity with the intestinal materials than with whole samples. Calibrated measurements did not affect participants’ decisions in any instance. Conclusions An international interlaboratory trial was conducted to assess the case for validation of PSL methods for shellfish. The results received from 5 laboratories gave 100% correct classification of 5 species for both whole and intestinal material, on the basis of whether a sample was or was not irradiated. No distinction was made between samples irradiated to 0.5 and 2.5 kGy, but even those with the lower dose were clearly identifiable as irradiated. On this basis, the case for validation of PSL as a quick low-cost screening method is clear. Whole samples are to be preferred, where available, because intestinal material gives consistently lower signal intensity. If only intestinal material is available, then sensitivity assessment by calibration is required. Acknowledgments Support for this work by MAFF under contract number 1B073 is gratefully acknowledged. We are also grateful to the following participants for the work and time they contributed to the study, without which it would not have been possible:
P. Brown and Y. Burton, Lincolne, Sutton and Wood Ltd., Norwich, UK H. Nootenboom, Food Inspection Service, Nijmegen, The Netherlands S. Pinnioja, University of Helsinki, Helsinki, Finland G.A. Schreiber and U. Wagner, BGVV, Berlin, Germany References (1) MAFF (1992) Detection of Irradiated Herbs and Spices, Validated Methods for the Analysis of Foodstuffs, V27, Ministry of Agriculture, Fisheries and Foods, Norwich, UK (2) MAFF (1993) J. Assoc. Public Anal. 29, 187–200 (3) European Standard Method (1997) Foodstuffs—Detection of Irradiated Food from which Silicate Minerals Can Be Isolated: Method by Thermoluminescence, BS EN 1788, British Standards Institute, London, UK (4) Carmichael, L.A., Sanderson, D.C.W., & Ni Riain, S. (1994) Radiat. Meas. 23, 455–463 (5) Sanderson, D.C.W. (1990) in Food Irradiation and the Chemist, D.E. Johnston & M.H. Stevenson (Eds), Royal Society of Chemistry, Cambridge, UK, pp 25–56 (6) Sanderson, D.C.W. (1991) in Potential New Methods of Detection of Irradiated Food, J. Raffi & J.J. Belliardo (Eds), EUR 13331, Luxembourg, pp 159–167 (7) Sanderson, D.C.W., & Clark, R.J. (1994) Radiat. Meas. 23, 633–639 (8) Clark, R.J., & Sanderson, D.C.W. (1994) Radiat. Meas. 23, 641–646 (9) Sanderson, D.C.W., Carmichael, L.A., Ni Riain, S., Naylor, J., & Spencer, J.Q. (1994) Food Sci. Technol. Today 8, 93–96 (10) Sanderson, D.C.W., Carmichael, L.A., & Naylor, J.D. (1995) Food Sci. Technol. Today 9, 150–154 (11) Sanderson, D.C.W., Carmichael, L.A., & Naylor, J.D. (1996) in Detection Methods for Irradiated Foods, C.H. McMurray, R. Gray, E.M. Stewart, & J. Pearce (Eds), Royal Society of Chemistry, Cambridge, UK, pp 140–148 (12) Sanderson, D.C.W. (1993) Detection of Irradiated Samples, UK Patent No. 93-8542930424
SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 989 (13) Sanderson, D.C.W. (1997) Detection of Irradiated Samples, UK Patent No. 2,291,707 (14) Sanderson, D.C.W., Carmichael, L.A., & Naylor, J.D. (1996) in Detection Methods for Irradiated Foods, C.H. McMurray, R. Gray, E.M. Stewart, & J. Pearce (Eds), Royal Society of Chemistry, Cambridge, UK, pp 124–138 (15) Sanderson, D.C.W., Carmichael, L.A., Spencer, J.Q., & Naylor, J.D. (1994) Photostimulated Luminescence and Thermoluminescence Techniques for Detecting Irradiated Foods, Final Report, Detection of Irradiated Shellfish, MAFF N2635, London, UK
(16) Schreiber, G.A., Hoffmann, A., Helle, N., & Bogl, K.W. (1993) Bundesgesundheitsblatt 36, 522–529 (17) MAFF (1997) Undeclared Irradiation of Foodstuffs, Working Party on Food Authenticity, Final Report, June 1997, Ministry of Agriculture, Fisheries and Food, London, UK (18) Sanderson, D.C.W., Carmichael, L.A., & Fisk, S. (1997) An International Collaborative Blind Trial of Thermoluminescence Detection of Irradiated Shellfish, Final Report, MAFF IB073, London, UK
