976 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 FOOD COMPOSITION AND ADDITIVES
Thermoluminescence 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. Christensen; H. Delincée; H. Nootenboom; J. Pfordt; S. Pinnioja; G.A. Schreiber; and U. Wagner
An international interlaboratory trial was conducted using thermoluminescence for the detection of irradiated shellfish, aimed at validating the method for routine use. Nephrops norvegicus, mussels, brown shrimps, black tiger prawns, and king scallops were presented as nonirradiated and irradiated to 0.5 and 2.5 kGy. The protocol called for the use of 3 preparation methods: extraction of silicates from whole shellfish by acid hydrolysis and physical separation, and of carbonates from powdered shells. Homogeneity was tested on each product and each treatment. Results verified that all methods were able to distinguish between nonirradiated and irradiated samples regardless of dose. Silicate methods produced better discrimination than powdered shell, and acid hydrolysis showed some evidence of better separation between the 2 doses than the physical method. Participants received each product in each treatment category for blind analysis. Six participants returned results for acid hydrolysis, 7 for physical separation, and 5 for the powdered shell method. Their results confirmed the homogeneity testing. Qualitative results gave 100% correct classification for both silicate methods and 85.3% for powdered shell. Silicate methods are therefore preferable unless only shell is available. Overall, the results confirmed the case for validation.
t is well established (1, 2) that thermoluminescence (TL) signals are associated with the silicate minerals present as contaminants in herbs and spices. These findings formed the basis, following interlaboratory trials, for the UK-validated and current European standard methods (3–16) and led to the inclusion of shrimps into the European standard method EN 1788 (16). This has also been adopted by the Codex Alimentarius Commission.
I
Received February 27, 2003. Accepted by SG May 23, 2003. Corresponding author’s e-mail:
[email protected].
The intestines of crustaceans contain small quantities of inorganic grits which, provided that there are sufficient quantities, can be used for TL analysis, following the standard density separation method (8, 16). Investigations in Finland (17) have shown good results from a small number of shellfish. A blind trial organized by the German Federal Health Office in 1994, on TL detection of irradiated Vietnamese shrimps was very promising (18). The physical recovery of intestinally trapped silicates is a laborious task, which may be prone to cross-contamination. Investigation at Scottish Universities Research and Reactor Centre (SURRC) of the use of acid hydrolysis (19) to simplify the method, suggested that this may have potential. The exoskeletal material of shellfish consists of an organic matrix containing inorganic minerals, predominantly calcite with minor apatite ingrowths. Calcite has well-established TL characteristics (20–22), which have mainly been studied for the purpose of dating. A study was conducted at SURRC (19) to examine the potential for measuring TL signals directly from shells to detect irradiation of shellfish for the enforcement of food labeling. The initial results from 9 species of shellfish with soft and hard shells indicated that the method is worth including in interlaboratory trials with the aim of validation. The present study was conducted in parallel with a TL study on fruit and vegetables, with which it shared prestudy material (23). The protocol was drafted in accordance with international validation criteria (24–26). The aims of this part of the study were to extend the range of species for which TL had been validated and to examine the success of different methods of preparation. With the carbonate method, included because the chosen products all had shell, establishment of an optimum classification threshold was an additional aim. To assess the case for validation, interlaboratory trials were initiated in 1995/1996, and have been completed. Samples of Nephrops norvegicus, mussels, black tiger prawns, brown shrimps, and king scallops, representing a mixture of warm and cold water species, were presented to participants in 3 conditions: nonirradiated and irradiated to 0.5 and 2.5 kGy. Each laboratory received one sample of each product in each category (total of 15) and was asked to implement 3 different preparation methods—dried powdered shell (carbonates), silicate extrac-
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mental and blanks performance. Results remain valid for this study. Homogeneity Testing Homogeneity testing was conducted at SURRC on a randomly selected excess material identical to that prepared for distribution to participants to characterize the TL response and assess the intrinsic variability of all samples for all methods within a single laboratory. Ten samples of each of the 5 products in each of their 3 categories, using all 3 methods, were analyzed (a total of 450 analyses). Choice of Samples and Predistribution Treatment Shellfish are candidates for irradiation preservation because they are high-value, easily perishable products, and radiation processing reduces pathogenic microorganisms. The samples chosen for this trial included a mixture of cold and warm water species: Norwegian N. norvegicus, Scottish mussels, English king scallops, Malaysian black tiger prawns, and Indian brown shrimps, purchased from a Glasgow shellfish wholesaler.
Figure 1. Glow ratios for each product [acid hydrolysis (a) and physical dissection (m) separation methods]. Homogeneity testing.
tion by acid hydrolysis, and silicate extraction by physical methods—followed by TL analysis. Experimental Protocol The analytical protocol was drafted in international format and sent to participants for comment. It gives instructions for sample preparation, measurement, and assessment. Three separation methods are featured: two silicate extractions (physical dissection and acid hydrolysis), and one using powdered shell (carbonates). Prestudy Materials The prestudy materials (LiF, International Atomic Energy Agency feldspars and 14C light source) were sent to participants for the fruit and vegetable study (23). The prestudy results were used to assess the individual laboratories’ instru-
Figure 2. Glow ratios for each product using powdered shell method. Homogeneity testing.
978 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003
On arrival at SURRC, the samples were immediately bagged into ca 500 g aliquots to avoid cross-contamination. Samples were randomly selected for irradiation at SURRC using 60Co, to doses of 0.5 and 2.5 kGy. Harwell Amber (Oxfordshire, UK) perspex dosimeters were positioned throughout the packages to ensure the correct dose. The 0.5 kGy samples received a mean dose of 0.49 ± 0.007 kGy, and the 2.5 kGy samples received a mean dose of 2.504 ± 0.09 kGy. The dosimetry system had been calibrated relative to National Physical Laboratory (NPL) standards in January 1995. Nonirradiated control samples were freezer-stored (–4°C) until the irradiation process was completed and the samples were ready for dispatch to participants. Excess material had been purchased and prepared to enable redistribution of samples to participants to replace spoiled consignments.
The protocol describes the use of 3 methods for the detection of irradiated shellfish: 2 silicate methods and a carbonate method. Minerals can be associated with both the exterior (shell) and the interior (flesh) of shellfish. The intestines of shellfish contain small quantities of inorganic grits, mainly silicates. The intestines are found as a tube (often dark) on the convex side of shrimps and prawns and in the interior of
molluscs. The shell consists of calcite together with organic material. (a) Acid hydrolysis (silicates).—Peeled whole samples or intestinal tracts were refluxed in 6M HCl for 2–3 h. After digestion was complete the solution was cooled, diluted with deionized water, and left to settle. The solution was decanted, leaving the minerals behind. These were then rinsed with water and washed with acetone to dry minerals. The separated minerals were then deposited onto stainless steel disks for TL readout. (b) Physical method (silicates).—The intestinal tract, where most of the minerals are found, was separated from the flesh of the prawn and placed in a beaker containing a small amount of deionized water. This step was repeated for several shellfish to gain sufficient material. To release the minerals, the solution was sieved after ultrasonic agitation; the minerals were then separated from organic constituents with heavy liquid (sodium polytungstate) and cleaned in HCl. Separated silicates were deposited onto stainless steel disks for TL readout. Glassware and process blanks were run in parallel with samples. (c) Carbonates.—Shells were dried in an oven at 50°C, prior to pulverizing in a ball mill or grinder. The sample was then sieved through a 125 mm mesh. The powder was freeze-dried
Figure 3. Participants’ results—acid hydrolysis.
Figure 4. Participants’ results—manual dissection.
Preparation of Blind Samples
SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 979
overnight to remove any bound water, and then dispensed onto stainless steel disks using silicone grease, for TL readout. TL Measurement and Recording of Results (a) Silicates.—Participants were asked to measure the first glow (G1) TL from room temperature to 400°C at 5°C/s, and then to irradiate the samples, using either a 60Co or a 90Sr source, prior to recording second glow (G2) under the same conditions. Participants were to report their TL data (G1 and G2 signals integrated over 20°C bands, and G1/G2) together with their qualitative decision for each sample. Their decision was to be based on both G1/G2 (glow ratio) and G1 glow shape. Where G2 did not exceed 10´ the minimum detectable level (MDL) as defined for each laboratory from the prestudy, the sample was to be rejected. The TL glow ratios for irradiated samples are usually greater than unity, while those from nonirradiated samples are generally 0.1 were to be classified as irradiated, to accommodate product-to-product interlaboratory variation. Results and Discussion Prestudy Results As with the fruit and vegetable study, prestudy results showed that there are still significant errors in temperature calibration in a number of laboratories. These did not affect qualitative results. MDLs were carried across from the prestudy to the blind analyses, but no rejections were necessary in addition to those identified by the participants. Homogeneity Testing The results from the homogeneity testing confirmed that both the silicate methods and the carbonate method are capable of distinguishing between nonirradiated and irradiated samples, at both doses. (a) Silicates.—The valid glow ratio data for both acid hydrolysis (a) and physical separation (m) methods from each product are presented in Figure 1. The glow ratios separate the nonirradiated and irradiated samples from each other, regardless of mineral separation technique. Lower glow ratios were observed for products that received the lower dose, which is consistent with expectations. The glow ratios for all irradiated samples typically were between 0.1 and 1. The response of product types to irradiation did not vary significantly from product to product. (b) Carbonates.—The integrated glow ratios from the 190°–200°C band for powdered shell are shown in Figure 2. There was good separation between nonirradiated and irradiated for all products. In all cases except brown shrimp, there was also clear distinction between the 0.5 and 2.5 kGy doses. It is notable for each product and category that there was significant variation in the range of glow ratios, and the application of a single threshold for all products was not possible, reinforcing earlier work (19). Participants’ Quantitative Results
Figure 5. Participants’ results—powdered shell.
Seven laboratories returned results of analysis, in the form of first and second glow values and glow ratios, and qualitative classification. There were some deviations from the procedure where some participants performed only single aliquot analysis, some did not return second glow data for samples which were below their own 10 MDL, and some did not return results for all 3 methods. All laboratories returned glassware and full process blanks to support their MDL values, which had not significantly changed since the prestudy. (a) Silicates–acid hydrolysis.—Figure 3 shows the glow ratios for the 220°–240°C temperature band for the results received for all products from 6 laboratories. Participants’ data resembled homogeneity testing. In some products, there was a greater spread of results than in the homogeneity testing. This is particularly noticeable in the soft shell products. Participants’ results showed that the irradiated samples are well dis-
980 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 Table 1. Qualitative results from 6 laboratories for acid hydrolysis method—a total of 90 blind coded samplesa
Product
No. of nonirradiated samples
0.5 kGy
+
–
+
–
No. sent
All irradiated samples
2.5 kGy +
–
Total
+
–
+
–
F
18
6
6
0
5
5
0
6
6
0
11
11
0
17
17
0
G
18
6
6
0
6
6
0
6
6
0
12
12
0
18
18
0
H
18
6
6
0
6
6
0
6
6
0
12
12
0
18
18
0
I
18
5
5
0
4
4
0
5
5
0
9
9
0
14
14
0
J
18
6
6
0
6
6
0
6
6
0
12
12
0
18
18
0
90
29
29
0
27
27
0
29
29
0
56
56
0
85
85
0
Total a
+ = Correct; – = incorrect; F = Nephrops; G = mussels; H = brown shrimps; I = black tiger prawns; J = king scallops. Total percentage of analyses leading = 94.5%; total percentage of results correct = 100%.
Participants’ Qualitative Results
tinguished from the nonirradiated samples within each laboratory, but separation between the 2 doses was not well resolved. Because of different laboratory conditions, there is some overlap of the absolute values of the ratios observed. This reinforces the importance of the homogeneity testing in distinguishing between inter- and intralaboratory variation. (b) Silicates–physical method.—Figure 4, the 220°–240°C temperature band glow ratio plot, shows the 7 participants’ results for all products using the physical separation method. As with the results from the acid hydrolysis method, the spread of glow ratio data is similar to that from the homogeneity testing. Again, results show the ability to distinguish between irradiated and nonirradiated products (c) Carbonates.—Five laboratories returned data for powdered shell. The results are presented in Figure 5 as glow ratios for all products for the 190°–200°C band. Distinction between all products and categories is less well-defined than that in the homogeneity testing data, most likely as a result of fading during the period between irradiation and measurement. Most participants’ data did, however, show separation between irradiated and nonirradiated products.
The qualitative results for each coded sample were compared with the true identity of each sample. When interpreting the data, participants followed the guidelines in the protocol; they also stated that glow shape was considered along with glow ratios. (a) Acid hydrolysis.—From the 6 laboratories, 85 results were reported from a total of 90 blind samples. All reported results were correctly identified. For the 5 cases where no conclusion was achieved, mineral yields were inadequate. Table 1 summarizes the participants’ results where 100% correct classification was achieved (b) Physical method.—The qualitative results from the 7 participants are displayed in Table 2. From a total of 105 blind samples, 103 results were received, all of which were correctly classified. The other 2 analyses failed sensitivity criteria. (c) Carbonates.—The results from the 5 laboratories for powdered shell are summarized in Table 3. From a total of 75 blind samples, 64 were correctly identified (85.3%). Eight samples from the 0.5 kGy category were misclassified. These were the only misclassifications in the entire study. The samples satisfied sensitivity criteria but did have low signal inten-
Table 2. Qualitative results from 7 laboratories for physical dissection method—a total of 105 blind coded samplesa No. of nonirradiated samples Product No. sent
+
–
0.5 kGy
2.5 kGy
+
–
+
–
All irradiated samples
Total
+
–
+
–
F
21
7
7
0
7
7
0
7
7
0
14
14
0
21
21
0
G
21
7
7
0
7
7
0
7
7
0
14
14
0
21
21
0
H
21
7
7
0
7
7
0
7
7
0
14
14
0
21
21
0
I
21
7
7
0
6
6
0
6
6
0
12
12
0
19
19
0
J
21
7
7
0
7
7
0
7
7
0
14
14
0
21
21
0
105
35
35
0
34
34
0
34
34
0
68
68
0
103
103
0
Total a
+ = Correct; – = incorrect; F = Nephrops; G = mussels; H = brown shrimps; I = black tiger prawns; J = king scallops. Total percentage of analyses leading = 98.1%; total percentage of results correct = 100%.
SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 981 Table 3. Qualitative results from 5 laboratories for powdered shell—a total of 75 blind coded samplesa No. of nonirradiated samples Product F
No. sent 15
5
0.5 kGy
All irradiated samples
2.5 kGy
Total
+
–
+
–
+
–
+
–
+
–
5
0
5
5
0
5
5
0
10
10
0
15
15
0
G
15
5
5
0
5
5
0
5
5
0
10
10
0
15
15
0
H
15
5
4
1
5
3
2
5
5
0
10
8
2
15
12
3
I
15
5
4
1
5
3
2
5
4
1
10
7
3
15
11
4
J
15
5
4
1
5
4
1
5
3
2
10
7
3
15
11
4
75
25
22
3
25
20
5
25
22
3
50
42
8
75
64
11
Total a
+ = Correct; – = incorrect; F = Nephrops; G = mussels; H = brown shrimps; I = black tiger prawns; J = king scallops. Total percentage correct = 85.3%.
sities. Residual organic material can also affect the signal, and it is not absolutely clear whether participants changed their instrument filters for this part of the study. Conclusions A blind international interlaboratory trial was conducted with the aim of extending the validation of the TL detection method to irradiated shellfish. Five species of shellfish were selected and presented in 3 blind categories: nonirradiated and irradiated with 0.5 and 2.5 kGy. Seven laboratories returned results for the blind samples, of which 5 laboratories reported results for all 3 methods and 6 reported results for both silicate methods. Results were returned as first and second glow integrals, glow ratios, and qualitative classifications for all products. Glow ratio and glow shape were considered by participants when classifying samples. Participants’ results for the silicate methods confirmed the homogeneity testing results in their ability to distinguish between nonirradiated and irradiated products. Rejection criteria were rigorously applied: 94.4% of acid hydrolysis and 98.1% physical separation yielded valid results, as did 96% of carbonate analysis. Qualitative results for silicates were 100% correct. With the powdered shell method, 85.3% were correctly identified. The study demonstrated the case for extending validation to these products. The results for silicate analysis were very good, and thoroughly validated the methods. The performance of the powdered shell method satisfied international criteria (80%) for validation, but participants’ results did not match those from homogeneity testing, and the silicate methods should be used in preference, when only shell is available. In general, it was noted that the acid hydrolysis method gave slightly improved discrimination compared with physical separation, although both methods gave satisfactory qualitative performance.
Acknowledgments Support for this work by the Ministry of Agriculture Fisheries and Food (MAFF) under contract No. 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. Christensen, Risø, Roskilde, Denmark H. Delincée, Federal Research Centre for Nutrition, Karlsruhe, Germany H. Nootenboom, Food Inspection Service, Nijmegen, The Netherlands J. Pfordt, Staatliches Lebensmitteluntersuchungsamt, Oldenburg, Germany S. Pinnioja, University of Helsinki, Helsinki, Finland G.A. Schreiber and U. Wagner, BGVV, Berlin, Germany References (1) Sanderson, D.C.W., Slater, C., & Cairns, K.J (1989) Nature 340, 23–24 (2) Sanderson, D.C.W., Slater, C., & Cairns, K.J. (1989) Radiat. Phys. Chem. 34, 915–924 (3) Sanderson, D.C.W., Slater, C., & Cairns, K.J. (1989) Int. J. Radiat. Biol. 55, 5 (4) 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 (5) Sanderson, D.C.W., Carmichael, L.A., Ni Riain, S., Naylor, J., & Spencer, J.Q. (1994) Food Sci. Technol. Today 8, 93–96 (6) Sanderson, D.C.W., Carmichael, L.A., & Naylor, J.D. (1995) Food Sci. Techno. Today 9, 150–154 (7) 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 (8) MAFF (1992) Detection of Irradiated Herbs and Spices, Validated Method for the Analysis of Foodstuffs, V27, Ministry of Agriculture, Fisheries and Foods, Norwich, UK (9) MAFF (1993) J. Assoc. Public Anal. 29, 187–200
982 SANDERSON ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 5, 2003 (10) Sanderson, D.C.W., Schreiber, G.A., & Carmichael, L.A. (1991) A European Interlaboratory Trial of TL Detection of Irradiated Herbs and Spices, Phase 1, SURRC report to BCR, European Commission, Brussels, Belgium (11) Raffi, J., Fakirian, A., & Lesgards, G. (1994) Ann. Falsif. Expert. Chim. Toxicol. 87, 125–134 (12) Pinnioja, S. (1993) Radiat. Phys. Chem. 42, 394–400 (13) Delincée, H. (1993) Radiat. Phys. Chem. 42, 351–357 (14) Roberts, P.B., & Hammerton, K.M. (1994) in Detection Methods for Irradiated Foods, C.H. McMurray, R. Gray, E.M. Stewart, & J. Pearce (Eds), Royal Society of Chemistry, Cambridge, UK, pp 178–181 (15) Hammerton, K.M., & Banos, C. (1994) in Detection Methods for Irradiated Foods, C.H. McMurray, R. Gray, E.M. Stewart, & J. Pearce (Eds), Royal Society of Chemistry, Cambridge, UK, pp 168–171 (16) European Standard Method (1997) Foodstuffs—Detection of Irradiated Food from which Silicate Minerals Can Be Isolated: Method by Thermoluminescence, BS EN 1788, BSI, London, UK (17) Autio, T., & Pinnioja, S. (1992) in Recent Advances on the Detection of Irradiated Food, EUR/14315/EN, Luxembourg, pp 177–185 (18) Schreiber, G.A., Wagner, U., Ammon, J., Buchholtz, H.V., Delincée, H., Estendorfer, S., von Grabowski, H.U., Kruspe,
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