an international collaborative blind trial on electron spin resonance (esr) identification of...

J Sci Food Agric 1997, 74, 473È484

An International Collaborative Blind Trial onElectron Spin Resonance Identiücation of(ESR)Irradiated Crustacea

Eileen M Stewart1,* and David J Kilpatrick2

1 Department of Food Science and 2 Department of Biometrics, The QueenÏs University of Belfastand The Department of Agriculture for Northern Ireland, Newforge Lane, Belfast, BT9 5PX, UK

(Received 12 July 1996 ; revised version received 20 November 1996 ; accepted 19 February 1997)

Abstract : The results of an interlaboratory collaborative trial are presentedwhere 15 participating laboratories used the technique of ESR spectroscopy todetect three species of Crustacea irradiated in the frozen state and stored at[20¡C. Samples of Norway lobster (Nephrops norvegicus), pink shrimp(Pandalus montagui) and crevettes (Penaeus (Penaeus) semisulcatus) were irradi-ated at dose levels of approximately 1, 3 and 5 kGy or left unirradiated. Eachlaboratory analysed nine samples of each species approximately 2 weeks follow-ing irradiation and the same number after storage for 2 months at [20¡C. Intotal, 270 samples were analysed for each of the three species of Crustacea. A95% correct identiÐcation rate was achieved for the crevette samples where 256of the 270 samples examined were correctly identiÐed. Two-hundred and twenty-six Norway lobster samples were correctly identiÐed, giving a correct identiÐca-tion rate of 84%. The most disappointing results were associated with pinkshrimp, where only 54% of the samples were correctly identiÐed. Storage at[20¡C was not found to have a signiÐcant e†ect on detection of the irradiatedsamples.

J Sci Food Agric 74, 473È484 (1997)No. of Figures : 4. No. of Tables : 5. No. of References : 28

Key words : Crustacea, shrimp, prawn, crevette, scampi, cuticle, c-irradiation,ESR, interlaboratory trial, irradiation detection

INTRODUCTION

At present a number of tests are being developed for theidentiÐcation of irradiated Crustacea and these includethermoluminescence (Schreiber et al 1995 ; Sanderson etal 1996), detection of 2-alkylcyclobutanones (McMurrayet al 1995) and electron spin resonance (ESR) spectros-copy (Desrosiers 1989 ; Stewart et al 1992 ; Helle et al1993 ; Morehouse and Desrosiers 1993 ; Stewart et al1994).

The technique of ESR spectroscopy is used to detectunpaired electrons in reactive entities, such as free rad-

* To whom correspondence should be addressed.

icals, which are normal components of many biologicalsystems. Irradiation, like other processing technologieswhich impart energy to food, also produces free rad-icals, but generally the latter have such a short life-spanthat they cannot be detected. However, if a foodstu†contains hard, dry components, such as bone, shell orseeds, the radicals can be trapped and their presenceconÐrmed by ESR spectroscopy (Stevenson and Gray1990 ; Raffi et al 1992).

In 1985, Dodd et al reported that ESR spectroscopyhad potential as an identiÐcation method for irradiatedCrustacea using the signal induced in the cuticle, whichhas a relatively high dry matter concentration. This

4731997 SCI. J Sci Food Agric 0022-5142/97/$17.50. Printed in Great Britain(

474 E M Stewart, D J Kilpatrick

Ðnding was later conÐrmed by workers such asGoodman et al (1989), Desrosiers (1989), Raffi andAgnel (1990), Stewart et al (1992, 1994), Morehouse andKu (1992) and Helle et al (1993). However, it was highlyevident from reported results that the ESR signalinduced in prawn or shrimp cuticle by ionising radi-ation is species-dependent. Therefore, it was recom-mended (Anon 1992) that, in all experimental work, thespecies of prawn or shrimp being examined should beidentiÐed by its scientiÐc name in order to avoid dis-crepancies in the results.

As with other detection methods, the usefulness ofESR spectroscopy as a test for the detection of irradi-ated Crustacea will have to be veriÐed in inter-laboratory blind trials where the history of the samplesis unknown to the analyst. To date the technique hasbeen evaluated as a detection method in a number ofintercomparisons organised by the Community Bureauof Reference (BCR) (Raffi et al 1992), the InternationalAtomic Energy Agency (IAEA) (Anon 1992 ; Desrosierset al 1994, 1996), the Ministry of Agriculture, Fisheriesand Food (MAFF) in the UK (Scotter et al 1990), theInstitut fu� r Sozialmedizin und Epidemiolgie des Bun-desgesundheitsamtes (BGA) in Germany (Schreiber et al1993, 1995, 1996) and the National Institute for Stan-dards and Technology (NIST) in the USA (Desrosiers etal 1990).

An in-house trial carried out in Belfast showed thepotential of using ESR spectroscopy for the identiÐca-tion of irradiated Norway lobster in a blind situation(Stewart and Stevenson 1997). In this trial, all 72 blindcoded samples received for analysis were identiÐed witha 100% success rate. The samples were either non-irradiated or had received mean irradiation doses ofapproximately 1, 2 or 3 kGy and were stored for 0, 7 or14 days at 1¡C.

In 1993/94 the Ðrst interlaboratory collaborative trialfor the detection of irradiated Crustacea was carried outby the BGA (Helle et al 1994 ; Schreiber et al 1996). Inthis trial, each of 20 participating laboratories receivedeight samples each of Norway lobster (origin : Mediter-ranean Sea) and brown shrimp (Crangon crangon),which were either non-irradiated or had been treatedwith doses of 2, 4 or 6 kGy. Of the 168 results reportedfor Norway lobster, one irradiated sample was incor-rectly identiÐed as non-irradiated (false negative) andthree non-irradiated samples were classiÐed as irradi-ated (false positive), while 98% were correctly identiÐed.In the case of brown shrimp, there was one false nega-tive, with 166 being successfully detected, giving a 99%correct identiÐcation rate.

This paper details an interlaboratory trial carried outin 1994/95, funded by MAFF. The objective of theintercomparison was to examine the potential of ESRspectroscopy for the identiÐcation of Norway lobster(Nephrops norvegicus) and two species of Crustacea notpreviously examined in a blind trial, ie pink shrimp

(Pandalus montagui) and crevettes (Penaeus (Penaeus)semisulcatus). In order to establish whether storage at[20¡C a†ected the results, samples were to be analysedduring December 1994 and 2 months later in February1995.

EXPERIMENTAL

Laboratories involved

Initially, 17 laboratories from a number of countrieswere approached to participate in the intercomparisonand, subsequently, 15 laboratories agreed to do so.

Sample preparation

It was decided to prepare and treat the Crustaceasamples in the frozen state, as it was not possible toobtain all three species at the same time and this wouldhave resulted in problems with maintaining the samplesin the fresh state prior to irradiation. Results from pre-vious work (Stewart et al 1993a) have shown that irra-diation, eg of Norway lobster, in the frozen state doesnot have a signiÐcant e†ect on the detection of irradi-ated samples or on the intensity of the radiation-induced ESR signal.

Norway lobsterWhole tails (meat with tail shell attached) of Norwaylobster, freshly caught in the Irish Sea, were purchasedfrom a local Ðsh merchant in Northern Ireland andtransported to the laboratory in a refrigerated vanmaintained at 4 ^ 1¡C. One whole tail was placed ineach of 180 resealable polythene bags. The samples werethen randomly allocated to the various treatments(irradiated or non-irradiated), coded and stored at[20¡C prior to irradiation.

Pink shrimpFresh whole pink shrimp (meat with full exoskeletonattached) were obtained from a local Ðsherman inNorthern Ireland, and as for the Norway lobstersamples were caught in the Irish Sea. On arrival at thelaboratory, Ðve shrimp were placed in each of 180resealable polythene bags after which they were treatedin a similar manner to the Norway lobster samples.

CrevetteWhole crevettes (meat with full exoskeleton attached)were purchased in the frozen state from a retail outlet in

International blind trial on ESR identiÐcation of irradiated Crustacea 475

London. These crevettes were of the farmed variety andoriginated from Guatemala. The samples arrived at thelaboratory by express post and were still in the frozenstate. One whole crevette was placed in each of 180resealable polythene bags and treated as for the pre-vious two species.

Irradiation

Samples were either not irradiated or given doses ofapproximately 1, 3 or 5 kGy. Cobalt 60 was used as thesource of ionising radiation (Gammabeam 650, NordionInternational Inc., Kanata, Canada) at a dose rate of1É3 kGy h~1. In order to keep the samples in the frozenstate, the temperature of the irradiation room wasmaintained at [4 ^ 1¡C. Following irradiation, thesamples were packed in polystyrene boxes containingdry ice and sent to each of the participating laboratoriesby express post. It was anticipated that the Europeansamples should arrive within 48 h of dispatch, whilethose destined for the USA would arrive within 72 h.

Dosimetry

In order to measure the irradiation doses received bythe samples, perspex gammachrome YR dosimeters(Atomic Energy Agency (AEA) Technology, Harwell,UK) were attached to the samples being given an irra-diation dose of 1 kGy, while amber perspex dosimeters(Type 3042C, AEA Technology) were used for thosereceiving 3 and 5 kGy. In total, 42 dosimeters were usedper dose. The change in absorbance of the gammach-rome and amber perspex dosimeters was measuredspectrophotometrically at 530 and 603 nm, respectively.The corresponding doses were calculated using cali-bration graphs provided by the National PhysicalLaboratory (NPL), Teddington, UK. In order to furthercheck the dosimetry, four alanine dosimeters (NPL,Teddington, UK) were also included with the samplesat each irradiation dose and the dose received by thesedosimeters was measured at the NPL.

Samples for analysis

Each laboratory received 54 randomly coded samplesfor analysis, 27 of which were identiÐed for assessmentimmediately and the remaining 27 to be analysed fol-lowing storage for 2 months at [20¡C. Within eachbatch of 27 samples, there were nine samples of eachspecies, with two at each of the four irradiation doses, ie0, 1, 3 or 5 kGy; the ninth sample was irradiated ateither 1 or 3 kGy and assigned to the various labor-atories in order to give an equal number of samples atthese two doses over all laboratories. Thus, the totalnumber of samples was 810, of which 180 were at0 kGy, 225 at 1 kGy, 225 at 3 kGy and 180 at 5 kGy.

Protocol for sample analysis

The following instructions were sent to each partici-pating laboratory prior to receiving the test samples.

Remove the tail section of each sample and cleanaway any adhering meat from the cuticle. Freeze-drythe cuticle for 18È22 h or dry in a vacuum dryingoven at 40¡C for 2 h. Crush the dried cuticle, egbetween two sheets of paper. Transfer approximately100 mg of the sample to an ESR tube of 5È6 mminternal diameter.

Place the ESR tube in the cavity of the ESRspectrometer, making sure that the sample is posi-tioned exactly in the centre of the cavity in order toobtain the optimum ESR signal. Derive the ESRspectrum of each sample at room temperature usingthe operating conditions given (Table 1). The oper-ating conditions may be adjusted to obtain theoptimum ESR signal.

Use the results sheet supplied to make a note of thesample number, the date on which the sample wasmeasured and the sample weight used. Due to di†er-ences in the shape of the ESR signals derived fromdi†erent varieties of prawns and shrimp, it seemshighly probable that the ESR signal is species-depen-dent. Therefore, identiÐcation of irradiated sampleshas to be performed by comparison of spectra fromsamples of the same species. The ESR spectra shownin Figs 1 to 3 (supplied with the protocol) are typicalof the species being analysed.

Norway lobsterThe ESR spectrum obtained from the cuticle of non-irradiated Norway lobster (Fig 1a) is typical of man-ganese (Mn2`), while irradiated samples (Fig 1b and 1c)show an additional unsymmetrical line with g-values of2É0010 ^ 0É0010 (main signal and 1É9970 ^ 0É0010g1)(shoulder It is this additional peak which indicatesg2).that the Norway lobster sample has been irradiated.

TABLE 1ESR spectrometer operating conditions

Centre of magnetic Ðeld 3494 gauss \ 349É4 mTSweep width 200 gauss \ 20É0 mTMicrowave power 15É8 mWMicrowave frequency 9É77 GHzModulation amplitude 3 gauss \ 0É3 mTConversion time 40É96 msTime constant 163É84 msSweep time 41É94 sResolution of Ðeld axis 1024Receiver gain 4É0 ] 104Number of scans 4

Note : Operating conditions for the Bruker EMS104 were thesame, with the exception of receiver gain which was 55 dB.


Fig 1. ESR spectra derived from Norway lobster cuticle : (a)non-irradiated, (b) irradiated at 1 kGy and (c) irradiated at5 kGy: g1\ 2É0010 ^ 0É0010 ; g2 \ 1É9970 ^ 0É0010 ;

H \ 2É0 mT; ] \ increasing H.

Fig 2. ESR spectra derived from pink shrimp cuticle : (a) non-irradiated, (b) irradiated at 1 kGy and (c) irradiated at 5 kGy:

H \ 2É0 mT;g1\ 2É0010 ^ 0É0010 ; g2 \ 1É9970 ^ 0É0010 ;] \ increasing H.

Fig 3. ESR spectra derived from crevette cuticle : (a) non-irradiated, (b) irradiated at 1 kGy and (c) irradiated at 5 kGy:g1\ 2É0040 ^ 0É0010 ; g2 \ 2É0010 ^ 0É0010 ; g3 \ 1É9970

H \ 2É0 mT; ] \ increasing H.^ 0É0010 ;

Pink shrimpThe ESR spectrum from non-irradiated pink shrimp(Fig 2a) consists of a singlet with little evidence ofMn2` present. The ESR spectrum derived from thecuticle of irradiated pink shrimp (Figs 2b and 2c) con-sists of several components. The lines indicated with g1(2É0010 ^ 0É0010) and (1É9970 ^ 0É0010) are due tog2an irradiation speciÐc radical and are not present in thenon-irradiated spectrum.

CrevetteThe ESR spectrum derived from the cuticle of non-irradiated crevettes (Fig 3a) is a weak singlet, while thatfrom irradiated shell is more complicated (Figs 3b and3c). The radiation-induced ESR signal can be identiÐedby g-values of 2É0040 ^ 0É0010 2É0010 ^ 0É0010(g1),

and 1É9970 ^ 0É0010 As for the pink shrimp,(g2) (g3).there is little evidence of Mn2` present.

The irradiated spectra given in the protocol are at doselevels of 1 and 5 kGy. Thus, in addition to making aqualitative assessment of the samples, participants arealso asked to attempt a visual classiÐcation of theabsorbed dose, indicating whether the samples identi-Ðed as irradiated have received a low, medium or highdose.

Statistical analysis

For each Crustacea species and actual irradiation dose,the results were tabulated in contingency tables classi-


Ðed by laboratory, storage time and estimated irradia-tion state. Two sets of tables were calculatedcorresponding to a qualitative (non-irradiated/irradiated) or a quantitative (zero/low/medium/high)estimated irradiation state. From these tabulations, theprobabilities of mis-identiÐcation were calculated. Alog-linear model was Ðtted to these contingency tablesin order to test for independence between the classifyingfactors, ie whether the classiÐcation to estimated irra-diation status group di†ered for laboratories averagedover storage times, similar to storage times averagedover laboratories and also for interaction betweenlaboratories and storage times.

RESULTS

The mean doses determined using the perspex andalanine dosimeters were in close agreement with eachother (Table 2). The total number of unknown samplesexamined together with those which were correctlyidentiÐed on a qualitative basis at each time period aregiven in Table 3. The number of samples correctly iden-

TABLE 2Mean irradiation doses received by the Crustacea samples

determined using both perspex and alanine dosimeters

Nominal Measured dose (kGy) Standard deviationdose(kGy) Perspex Alanine Perspex Alanine

1É0 1É18 1É13 0É072 0É0733É0 3É32 3É39 0É201 0É0245É0 5É61 5É67 0É206 0É043

tiÐed by the individual laboratories at each samplingtime are presented in Table 4.

Norway lobster

Of the 135 samples analysed initially, 111 were correctlyidentiÐed, while 115 of the 135 samples examined fol-lowing 2 months storage at frozen temperatures werealso identiÐed correctly (Table 3). In total, there werefour false positives and one sample which could not be

TABLE 3Total number of Crustacea samples analysed at each time period together with the number of correct identiÐca-

tions

Crustacea species Dose received Number of samples T ime after irradiation Correct identiÐcations(kGy) (W eeks)

Norway lobster 0É00 33 2 3232 10 28

1É13 35 2 1635 10 21

3É32 35 2 3435 10 35

5É61 32 2 2933 10 31

Total 270 226

Pink shrimp 0É00 32 2 2233 10 21

1É13 35 2 1435 10 13

3É32 35 2 1635 10 23

5É61 33 2 1932 10 18

Total 270 146

Crevette 0É00 33 2 3133 10 30

1É13 35 2 3235 10 32

3É32 35 2 3435 10 34

5É61 32 2 3132 10 32

Total 270 256


TABLE 4Number of Crustacea samples correctly identiÐed by each participating laboratory at each

time period

Participating Crustacea species

laboratoryNorway Norway Pink Pink Crevette Crevettelobster lobster shrimp shrimp 2 weeks 10 weeks2 weeks 10 weeks 2 weeks 10 weeks

A 7 9 ” 6 8 9B 9 6 ” ” 9 9C 6 8 ” ” 9 9D 7 8 2 2 9 9E 7 7 3 2 9 8F 7 9 5 4 7 7G 7 8 2 2 9 9H 7 8 8 7 9 9J 9 9 9 7 9 9K 5 2 5 8 5 6L 8 8 7 8 9 9M 8 9 8 8* 9 9N 7 9 6 8 9 9P 9 8 9 6 9 9Q 8 7 7 7 9 8a

A total of nine samples were analysed for each Crustacea species at each time period.* One blind coded sample was not present in the batch received by the participating labor-atory.” Participating laboratory did not attempt to identify the samples.

identiÐed. At the lowest dose level of 1 kGy, there were37 false negatives, along with two samples for which noidentiÐcation was made. All samples which had beengiven an irradiation dose of 3 kGy and stored for 2months at frozen temperatures were correctly identiÐed,while only one sample was identiÐed incorrectly in thebatch of samples examined after the 2 weeks. A total ofÐve samples were incorrectly identiÐed as non-irradiated at the highest dose level of 5 kGy, four ofthese being from the same laboratory (laboratory K,Table 4) which could not identify samples at the 0 and1 kGy level and incorrectly identiÐed a 3 kGy sample.The latter laboratory could not identify, or incorrectlyidentiÐed, 11 out of the total 18 Norway lobster samplesanalysed. The other sample incorrectly judged as non-irradiated at the 5 kGy dose level was due to misinter-pretation of the ESR spectrum.

Overall, 92% of the non-irradiated samples were cor-rectly identiÐed, as were 82% of the irradiated samples.If the results of laboratory K, which had the highestnumber of incorrect identiÐcations, were excluded, anoverall correct identiÐcation rate of 87% would havebeen achieved. Statistical analysis of these qualitativeresults showed that the probability of making a correctidentiÐcation of samples as either irradiated or non-irradiated did not signiÐcantly di†er between storagetimes or between laboratories.

Figure 4a shows the dose classiÐcation made by thevarious laboratories for both the irradiated and non-irradiated Norway lobster samples over both storageperiods. The participating laboratories were required, ifpossible, to classify the samples as having receivedeither a zero, low, medium or high irradiation dose. Itcan be seen that, in the case of the samples which hadreceived 3 and 5 kGy, approximately 50 and 54% of thesamples, respectively, were correctly classiÐed. In thecase of the 1 kGy samples, only 34% were correctlyclassiÐed as having received a low dose of irradiation,with 46% incorrectly classiÐed as non-irradiated. Theseresults were improved when the data of laboratory Kwere ignored, with 40, 54 and 57% of the 1, 3 and5 kGy samples being correctly classiÐed.

There was no signiÐcant e†ect of storage time at anyof the dose levels. However, a signiÐcant laboratorye†ect (P\ 0É05) was found at all doses except zero.When the results of laboratory K were excluded, thissigniÐcant e†ect was still found at the 1 and 3 kGy doselevels, but not at the 5 kGy level.

Pink shrimp

Overall, of the 270 samples analysed, only 50% of theirradiated samples and 66% of the non-irradiated


Fig 4. Dose classiÐcation of (a) Norway lobster, (b) pink shrimp and (c) crevette samples over both storage periods.


samples were correctly identiÐed (Table 3). Two of theparticipating laboratories did not attempt to interpretthe spectra derived from this species of Crustacea oneither occasion, while three laboratories identiÐed all ofthe samples as being non-irradiated (Table 4). Of the 15participants only one laboratory (L) derived spectrasimilar to those given in the working protocol. Labor-atory L correctly identiÐed 15 out of the 18 pink shrimpsamples examined (Table 4). However, when the ESRspectra from the three samples incorrectly identiÐedwere examined, it was found that one irradiated (1 kGy)spectrum had been misinterpretated as non-irradiated,while two samples which were non-irradiated controlshad unexpectedly given ESR signals typical of thosederived from irradiated cuticle. The latter two sampleswere re-analysed and, once again, the characteristic irra-diated spectra were derived ; therefore, it is assumed thatthe samples had been miscoded.

Statistical analysis showed that storage at frozen tem-peratures did not inÑuence detection of the irradiatedsamples. However, a highly signiÐcant (P\ 0É001) dif-ference was found between the participating labor-atories for both non-irradiated and irradiated samples.

Figure 4b shows that, with such a high number ofincorrect identiÐcations being made, it was clear thatthe laboratories had great difficulty in making a correctquantitative assessment of the dose received by thesamples. As an example, at the 3 kGy dose level, only22% of the samples were classiÐed as having received amedium dose of irradiation.

On a quantitative basis there was no signiÐcant e†ectof the length of storage. A very highly signiÐcant(P\ 0É001) di†erence was observed between labor-atories at the higher dose levels of 3 and 5 kGy but notat the lower doses.

Crevette

On both occasions, 128 of the 135 samples examinedwere identiÐed correctly (Table 3). Laboratory A (Table4) identiÐed one non-irradiated crevette sample as beingirradiated ; however, a check on the ESR spectrum ofthis sample showed that it had been misinterpreted.Laboratory F also incorrectly identiÐed four samples,with two non-irradiated crevettes being identiÐed asirradiated and two irradiated samples as non-irradiated.However, it should be pointed out that, in the case ofthree of these samples, the analyst did signify that theidentiÐcations made were questionable due to the noisybaseline of the spectra. The sample incorrectly identiÐedby laboratory E (Table 4) was at the lowest irradiationdose of 1 kGy and the derived spectrum had a lowsignal-to-noise ratio making interpretation difficult. Ofthe 14 samples which were incorrectly identiÐed, sevenwere analysed by the same laboratory (K) that had alsoencountered problems with the Norway lobster. Two of

the samples were non-irradiated, three had received adose of 1 kGy, one a dose of 3 kGy and the Ðnal samplewas at the highest dose level of 5 kGy. The ESR spectraderived for the crevette samples by this laboratoryexhibited numerous interfering lines, making qualitativeassessment of the spectra extremely difficult. Thesespectra did not resemble those given in the workingprotocol for this species and could not be used to makean unequivocal identiÐcation of irradiated samples.

Overall, 92% of the non-irradiated crevette sampleswere correctly identiÐed and 96% of the irradiatedsamples. If the results of laboratory K, which incorrect-ly identiÐed seven of the samples, were discounted, anoverall correct identiÐcation rate of 98% was achieved.It is also noteworthy that, on each of both storageoccasions, a total of 12 laboratories achieved a 100%correct identiÐcation rate (Table 4).

As for the Norway lobster, a statistical analysis of thequalitative results showed that the probability ofmaking a correct identiÐcation of irradiated sampleswas not signiÐcantly a†ected by either storage or testinglaboratory.

The dose classiÐcation made by the 15 participatinglaboratories over both storage periods are presented inFig 4c. Sixty-two per cent of the 1 kGy samples werecorrectly classiÐed as having received a low dose, 46%of the 3 kGy samples as having been given a mediumdose of irradiation and 51% of the 5 kGy samples asbeing irradiated with a high dose.

On a quantitative basis there was a signiÐcant(P\ 0É05) e†ect of storage time at the 5 kGy dose levelonly. A signiÐcant di†erence (P\ 0É05) was foundbetween participating laboratories at the 1 kGy dosebut not at the other doses. The latter e†ect was notobserved when the results of laboratory K wereexcluded from analysis.

DISCUSSION

Of the three species tested in the trial, the irradiatedcrevette samples exhibited the highest correct identiÐca-tion rate (96%), while the irradiated pink shrimpsamples proved to be the most difficult to detect (50%).

There was a higher probability of making an incor-rect identiÐcation of samples irradiated at the lowestdose level, and this applied especially to the Norwaylobster, where only 53% of the samples given 1 kGywere identiÐed as irradiated. In the case of the lattersamples, it was found that the radiation-induced peakwas not as well deÐned as it was at the higher doselevels of 3 and 5 kGy. However, it has been found(Stewart 1993) that, if only one sliver or fragment of tailcuticle (approximately 10È20 mg in weight) is taken, theESR spectrum derived exhibits a more pronounced freeradical peak which is easier to identify. It is possible toobtain such a sample from the Norway lobster exoskel-


eton, since the cuticle is more rigid compared to otherspecies of prawn or shrimp. In addition, if wholeNorway lobsters are received at a testing laboratory, itwould also be advantageous to use cuticle from theclaws/nippers for ESR analysis, as these are the hardestcomponents of the exoskeleton and have been shown inprevious work (Stewart et al 1993b) to exhibit the mostintense ESR signal.

The correct identiÐcation rate for Norway lobster inthis collaborative trial was somewhat lower than thatobtained for the in-house blind trial carried out pre-viously where a 100% correct identiÐcation rate wasachieved (Stewart and Stevenson 1997). However, thiswas most likely due to the fact that the analyst examin-ing the samples in the latter trial was familiar with theprotocol and the species in question, thereby decreasingthe possibility of making an incorrect identiÐcation.

Of the 14 crevette samples incorrectly identiÐed, sixwere at the 1 kGy dose level, with three of the latterbeing associated with one laboratory. The incorrectidentiÐcations were as a result of the poor spectraderived from the samples which exhibited low signal-to-noise ratios and numerous interfering lines. Over all thelaboratories, the spectra derived for the crevettes weresimilar in shape to those given in the working protocol,as were the spectra for the Norway lobster. However,the latter was not found to be the case for the pinkshrimp.

The e†ect of storage on the radiation-induced signalmay have accounted for the inability to detect the irra-diated pink shrimp samples. Previous experimentalwork carried out by Stewart and Gray (1996) showedthat storage at [20¡C does have a highly signiÐcante†ect on the signal stability of this particular species ofpink shrimp. The most signiÐcant decrease in signalstrength occurs during the initial 7 days of storage, afterwhich there is a more gradual decline. It may be thecase that, by the time the participating laboratoriesreceived and analysed the samples, the radiation-induced signal had decreased to such an extent that itcould not be detected. In contrast to the ESR signalgiven for irradiated pink shrimp in the working proto-col, a number of laboratories derived an intense broadunspeciÐc singlet. Other laboratories also derived thelatter signal, along with other resonances which werenot present in non-irradiated samples, thereby makingqualitative identiÐcation possible. The radiation-induced signal was present in the latter samples,although it had decayed and was being overlapped bythe broad unspeciÐc resonance line previously men-tioned.

It had been suggested that thawing of the frozensamples during transport to the laboratories followedby re-freezing on arrival at their destination may havea†ected interpretation of the ESR spectra. However,work carried out by Desrosiers (1989) on pink shrimpstored at [20¡C showed that the ESR signal survived

four freezeÈthaw cycles and could be detected after 43days, although the intensity was reduced. Therefore, itseems unlikely that freezing and thawing of the sampleshad a signiÐcant e†ect on the ESR spectra of the pinkshrimp in this trial.

The sensitivity of the ESR spectrometer magneticcavity being used to derive the spectra may also haveaccounted for the variability in the ESR spectra derivedfrom the pink shrimp. The laboratory which derived theESR spectra similar to those given in the protocol useda Bruker ECS106 spectrometer Ðtted with a TMH ECS4108/9105 single resonance cavity. In general, it wasfound that the laboratories with the ESP300 andECS106 spectrometers produced spectra which couldnot be used to identify irradiated pink shrimp samples.The latter was not the case for Norway lobster or cre-vettes, where the type of spectrometer did not appear tohave a signiÐcant e†ect on qualitative identiÐcation ofthe samples. Raffi et al (1992) also suggested the sensi-tivity of the ESR cavity may have had an inÑuence onthe results of the BCR collaborative trial, especially ona quantitative basis.

One participating laboratory (laboratory P, Table 4)undertook an alternative approach for derivation of theESR spectra for the pink shrimp samples. On initialexamination the spectra derived were composed of theunspeciÐc broad singlet as described previously. Thelaboratory in question then recorded the spectra as thesecond derivative using a higher power, and the sampleswere assessed on the appearance of two high Ðeld peaksin the spectra. The latter approach provided a bettercontrast from sample to sample on initial analysis,where all nine samples were correctly identiÐed, butfailed to do so following storage for 10 weeks, as threeidentiÐcations were incorrect.

Laboratory K was found to have the highest numberof incorrect identiÐcations in the trial, especially in thecase of the Norway lobster samples. It came to light,however, that the laboratory did not adhere strictly tothe working protocol, especially in terms of samplepreparation. The protocol stated that the samples couldeither be freeze-dried for 22 h or air-dried in a vacuumoven at approximately 40¡C. The participating labor-atory air-dried the cuticle samples in an oven at 100È110¡C for 15È30 min, and such a high temperaturewould certainly have had a detrimental e†ect on theradiation-induced ESR signal (Stewart et al 1993a),thereby a†ecting spectral interpretation.

As only four of the 15 participating laboratories hadsubstantial experience in using the technique of ESRspectroscopy to detect irradiated Crustacea, the resultsobtained for Norway lobster and, especially, the cre-vettes were very acceptable. It was also observed thateight laboratories correctly identiÐed more Norwaylobster samples after the 10 week storage period thanafter the initial 2 weeks, possibly as a result of increasedfamiliarity with the protocol following analysis of the


TABLE 5List of participants

Preparation of samples

Dr E M Stewart, Mr W D Graham Food Science Division, The Department of Agriculture for NorthernIreland, Newforge Lane, Belfast, BT9 5PX, UK

ParticipantsDr J Ammon Chemische Landesuntersuchungsanstalt, Karlsruhe, Ho†stra�e 3,

76133 Karlsruhe, GermanyDr D Erning Chemisches Untersuchungsamt der Stadt Hamm, Sachsenweg 6,

59073 Hamm, GermanyDr B Goodman, Dr N Deighton Scottish Crop Research Institute, Invergowrie, Dundee, Scotland,

UKDr R Gray Food Science Division, The Department of Agriculture for Northern

Ireland, Newforge Lane, Belfast, BT9 5PX, UKDr N Helle, Dr U Ballin Staatliches Veterina� runtersuchungsamt fu� r Fische und Fischwaren,

Schleusenstra�e, 27472 Cuxhaven, GermanyDr T Ku� hn Hygienisches Institut Chemische und

Lebensmitteluntersuchungsanstalt, Postfach 261551, 20505Hamburg, Germany

Dr E Marchioni Centre Re� gional DÏInnovation et de Transfert de Technologie, 19rue de Saint-Junien, BP 23, 67305 Schiltigheim Ce� dex, France

Dr W Meier Kantonales Laboratorium Zu� rich, Postfach, 8030 Zu� rich,Switzerland

Dr K Morehouse US Food and Drug Administration, Centre for Food Safety andApplied Nutrition, Chemistry Methods Branch, HFS-248, 200CStreet SW, Washington, DC 20204, USA

Dr S Onori, Dr M Pantaloni Instituto Superiore Ai Sanita, Physics Laboratory, Viale ReginaElena 299, 00161 Rome, Italy

Dr J Pfordt Staatliches Lebensmitteluntersuchungsamt, Oldenburg, Postfach2462, 26014 Oldenburg, Germany

Prof J J Ra†i LARQUA, Faculte� de Sainte-Je� roü me, Avenue Escadrille NormandieNie� men, F-13397 Marseille Ce� dex 20, France

Dr G Schreiber, Frau B Linke Bundesinstitut fu� r gesundheitlichen Verbraucherschutz undVeterina� rmedizin, General Pape-Str. 62, D-12101 Berlin, Germany

Dr C Schleich Chemisches Untersuchungsamt Mainz, Emy-Roeder-Stra�e 1,55129 Mainz, Germany

Prof W Stachowicz Institute of Nuclear Chemistry and Technology, Department ofRadiation Chemistry and Technology, Dorodna 16, 03-195 Warsaw,Poland

Ðrst set of samples, thus verifying the earlier observationmade for Norway lobster results.

The majority of irradiated samples identiÐed as non-irradiated were at the 1 kGy dose level. However, on acommercial basis, as prawn and shrimp samples areirradiated at doses of 2 kGy in the raw chilled state or2É5 kGy when frozen, failure to detect irradiatedsamples at the lowest dose level should not present amajor problem. In the interlaboratory blind trialorganised by the BGA (Helle et al 1994 ; Schreiber et al1996), it was demonstrated that samples of Norwaylobster and brown shrimp given a 2 kGy dose could beeasily detected.

On a quantitative basis, at present, it is not possibleto estimate the actual irradiation dose received by irra-diated Crustacea samples, although results reported byStewart and Stevenson (1997) for Norway lobster were

encouraging. Nevertheless, the results of this trial haveshown that if ESR spectra irradiated at a range of dosesare available for comparison purposes, it should be pos-sible to determine if samples have received a low,medium or high dose of irradiation.

CONCLUSIONS

The results of this interlaboratory blind trial clearlydemonstrate that the usefulness of ESR spectroscopy asa detection method for irradiated Crustacea variesaccording to the species being examined. The method-ology can certainly be used for the qualitative detectionof irradiated Norway lobster and crevettes but couldnot be used to unequivocally identify irradiated samplesof the particular species of pink shrimp tested in this


trial. Additional interlaboratory trials are required tofurther validate the ESR method for a range of prawnand shrimp species due to the fact that the radiation-induced ESR signal is species-dependent and alsoappears to be inÑuenced by the geographical origin ofthe samples. At a meeting of the European Committeefor Standardisation (CEN) Working Group on Irradi-ated Foodstu†s held in Lisbon in 1995, it was con-cluded that there is not enough fundamental knowledgeof the origin of the ESR signals derived from the cuticleat present to enable the ESR method to be standardisedfor the detection of irradiated Crustacea (Anon 1995).Therefore, it would seem that such basic research needsto be carried out before other interlaboratory trials areundertaken and the methodology Ðnally standardised.

DEDICATION

T he authors wish to dedicate this paper to their friendand colleague, the late Dr M Hilary Stevenson OBE(1947È1994).

ACKNOWLEDGEMENTS

The authors would like to thank the Ministry of Agri-culture, Fisheries and Food (MAFF) who funded thetrial and to all those who participated so willingly(Table 5). Thanks are also due to Mr William DGraham of the Food Science Division of the Depart-ment of Agriculture for Northern Ireland for assistancein preparation of the samples.

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an international collaborative blind trial on electron spin resonance (esr) identification of...

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