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Chemiluminescence measurements as an identification method for gamma-irradiated foodstuffs
Radiat. Phys. Chem, Vol, 25. Nos. 1-3. pp. 173-185. 1985
I)146-5724/85 $3.00 + .00 Pergamon Press Ltd
Printed in Great Britain.
CHEMILUMINESCENCE MEASUREMENTS AS AN IDENTIFICATION METHOD FOR GAMMA-IRRADIATED FOODSTUFFS W. BGgl and L. Heide Federal Health Office, D-8042 Neuherberg,
Institute for Radiation Hygiene, Federal Republic of Germany
ABSTRACT Samples of 19 different spices, milk powder, whole onions and frozenn chicken were exposed to a Co-60 source with radiation doses up to 10 ~ Gy. The subsequent reaction of the irradiated foodstuffs in a luminol solution resulted in light emission (chemiluminescence). This effect can be used as an indicator of radiation treatment. KEYWORDS Food irradiation; identification.
spices;
milk powder;
onions;
chicken;
chemiluminescence;
INTRODUCTION The treatment of foodstuffs by ionizing radiation has again become the centre of lively discussion when a joint committee of experts of the FAO/ IAEA/WHO at a meeting on 27 October - 3 November 1980 had passed a recommendation stating that the irradiation of all foodstuffs with an average total dose up to 10 kGy would not result in any toxicologic effects (I). In the Federal Republic of Germany, contrary to many other countries, it is at present not permitted to expose foodstuffs to ionizing radiation for the purpose of germ-reducing (e.g. pasteurlsation or sterilization). In order to ensure the observance of the food law, analytical procedures have become necessary for identification of irradiated foodstuffs. The purpose of this study was to investigate to what extent chemiluminescence measurements of irradiated foodstuffs are suitable for a speedy and reliable identification of radiation treatment. In this connection not only the possibility of being able to identify radiation treatment after a longer period of storage had to be considered, but also the probability of a quenching or fading of the effect used for identifying irradiation, or the stimulation of the identifying radiation effect by other treatment methods for the foodstuffs under investigation, respectively. EXPERIMENTAL General: When treating solid samples of various substances with ionizing radiation, light is emitted by these samples as soon as they are being dissolved in water. The light is emitted in short pulses whereby the pulse length is determined mainly by the time interval during which the irradiated sample is dissolved in the solution. The integral light yield depends on the administered radiation dose and, in many cases, increases with the dose until a saturation value is reached (2,3). The light yield of the irradiated substance can be essentially increased by using a photosensitizer during the process of dissolution. One of the most frequently used photosensitizers is luminol, a cyclic hydrazide of 3-amlnophthalic acid capable of giving off in alkaline solution two protons from the hydrazide structure. The molecule disintegrates during oxidation and creates a chemiluminescence that is particularly pronounced at pH-values ~I0. The emission spectrum of the emitted light in aqueous solution reaches its maximum at a wave lenght of 424 nm (2,3). Where the mechanism causing the single molecule to be luminescent is concerned, it may be stated that a triplet excited level of 3-amlnophthalate demonstrates the species responsible for luminescence and that 3-aminophthalate and molecular nitrogen are forming in the final oxidation phase (2-7). 173
174
The reaction process wing diagram:
W. BOGL
leading
L. HEIDE
AND
to light emission
O
2OH-
".~.c/NH
2H~O
~..~.....c/N-,
II
O
o
NH~
II
C~ /
C~O-
I.
O
j
II
_
bvox~l~i~laie.~ , x~.
NH2
by the follo
O
C'~T H
NH2
is demonstrated
R- (~H-R'
O
/
L NH=
"O2
+ N2
llKCrllld
o]. "OO
o
C"O-
II
II
*
NH2
O
R-CH2-R'
.
NH 2
J
-
~iziq ..~kltOn
R-CH--R' + HTO 2 OH"
h.v
O
R-CH-R' c~
R-CH--R' + 0 2 -_
+
Pad~
R-CH-R' I "O2 -
R-CH2.-R' Jr OH" H202
Luminol reacts in solution containing oxidation media, such as H909 or peroxide radicals, by formation of molecular nitrogen. 3-Aminophth~late produced at the excited level is reversed to its ground level during light emission. When solid organic substances are irradiated or, subsequently, come in contact with water, hydrogen peroxide and organic peroxide radicals among other oxidizing agents - may develop as follows: at first, among others, carbon radicals will develop from irradiation. These can be converted by oxygen into peroxide radicals or may react in the luminol solution with water by forming OH-radicals. H~O~ will then be produced via the OH-radicals. If the irradiated substances are insoluble solids, e.g. pulverized spices, then upon contact with water the radicals will at first react only on the surface. Therefore, at a specific radiation dose, the amount of emitted light is very much larger when dealing with soluble substances in luminol solution, since all reaction products with oxidizable properties are thus able to react with luminol in a short period of time. -
Foodstuffs: Cinnamon, Curry, Red Pepper (Paprika), Anise, Basil, Cumin, Turmeric, Tarragon, Fennel, Garlic, Coriander, Cloves, Pepper, Pimento, Sage, Celery, Sesame, Junipers and Onions were purchased from different producers unselectedly in dried form as powder, or kernels and plants, respectively, and used for irradiation experiments normally without further pre-treatment. The finely pulverized milk powder contained 17% fat and was enriched by milk sugar. In addition, deep frozen chicken and whole onions were used for irradiation purposes. Irradiation: Irradiation was done by using a Co-60 source.
During
irradiation
all but
175
Chemiluminescence Measurements
the chicken samples were placed in glas tubes of about 10 cm height (0 1.6 cm; wall strength 0.1 cm). The tubes were sealed by parafilm. The chicken samples were irradiated in deep frozen condition in Dewarcontainers. Each irradiated sample quantity weighed approximate1 3 to 10 g. Atmospheric oxygen was not excluded during irradiation. I he dose rate of the co-60 source used was within a range of 5.5 to 5.8 kGy/h. The maximum temperature increase in the samples during irradiation was about 10°C. The absorbed dose data indicated in the following description of the experiments contain a maximum error of 2 10%.
10kGy
16,37 16,02
4.22 44’:; _
16,28 15,O8 16,13 ,16,50 r13,19
4:13 .4,47 4,21 b 3.62
15,98$,52
4,22+0,10
. ,
Chamiluminoswnw memurennntl of unirdiatd and irradiated cinnrman powder.
1143 704 l 58317 1255 .I305 868,7
1198
c
Fig. 1.
mV
mV/fisac
mV
mV/ssec
10342237
1882 1110
b
966,1 1937 .1974
1438
1477 1569+343
w.
IT6
BOGL AND L. HEIDE
Reaction with !uminol: Light emission (chemiluminescence) was initiated by automatic dispension of the irradiated food samples weighted in polystyrene cuvettes each with 0.2 mi of luminol reagent. The !uminol solution is a combination of: 125 mg luminol (0.7 mM) 2.5 mg Hemin (3.8 luM) 1.25 g Na~CO~ (11.8 mM) ad 1000 ml H~0. The solution was adjusted with I M HCI to pH 10 or 11, respectively. The weighted sample quantity in most experiments rement. All tests were done at room temperature. Chemiluminescence measurements: To measure the emitted light (luminescence), of LKB Co. (LKB 1215) was used.
was 3 to 30 mg per measu-
a luminescence
photometer
Demonstration and evaluation of the results: Using cinnamon powder as an example, figure I shows the evaluation of the results. All analyses were done five-fold to seven-fold of each one of the irradiated sample and the non-irradlated control sample. The integral chemiluminesc e n c e i ~ e n s i t y was registered during the first 5 seconds after injecting luminol into the test tube containing the food sample (unit: V/5sec or mV/5sec) as well as the maximum of the chemiluminescence intensity during the first 5 seconds (unit: V or mV) and the light emission function of the chemiluminescence during the first seconds after luminol injection (the time scale in fig. I is valid for all recorder diagrams shown in this study). Considerung the often relatively large dispersion range of the values of individual trials, the following procedure was found to be of merit: as already mentioned, multiple analyses were always performed. In addition, the lowest as well as the highest measuring values were cancelled (those values are marked by a black triangle in the figs.). Mean values and standard deviations were calculated from the remaining values. The error bars of all diagrams presented as follows are thus representing the standard deviation (68,3%). RESULTS
(8 - 12)
Spices: TABLE
I
Chemiluminescence measurements of the most important results
Spices
Anise Basil Cumin Turmeric Tarragon Fennel Garlic Coriander Cloves Pepper Pimento Sage Celery Sesame Junipers Onions Cinnamon Curry Red Pepper
with
Chemiluminescence Intensity (factor: irradiated spices 110 kGy)/unirradiated spices) immediately I month after after irradiation irradiation 12,3 6,5 7,8 10 4 8 1 34 4 5 2 16 0 6 4 8 1 6 4 I 225 44,1 933 9,9 648 158 2
27,4 5,6 2,8 7,4 1,6 8,6 1,7 4,6 I 16,1 1,9 370 9O 2
irradiated
spices;
summary
Identification of irradiation is possible (factor ~ 2) after irradiation at times: (days) >56 ~56 >17 >11 >56 >38 3 >56 >27 5O 40 ~13 >38 ~27 37 >>6O >>6O (4o)
ChemiLuminescence Measurements
177
In table I the most significant results are compiled from chemiluminescence measurements of 19 irradiation spices. It is evident that with the exception of sage, garlic and red pepper (Paprika) powder, radiation treatment can be easily identified after a long period of time. For most of the spices, radiation treatment is identifiable even for a time period of more than 2 months after the irradiation. Figure 2 demonstrates the chemiluminescence intensity as a function of the radiation dose, as exemplified by fennel. Figure 3 shows chemiluminescence intensity as function of storage time after irradiation, as demonstrated by basil.
35 --
72
~ s4
30
E
~ 56 •~ 48 ==
> 25 ~
40
zo
32 24
._~ lO
~- 16
E = E
"~ 8
5
=E 0
r ....
0 -~L-,
,
,
,
,
0 0,1 0250,6 1,5 4,0
, 10(log)
Radiation dose [kGy] Fig. 2. Chemiluminescence intensity of irradiated fennel as a function of radiation dose.
i
0 8' 1; 2432 40 48 56
C.3
Time after irradiation [days] Fig. 3.
Chemiluminescence intensity of irradiated basil as a function of storage time after irradiation.
It is a known fact that heat treatment can repair radiation damage in many solid substances. Therefore, it was first interesting to see whether a 16-hour heat treatment may decrease the chemiluminescence intensity of the irradiated spice samples during the consecutive luminol reaction.
1,0
¢J
-= 1,5 E
._~
h
e-
c
1,0
._= 8
~ ~ ~ \
== 0,5
=
.E
E
o--
_E 0,5
\.
-
0,0
• !
20 Fig. 4.
,o I
0 Gy
• I
50 100 Temperature [°C]
o--
E
x-
¢J
-O u
130
Chemiluminescence intensity of irradiated cinnamon powder as a function of the temperatureof a heat treatment (16h) after irradiation.
0,0
0 Gy'~~ i 1
2
Incubation time [h] Fig. 5. Chemiluminescence intensity of irradiated cinnamon powder (104 Gy) as a function of the duration of a watervapor treatment.
W . BOGL ~.-~D L. HEIDE
178
Figure 4 shows
the results of the examination.
Radiation treatment can still be identified after a 16-hour heat treatment in a drying oven at various temperatures. It is further known that radicals in dry substances remain stable for a prolonged period of time at room temperature. Upon contact with water they react with the water by forming substances without unpaired electrons. As a second step it was consequently examined whether the chemiluminescence intensity could be decreased during the consecutive reaction with luminol solution when treating the irradiated food samples with water-vapor Again taking cinnamon powder as an example, fig. 5 shows the results from this examination. After the steam treatment (2 h at 25°C in a water-vapor saturated atmosphere; afterwards the samples were sticky and could no longer be dispersed) it was impossible to identify the earlier irradiation treatment. Furthermore it had to be examined to which extent an UV-irradiation of the spices could initiate a reaction in luminol solution comparable to that resulting from gamma irradiation. Figure 6 demonstrates the results from the respective examination of cinnamon powder. The exposure of untreated samples to UV-radiation results in an increase of the chemiluminescence intensity (only at the foodstaff surface due to the low range of UV-radiation). Since the shape of the light-emissioncurve varies, it is sometimes possible to make a distinction between gammaand UV-irradiation.
I.O
100
--. >
>
E
E 8o
5000 4000
~ 60 ._~
3000
'-
40
•~
20
E
0
2000 ....
,=~
6000
---
',
0
Fig. 6.
- i ' ' ' "
UV-C
1000
=
,
7,2
Hilk powder: Figure 7 shows the chemiluminescence as a function of the radiation dose.
0
0
i
14,4 UV-lrradiation [J/cm2] Chemiluminescenceintensity of untreated cinnamon powder after UV -irradiation. 2,4
=E
Fig. 7.
0,1 0,6 1,5 4,0 10(log) Radiation dose [kGy]
The integral chemiluminescence intensity of irradiated milk powder as a function of the radiation dose.
intensity of irradiated milk powder
In Figure 8 all the chemiluminescent intensity measurements of the irradiated milk powder after different times of storage are summarized (the milk powder was packaged during storage). Similarly, compared with the spices, the irradiated milk powder was also heated or treated with water-vapor, respectively. The results of the heat treatment (16 hours for each sample) are summarized in figure 9. A probable explanation for the increased chemiluminescence intensities after heat treatment is the formation of fat-oxidation products. Water-vapor treatment showed the same results as those with cinnamon; irradiation could not be identified.
prior
In addition, it had to be examined to which extent an UV-irradiation of milk powder could initiate a reaction in the luminol solution similar to that resulting from gamma radiation. The results are summarized in figure 10.
Chemiluminescence Measurements
179
In the case of milk powder, the form of the l i g h t - e m i s s i o n - c u r v e tical when using gamma- or UV-irradiation.
= ~ ="
20
~.-~
•-=
10 o
~
Gy
E
4
e" m
14
12
E~ ~'~ -~
.~
is iden-
E •
,
,
,
,
|
|
m
!
2
U
,
0
10 20 30 4[) 5() 60 Time after irradiation [days] Fig. 8. The integral chemiluminescence intensity of irradiated milk powder as a function of storage time after irradiation.
o z6 56 Fig. 9.
IHO
Temperature [°C] The integral chemiluminescence intensity of irradiated milk powder as a function of the heat treatment after irradiation.
In a final experiment, untreated milk powder in a thin layer was exposed to air. Figure 11 shows the chemiluminescence intensity of the milk powder (non-irradiated) as a time function of air exposure. Due to fat-oxidation, a rapid increase of the chemiluminescence intensity occured. Because of similar c h e m i l u m i n e s c e n c e effects of milk powder treated with air, UVradiation or gamma radiation respectively (compare figures 7, 9, 10 and 11), a sure identification of gamma irradiated milk powder is not possible at the moment.
t.)
4
i.n
"~
3
j
2000
H~
1000
E~ ~.-=
5 0 2,'4 7~2 UV- irradiation [J/cm 2 ] Fig. 10. The integral chemiluminescence intensity of untreated milk powder after UVirradiation.
0
, 246
, 10
, Tlmeo24irexposure[h]__
48
Fig. 11. The integral chemiluminescence intensity of unirradiated milk powder as a function of the time of air exposure.
Whole onions: In figure 12, some results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of unirradiated and irradiated white onion-skln are demonstrated. The m e a s u r e m e n t s were performed immediately after the irradiation, but also some weeks later the detection of the irradiation process is still possible. Figure 13 demonstrates the c h e m i l u m i n e s c e n c e intensity of irradiated brown onion-skin as a function of the radiation dose. Compared with figure 12 it can be seen that the c h e m i l u m i n e s c e n c e intensity of the brown onion-skin
I~0
W
BOOL
~NC)
k. HEIDE
10kGy
unirradiat~
mV/Ssec 28,38 30,33 46,98 •50,67 24,70 45,24 •17,57
Fig. 12.
7
.
~ ' !
:
7,48 8,06 12,73 14,22 7,16 •18,89
2031 1150 897,1 •3407 1219 1338 • 893,7
b 5,07
Chemiluminescerme measurements of unirradiated and irradiated onion skin (white, lcm 2, immediately after irradiation).
. . . . . . . . . . .
......
7 .
:
.
.
.
.
.
.
.
.
.
.
.
.
.
mV
mv/Ssec
mV
"
782,3 537,4 • 360,4 ~1626 609,4 382,4 364,6
.
;i?~LT"_-'
.
.
.
.
L~L-_-;2L2.~I212.;~.
.
.
.
"
T-
;..;.
. . . . . .
.
i
~%
L
• i. . . . . . . . . .
T ! .......... . . . . . . . . . . . . . .
...................
,_=
?..-~LL
(10 kGy) is much lower compared to the results, m e a s u r e d with the white skin. The reason for this d e c r e a s e of the c h e m i l u m i n e s c e n c e intensity is a fast e x t r a c t i o n of the o n i o n - s k i n dye with the luminol s o l u t i o n and subsequent light q u e n c h i n g in the luminol solution. In figure 14, the results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of irradiated onion (without the skin) as a function of r a d i a t i o n dose are summarized. This figure quite clearly shows only a small d i f f e r e n c e in the c h e m i l u m i n e cence intensities b e t w e e n u n i r r a d i a t e d and i r r a d i a t e d onions.
Chemiluminescence M e a s ~ e m e n t s
;.8[
Using red onion-skin for the identification of gamma irradiation, it is impossible to see any difference between the irradiated and the untreated samples. The reason is again a fast extraction of the skin dye with the luminol solution. This extraction of red onion skin is demonstrated in figure 15 (extinction of the luminol solution as a function of extraction time). It can bee seen, that the extraction is very fast. The consequence is a complete quenching of the emitted light in the lumlnol solution.
160 ,_=
> E
120 c
.E > EE
300
._.R
8O
200
c
100
40
O
~ ¢ " ..... ;
,
I 0
0,3
1,0
3,0
•.~
-
0,5
._o <.J X t,M
0
(log)
Fig. 14. Chemiluminescence intensity of irradiated onion (without onionskin) as a function of radiation dose.
E tJJ
I 10
Radiation dose [ kGy ]
Fig. 13. Chemiluminescence intensity of irradiated onion-skin (brown) as a function of radiation dose.
1,0
re.', ......; ' ,
¢J
0 0,3 1,0 3,0 10 (Iogi Radiation dose [ kGy ]
1
!
I
0
5
20
Extraction time [sec]
Fig. 15, Extraction of red onion-skin with a luminol solution; extinction of the solution as a function of extraction time.
182
W. BOGL AND L. HEIDE
Frozen chicken: In figure 16, some results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of gamma irradiated and untreated frozen chicken bone are summarized. These m e a s u r e m e n t s were performed immediately after the irradiation. Using the c h e m i l u m i n e s c e n c e method for irradiation identification in frozen chicken, it is preferable to prepare and measure some chicken cartilage, as it is demonstrated in figures 17 and 18. The intensity difference of
10 kGy
unirradiated
mV/Ssec -
mV/Ssec"
mV >161,~ 74,7 92,4
•216,2 1£9,6 145,3 104,7 100,1 178,0 • 86,0
56,5 • 37,5 100,6 44,3
2415 2501 • 4394 • 22~0 3897 3970 4382
mV 1044 i~66 2099 1093 I921 1938 i,2293
I
'i!
": 4
'l;
'I'
Fig. 16. Chemiluminescencemeasurements of unirradiatedand irradiateddeep frozen chickenbone (immediately after irradiation)
2.
_
I + h
- !!
4----- ~-- ~'-7:-7 7 7 . . . . . . .
A :'Z
. . . .
_
]
~
_
L
Chcmituminescence Measurements
183
the emitted light between the untreated sample and the gamma irradiated sample is larger when using cartilage rather than chicken bone or meat. This can be seen expecially in figure 18. This figure also demonstrates quite clearly that some weeks after irradiation, the process can still be identified.
unir~di~
mV/Ssec 117,7 85,1 • 118,1 114,1 108,5 39,8 • 37,6
Fig. 17,
lOkGy
mV
mV/Ssec"
43,0 32,6 41,6 • 44,9 34,0 •12,5 13,6
•2085 4675 •7726 6156 4801 3709 3032
mV • 74o,5 1585 •23O5 1797 1665 1143 947,1
Chemiluminescence measurements of unirrediated and irradiated deep frozen chickan cartilage (16 days after irradiation)
.
.
.
.
.
.
.
.
.
Cocoa powder; parsley (preliminary results): In figures 19 and 20, preliminary results of irradiated and untreated cocoa powder and parsley are demonstrated. All of these measurements were performed immediately after irradiation. CONCLUSIONS To sum-up, we can say that measurement of chemiluminescence would seem to be a method which permits quick and reliable detection of ionizing radiation treatment with of a lot of foodstuffs. At least for some foodstuffs, it will be possible to identify the radiation treatment process as late
18~
VV. BOGL ,~:~D L. HEIDE
10
deepfrozenchicken
¢J
s
~-
'l~kGy
c
= 6 c
==
"~
4
bone10 kGy IJ.
~ ( ~ %unirradiated I
YTi
meatl0kGy
!
0
9111~I
<~
8 Timeafterirradiation[days]
I
16
Fig. 18. Chemiluminescence intensity of irradiated deep frozen chicken cartilage, bone and meat as a function of storage time after irradiation.
10kGy
unirradiated mV/5sec
17,1 16,5 17,1
mV
6,73 6,73 6,95
mVl5~c 66,7 62,8 64,0
i
mV
15,4 14,6 16,3
Fig.19. Chemiluminescence measurements of unirradiatedandirradiatedcacao powder.
as several month after irradiation. The chemical background of a lot of the measured chemiluminescence effects is not explicable at the moment. In spite of the probability of a quenching or fading of the effect used for the detection of irradiation, or the stimulation of chemiluminescence effects by other treatment methods, in the near future it will be possible to develop chemiluminescence measurement to a dependable, precise and rapid method of identifying irradiation treatment in at least some foodstuffs.
Chemiluminescence Measurements
lOkGy
unirradi~ed
mV~sec
8,70 9,34 8,49
Fig. 20.
185
mV
3,33 3,80 3,36
mV/5~
33,3 58,9 33,6
mV
14,7 27,1 14,8
Chemiluminescence meuumments of unirradiatod and irradiated parsley.
REFERENCES (I)
World Health Organization: Wholesomeness of irradiated food. Report of a Joint FAO/IAEA/WHO Expert Committee. Technical Report Series 659, World Health Organisation, Geneva 1981. (2) Ettinger, K.V., and K.J. Puite (1982). Int. J. Appl. Radiat. Isot., 33, 1115-1138. (3) Puite, K.J., and K.V. Ettinger (1982). Int. J. Appl. Radiat. Isot., .,~t~, 1139-1157. (4) White, E.H., and M.M. Bursey (1964). J. Amer. Chem. Soc.. 86, 941-942. (5) White, E.H., O.C. Zafiriou, W. K~gi, and J.H.M.'Hill (1964), J. Amer. Chem. Soc., 86, 940-941. (6) White, E.H., and M.M. Bursey (1966). J. Amer. Chem~ Soc., ~I, 19121917. (7) Roswell, D.F., and E.H. White (1978). Methods in Enz~mology, 57, Academic Press, Inc., 409-423. (8) B5gl, W., and L. Heide (1983). ISH-Berlcht ~2, Institute for Radiation Hygiene of the Federal Health Office. (9) Heide, L., and W. BGgl (1984). ISH-Heft 41, Institute for Radiation Hygiene of the Federal Health Office. (10) Heide, L., and W. BSgl (1984). ISH-Heft 5~, Institute for Radiation Hygiene of the Federal Health Office. (11) B6gl, W., and L. Helde (1984). Flelschwirtseh., 64(9), 1120-1126. (12) BGgl, W., and L. Heide (1984). Symposium handbook, ~rd International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence, Birmingham, GB, 17.-19.4.1984, 98.
I)146-5724/85 $3.00 + .00 Pergamon Press Ltd
Printed in Great Britain.
CHEMILUMINESCENCE MEASUREMENTS AS AN IDENTIFICATION METHOD FOR GAMMA-IRRADIATED FOODSTUFFS W. BGgl and L. Heide Federal Health Office, D-8042 Neuherberg,
Institute for Radiation Hygiene, Federal Republic of Germany
ABSTRACT Samples of 19 different spices, milk powder, whole onions and frozenn chicken were exposed to a Co-60 source with radiation doses up to 10 ~ Gy. The subsequent reaction of the irradiated foodstuffs in a luminol solution resulted in light emission (chemiluminescence). This effect can be used as an indicator of radiation treatment. KEYWORDS Food irradiation; identification.
spices;
milk powder;
onions;
chicken;
chemiluminescence;
INTRODUCTION The treatment of foodstuffs by ionizing radiation has again become the centre of lively discussion when a joint committee of experts of the FAO/ IAEA/WHO at a meeting on 27 October - 3 November 1980 had passed a recommendation stating that the irradiation of all foodstuffs with an average total dose up to 10 kGy would not result in any toxicologic effects (I). In the Federal Republic of Germany, contrary to many other countries, it is at present not permitted to expose foodstuffs to ionizing radiation for the purpose of germ-reducing (e.g. pasteurlsation or sterilization). In order to ensure the observance of the food law, analytical procedures have become necessary for identification of irradiated foodstuffs. The purpose of this study was to investigate to what extent chemiluminescence measurements of irradiated foodstuffs are suitable for a speedy and reliable identification of radiation treatment. In this connection not only the possibility of being able to identify radiation treatment after a longer period of storage had to be considered, but also the probability of a quenching or fading of the effect used for identifying irradiation, or the stimulation of the identifying radiation effect by other treatment methods for the foodstuffs under investigation, respectively. EXPERIMENTAL General: When treating solid samples of various substances with ionizing radiation, light is emitted by these samples as soon as they are being dissolved in water. The light is emitted in short pulses whereby the pulse length is determined mainly by the time interval during which the irradiated sample is dissolved in the solution. The integral light yield depends on the administered radiation dose and, in many cases, increases with the dose until a saturation value is reached (2,3). The light yield of the irradiated substance can be essentially increased by using a photosensitizer during the process of dissolution. One of the most frequently used photosensitizers is luminol, a cyclic hydrazide of 3-amlnophthalic acid capable of giving off in alkaline solution two protons from the hydrazide structure. The molecule disintegrates during oxidation and creates a chemiluminescence that is particularly pronounced at pH-values ~I0. The emission spectrum of the emitted light in aqueous solution reaches its maximum at a wave lenght of 424 nm (2,3). Where the mechanism causing the single molecule to be luminescent is concerned, it may be stated that a triplet excited level of 3-amlnophthalate demonstrates the species responsible for luminescence and that 3-aminophthalate and molecular nitrogen are forming in the final oxidation phase (2-7). 173
174
The reaction process wing diagram:
W. BOGL
leading
L. HEIDE
AND
to light emission
O
2OH-
".~.c/NH
2H~O
~..~.....c/N-,
II
O
o
NH~
II
C~ /
C~O-
I.
O
j
II
_
bvox~l~i~laie.~ , x~.
NH2
by the follo
O
C'~T H
NH2
is demonstrated
R- (~H-R'
O
/
L NH=
"O2
+ N2
llKCrllld
o]. "OO
o
C"O-
II
II
*
NH2
O
R-CH2-R'
.
NH 2
J
-
~iziq ..~kltOn
R-CH--R' + HTO 2 OH"
h.v
O
R-CH-R' c~
R-CH--R' + 0 2 -_
+
Pad~
R-CH-R' I "O2 -
R-CH2.-R' Jr OH" H202
Luminol reacts in solution containing oxidation media, such as H909 or peroxide radicals, by formation of molecular nitrogen. 3-Aminophth~late produced at the excited level is reversed to its ground level during light emission. When solid organic substances are irradiated or, subsequently, come in contact with water, hydrogen peroxide and organic peroxide radicals among other oxidizing agents - may develop as follows: at first, among others, carbon radicals will develop from irradiation. These can be converted by oxygen into peroxide radicals or may react in the luminol solution with water by forming OH-radicals. H~O~ will then be produced via the OH-radicals. If the irradiated substances are insoluble solids, e.g. pulverized spices, then upon contact with water the radicals will at first react only on the surface. Therefore, at a specific radiation dose, the amount of emitted light is very much larger when dealing with soluble substances in luminol solution, since all reaction products with oxidizable properties are thus able to react with luminol in a short period of time. -
Foodstuffs: Cinnamon, Curry, Red Pepper (Paprika), Anise, Basil, Cumin, Turmeric, Tarragon, Fennel, Garlic, Coriander, Cloves, Pepper, Pimento, Sage, Celery, Sesame, Junipers and Onions were purchased from different producers unselectedly in dried form as powder, or kernels and plants, respectively, and used for irradiation experiments normally without further pre-treatment. The finely pulverized milk powder contained 17% fat and was enriched by milk sugar. In addition, deep frozen chicken and whole onions were used for irradiation purposes. Irradiation: Irradiation was done by using a Co-60 source.
During
irradiation
all but
175
Chemiluminescence Measurements
the chicken samples were placed in glas tubes of about 10 cm height (0 1.6 cm; wall strength 0.1 cm). The tubes were sealed by parafilm. The chicken samples were irradiated in deep frozen condition in Dewarcontainers. Each irradiated sample quantity weighed approximate1 3 to 10 g. Atmospheric oxygen was not excluded during irradiation. I he dose rate of the co-60 source used was within a range of 5.5 to 5.8 kGy/h. The maximum temperature increase in the samples during irradiation was about 10°C. The absorbed dose data indicated in the following description of the experiments contain a maximum error of 2 10%.
10kGy
16,37 16,02
4.22 44’:; _
16,28 15,O8 16,13 ,16,50 r13,19
4:13 .4,47 4,21 b 3.62
15,98$,52
4,22+0,10
. ,
Chamiluminoswnw memurennntl of unirdiatd and irradiated cinnrman powder.
1143 704 l 58317 1255 .I305 868,7
1198
c
Fig. 1.
mV
mV/fisac
mV
mV/ssec
10342237
1882 1110
b
966,1 1937 .1974
1438
1477 1569+343
w.
IT6
BOGL AND L. HEIDE
Reaction with !uminol: Light emission (chemiluminescence) was initiated by automatic dispension of the irradiated food samples weighted in polystyrene cuvettes each with 0.2 mi of luminol reagent. The !uminol solution is a combination of: 125 mg luminol (0.7 mM) 2.5 mg Hemin (3.8 luM) 1.25 g Na~CO~ (11.8 mM) ad 1000 ml H~0. The solution was adjusted with I M HCI to pH 10 or 11, respectively. The weighted sample quantity in most experiments rement. All tests were done at room temperature. Chemiluminescence measurements: To measure the emitted light (luminescence), of LKB Co. (LKB 1215) was used.
was 3 to 30 mg per measu-
a luminescence
photometer
Demonstration and evaluation of the results: Using cinnamon powder as an example, figure I shows the evaluation of the results. All analyses were done five-fold to seven-fold of each one of the irradiated sample and the non-irradlated control sample. The integral chemiluminesc e n c e i ~ e n s i t y was registered during the first 5 seconds after injecting luminol into the test tube containing the food sample (unit: V/5sec or mV/5sec) as well as the maximum of the chemiluminescence intensity during the first 5 seconds (unit: V or mV) and the light emission function of the chemiluminescence during the first seconds after luminol injection (the time scale in fig. I is valid for all recorder diagrams shown in this study). Considerung the often relatively large dispersion range of the values of individual trials, the following procedure was found to be of merit: as already mentioned, multiple analyses were always performed. In addition, the lowest as well as the highest measuring values were cancelled (those values are marked by a black triangle in the figs.). Mean values and standard deviations were calculated from the remaining values. The error bars of all diagrams presented as follows are thus representing the standard deviation (68,3%). RESULTS
(8 - 12)
Spices: TABLE
I
Chemiluminescence measurements of the most important results
Spices
Anise Basil Cumin Turmeric Tarragon Fennel Garlic Coriander Cloves Pepper Pimento Sage Celery Sesame Junipers Onions Cinnamon Curry Red Pepper
with
Chemiluminescence Intensity (factor: irradiated spices 110 kGy)/unirradiated spices) immediately I month after after irradiation irradiation 12,3 6,5 7,8 10 4 8 1 34 4 5 2 16 0 6 4 8 1 6 4 I 225 44,1 933 9,9 648 158 2
27,4 5,6 2,8 7,4 1,6 8,6 1,7 4,6 I 16,1 1,9 370 9O 2
irradiated
spices;
summary
Identification of irradiation is possible (factor ~ 2) after irradiation at times: (days) >56 ~56 >17 >11 >56 >38 3 >56 >27 5O 40 ~13 >38 ~27 37 >>6O >>6O (4o)
ChemiLuminescence Measurements
177
In table I the most significant results are compiled from chemiluminescence measurements of 19 irradiation spices. It is evident that with the exception of sage, garlic and red pepper (Paprika) powder, radiation treatment can be easily identified after a long period of time. For most of the spices, radiation treatment is identifiable even for a time period of more than 2 months after the irradiation. Figure 2 demonstrates the chemiluminescence intensity as a function of the radiation dose, as exemplified by fennel. Figure 3 shows chemiluminescence intensity as function of storage time after irradiation, as demonstrated by basil.
35 --
72
~ s4
30
E
~ 56 •~ 48 ==
> 25 ~
40
zo
32 24
._~ lO
~- 16
E = E
"~ 8
5
=E 0
r ....
0 -~L-,
,
,
,
,
0 0,1 0250,6 1,5 4,0
, 10(log)
Radiation dose [kGy] Fig. 2. Chemiluminescence intensity of irradiated fennel as a function of radiation dose.
i
0 8' 1; 2432 40 48 56
C.3
Time after irradiation [days] Fig. 3.
Chemiluminescence intensity of irradiated basil as a function of storage time after irradiation.
It is a known fact that heat treatment can repair radiation damage in many solid substances. Therefore, it was first interesting to see whether a 16-hour heat treatment may decrease the chemiluminescence intensity of the irradiated spice samples during the consecutive luminol reaction.
1,0
¢J
-= 1,5 E
._~
h
e-
c
1,0
._= 8
~ ~ ~ \
== 0,5
=
.E
E
o--
_E 0,5
\.
-
0,0
• !
20 Fig. 4.
,o I
0 Gy
• I
50 100 Temperature [°C]
o--
E
x-
¢J
-O u
130
Chemiluminescence intensity of irradiated cinnamon powder as a function of the temperatureof a heat treatment (16h) after irradiation.
0,0
0 Gy'~~ i 1
2
Incubation time [h] Fig. 5. Chemiluminescence intensity of irradiated cinnamon powder (104 Gy) as a function of the duration of a watervapor treatment.
W . BOGL ~.-~D L. HEIDE
178
Figure 4 shows
the results of the examination.
Radiation treatment can still be identified after a 16-hour heat treatment in a drying oven at various temperatures. It is further known that radicals in dry substances remain stable for a prolonged period of time at room temperature. Upon contact with water they react with the water by forming substances without unpaired electrons. As a second step it was consequently examined whether the chemiluminescence intensity could be decreased during the consecutive reaction with luminol solution when treating the irradiated food samples with water-vapor Again taking cinnamon powder as an example, fig. 5 shows the results from this examination. After the steam treatment (2 h at 25°C in a water-vapor saturated atmosphere; afterwards the samples were sticky and could no longer be dispersed) it was impossible to identify the earlier irradiation treatment. Furthermore it had to be examined to which extent an UV-irradiation of the spices could initiate a reaction in luminol solution comparable to that resulting from gamma irradiation. Figure 6 demonstrates the results from the respective examination of cinnamon powder. The exposure of untreated samples to UV-radiation results in an increase of the chemiluminescence intensity (only at the foodstaff surface due to the low range of UV-radiation). Since the shape of the light-emissioncurve varies, it is sometimes possible to make a distinction between gammaand UV-irradiation.
I.O
100
--. >
>
E
E 8o
5000 4000
~ 60 ._~
3000
'-
40
•~
20
E
0
2000 ....
,=~
6000
---
',
0
Fig. 6.
- i ' ' ' "
UV-C
1000
=
,
7,2
Hilk powder: Figure 7 shows the chemiluminescence as a function of the radiation dose.
0
0
i
14,4 UV-lrradiation [J/cm2] Chemiluminescenceintensity of untreated cinnamon powder after UV -irradiation. 2,4
=E
Fig. 7.
0,1 0,6 1,5 4,0 10(log) Radiation dose [kGy]
The integral chemiluminescence intensity of irradiated milk powder as a function of the radiation dose.
intensity of irradiated milk powder
In Figure 8 all the chemiluminescent intensity measurements of the irradiated milk powder after different times of storage are summarized (the milk powder was packaged during storage). Similarly, compared with the spices, the irradiated milk powder was also heated or treated with water-vapor, respectively. The results of the heat treatment (16 hours for each sample) are summarized in figure 9. A probable explanation for the increased chemiluminescence intensities after heat treatment is the formation of fat-oxidation products. Water-vapor treatment showed the same results as those with cinnamon; irradiation could not be identified.
prior
In addition, it had to be examined to which extent an UV-irradiation of milk powder could initiate a reaction in the luminol solution similar to that resulting from gamma radiation. The results are summarized in figure 10.
Chemiluminescence Measurements
179
In the case of milk powder, the form of the l i g h t - e m i s s i o n - c u r v e tical when using gamma- or UV-irradiation.
= ~ ="
20
~.-~
•-=
10 o
~
Gy
E
4
e" m
14
12
E~ ~'~ -~
.~
is iden-
E •
,
,
,
,
|
|
m
!
2
U
,
0
10 20 30 4[) 5() 60 Time after irradiation [days] Fig. 8. The integral chemiluminescence intensity of irradiated milk powder as a function of storage time after irradiation.
o z6 56 Fig. 9.
IHO
Temperature [°C] The integral chemiluminescence intensity of irradiated milk powder as a function of the heat treatment after irradiation.
In a final experiment, untreated milk powder in a thin layer was exposed to air. Figure 11 shows the chemiluminescence intensity of the milk powder (non-irradiated) as a time function of air exposure. Due to fat-oxidation, a rapid increase of the chemiluminescence intensity occured. Because of similar c h e m i l u m i n e s c e n c e effects of milk powder treated with air, UVradiation or gamma radiation respectively (compare figures 7, 9, 10 and 11), a sure identification of gamma irradiated milk powder is not possible at the moment.
t.)
4
i.n
"~
3
j
2000
H~
1000
E~ ~.-=
5 0 2,'4 7~2 UV- irradiation [J/cm 2 ] Fig. 10. The integral chemiluminescence intensity of untreated milk powder after UVirradiation.
0
, 246
, 10
, Tlmeo24irexposure[h]__
48
Fig. 11. The integral chemiluminescence intensity of unirradiated milk powder as a function of the time of air exposure.
Whole onions: In figure 12, some results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of unirradiated and irradiated white onion-skln are demonstrated. The m e a s u r e m e n t s were performed immediately after the irradiation, but also some weeks later the detection of the irradiation process is still possible. Figure 13 demonstrates the c h e m i l u m i n e s c e n c e intensity of irradiated brown onion-skin as a function of the radiation dose. Compared with figure 12 it can be seen that the c h e m i l u m i n e s c e n c e intensity of the brown onion-skin
I~0
W
BOOL
~NC)
k. HEIDE
10kGy
unirradiat~
mV/Ssec 28,38 30,33 46,98 •50,67 24,70 45,24 •17,57
Fig. 12.
7
.
~ ' !
:
7,48 8,06 12,73 14,22 7,16 •18,89
2031 1150 897,1 •3407 1219 1338 • 893,7
b 5,07
Chemiluminescerme measurements of unirradiated and irradiated onion skin (white, lcm 2, immediately after irradiation).
. . . . . . . . . . .
......
7 .
:
.
.
.
.
.
.
.
.
.
.
.
.
.
mV
mv/Ssec
mV
"
782,3 537,4 • 360,4 ~1626 609,4 382,4 364,6
.
;i?~LT"_-'
.
.
.
.
L~L-_-;2L2.~I212.;~.
.
.
.
"
T-
;..;.
. . . . . .
.
i
~%
L
• i. . . . . . . . . .
T ! .......... . . . . . . . . . . . . . .
...................
,_=
?..-~LL
(10 kGy) is much lower compared to the results, m e a s u r e d with the white skin. The reason for this d e c r e a s e of the c h e m i l u m i n e s c e n c e intensity is a fast e x t r a c t i o n of the o n i o n - s k i n dye with the luminol s o l u t i o n and subsequent light q u e n c h i n g in the luminol solution. In figure 14, the results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of irradiated onion (without the skin) as a function of r a d i a t i o n dose are summarized. This figure quite clearly shows only a small d i f f e r e n c e in the c h e m i l u m i n e cence intensities b e t w e e n u n i r r a d i a t e d and i r r a d i a t e d onions.
Chemiluminescence M e a s ~ e m e n t s
;.8[
Using red onion-skin for the identification of gamma irradiation, it is impossible to see any difference between the irradiated and the untreated samples. The reason is again a fast extraction of the skin dye with the luminol solution. This extraction of red onion skin is demonstrated in figure 15 (extinction of the luminol solution as a function of extraction time). It can bee seen, that the extraction is very fast. The consequence is a complete quenching of the emitted light in the lumlnol solution.
160 ,_=
> E
120 c
.E > EE
300
._.R
8O
200
c
100
40
O
~ ¢ " ..... ;
,
I 0
0,3
1,0
3,0
•.~
-
0,5
._o <.J X t,M
0
(log)
Fig. 14. Chemiluminescence intensity of irradiated onion (without onionskin) as a function of radiation dose.
E tJJ
I 10
Radiation dose [ kGy ]
Fig. 13. Chemiluminescence intensity of irradiated onion-skin (brown) as a function of radiation dose.
1,0
re.', ......; ' ,
¢J
0 0,3 1,0 3,0 10 (Iogi Radiation dose [ kGy ]
1
!
I
0
5
20
Extraction time [sec]
Fig. 15, Extraction of red onion-skin with a luminol solution; extinction of the solution as a function of extraction time.
182
W. BOGL AND L. HEIDE
Frozen chicken: In figure 16, some results of c h e m i l u m i n e s c e n c e m e a s u r e m e n t s of gamma irradiated and untreated frozen chicken bone are summarized. These m e a s u r e m e n t s were performed immediately after the irradiation. Using the c h e m i l u m i n e s c e n c e method for irradiation identification in frozen chicken, it is preferable to prepare and measure some chicken cartilage, as it is demonstrated in figures 17 and 18. The intensity difference of
10 kGy
unirradiated
mV/Ssec -
mV/Ssec"
mV >161,~ 74,7 92,4
•216,2 1£9,6 145,3 104,7 100,1 178,0 • 86,0
56,5 • 37,5 100,6 44,3
2415 2501 • 4394 • 22~0 3897 3970 4382
mV 1044 i~66 2099 1093 I921 1938 i,2293
I
'i!
": 4
'l;
'I'
Fig. 16. Chemiluminescencemeasurements of unirradiatedand irradiateddeep frozen chickenbone (immediately after irradiation)
2.
_
I + h
- !!
4----- ~-- ~'-7:-7 7 7 . . . . . . .
A :'Z
. . . .
_
]
~
_
L
Chcmituminescence Measurements
183
the emitted light between the untreated sample and the gamma irradiated sample is larger when using cartilage rather than chicken bone or meat. This can be seen expecially in figure 18. This figure also demonstrates quite clearly that some weeks after irradiation, the process can still be identified.
unir~di~
mV/Ssec 117,7 85,1 • 118,1 114,1 108,5 39,8 • 37,6
Fig. 17,
lOkGy
mV
mV/Ssec"
43,0 32,6 41,6 • 44,9 34,0 •12,5 13,6
•2085 4675 •7726 6156 4801 3709 3032
mV • 74o,5 1585 •23O5 1797 1665 1143 947,1
Chemiluminescence measurements of unirrediated and irradiated deep frozen chickan cartilage (16 days after irradiation)
.
.
.
.
.
.
.
.
.
Cocoa powder; parsley (preliminary results): In figures 19 and 20, preliminary results of irradiated and untreated cocoa powder and parsley are demonstrated. All of these measurements were performed immediately after irradiation. CONCLUSIONS To sum-up, we can say that measurement of chemiluminescence would seem to be a method which permits quick and reliable detection of ionizing radiation treatment with of a lot of foodstuffs. At least for some foodstuffs, it will be possible to identify the radiation treatment process as late
18~
VV. BOGL ,~:~D L. HEIDE
10
deepfrozenchicken
¢J
s
~-
'l~kGy
c
= 6 c
==
"~
4
bone10 kGy IJ.
~ ( ~ %unirradiated I
YTi
meatl0kGy
!
0
9111~I
<~
8 Timeafterirradiation[days]
I
16
Fig. 18. Chemiluminescence intensity of irradiated deep frozen chicken cartilage, bone and meat as a function of storage time after irradiation.
10kGy
unirradiated mV/5sec
17,1 16,5 17,1
mV
6,73 6,73 6,95
mVl5~c 66,7 62,8 64,0
i
mV
15,4 14,6 16,3
Fig.19. Chemiluminescence measurements of unirradiatedandirradiatedcacao powder.
as several month after irradiation. The chemical background of a lot of the measured chemiluminescence effects is not explicable at the moment. In spite of the probability of a quenching or fading of the effect used for the detection of irradiation, or the stimulation of chemiluminescence effects by other treatment methods, in the near future it will be possible to develop chemiluminescence measurement to a dependable, precise and rapid method of identifying irradiation treatment in at least some foodstuffs.
Chemiluminescence Measurements
lOkGy
unirradi~ed
mV~sec
8,70 9,34 8,49
Fig. 20.
185
mV
3,33 3,80 3,36
mV/5~
33,3 58,9 33,6
mV
14,7 27,1 14,8
Chemiluminescence meuumments of unirradiatod and irradiated parsley.
REFERENCES (I)
World Health Organization: Wholesomeness of irradiated food. Report of a Joint FAO/IAEA/WHO Expert Committee. Technical Report Series 659, World Health Organisation, Geneva 1981. (2) Ettinger, K.V., and K.J. Puite (1982). Int. J. Appl. Radiat. Isot., 33, 1115-1138. (3) Puite, K.J., and K.V. Ettinger (1982). Int. J. Appl. Radiat. Isot., .,~t~, 1139-1157. (4) White, E.H., and M.M. Bursey (1964). J. Amer. Chem. Soc.. 86, 941-942. (5) White, E.H., O.C. Zafiriou, W. K~gi, and J.H.M.'Hill (1964), J. Amer. Chem. Soc., 86, 940-941. (6) White, E.H., and M.M. Bursey (1966). J. Amer. Chem~ Soc., ~I, 19121917. (7) Roswell, D.F., and E.H. White (1978). Methods in Enz~mology, 57, Academic Press, Inc., 409-423. (8) B5gl, W., and L. Heide (1983). ISH-Berlcht ~2, Institute for Radiation Hygiene of the Federal Health Office. (9) Heide, L., and W. BGgl (1984). ISH-Heft 41, Institute for Radiation Hygiene of the Federal Health Office. (10) Heide, L., and W. BSgl (1984). ISH-Heft 5~, Institute for Radiation Hygiene of the Federal Health Office. (11) B6gl, W., and L. Helde (1984). Flelschwirtseh., 64(9), 1120-1126. (12) BGgl, W., and L. Heide (1984). Symposium handbook, ~rd International Symposium on Analytical Applications of Bioluminescence and Chemiluminescence, Birmingham, GB, 17.-19.4.1984, 98.