Evaluation of Food Irradiation Procedures QUANTITATIVE CHEMICAL MEASUREMENTS UTILIZING HIGH ENERGY CATHODE RAYS SAMUEL A. GOLDBLITH, BERNARD E. PROCTOR, AND OLIVIA A. HAMMERLE Department of Food Technology, Massachusetts Institute of Technology, Cambridge, Mass.
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F BEAMS of high energy cathode rays are t o be used for the
sterilization of foods and pharmaceuticals, some rapid routine technique must be available t o determine the penetration of these radiations and the accelerating voltage of the electrons, the area covered by the radiations, and the variation of the electron density of the beam. A number of methods of measuring radiation doses other than ionization techniques have been proposed. Day and Stein (6) have recently reviewed this subject. Certain qualitative aspects of the use of methylene blue and other oxidation-reduction dyes as radiation indicators have been pointed out by Proctor and Goldblith (9),and research in this field has been continued by these investigators with the view of obtaining quantitative data. Since this research was completed, Day and Stein (6) have published their results with agar gels containing methylene blue and benzoate. This present paper describes qualitative and quantitative findings relating to the effects of high energy electrons on aqueous and nonaqueous solutions of methylene blue, the use of agar gels containing resazurin as an index of the penetration of high energy electrons, and the application of these data t o field distribution studies. Cathode rays ranging in voltage from 2.0 to 16 million electron volts were used as the source of radiation.
Solutions of methylene blue chloride were made in distilled water, in glycerol, and in absolute methanol. Samples (2 ml. each) of the methylene blue solutions of the desired concentration were pipetted into thin-walled, glass ampoules having a 5-ml. capacity. The ampoules were sealed with an oxygen flame and exposed to the cathode rays while lying on their long dimension. Thus the depth of the solution was within 0.6 cm. of the maximum range of the 3-m.e.v. electrons. This allowed a fairly uniform distribution of the radiation dose. The concentration of the methylene blue solution was measured before and after irradiation by means of a Lumetron photoelectric colorimeter, Model 400A, provided with a 650-mp filter. A standard curve for calibration of the colorimeter was determined for known concentrations of methylene blue in distilled water. This curve was found to obey Beer's law in concentrations ranging up to 4 micrograms per ml. Irradiated solutions were appropriately diluted t o this concentration range and the unchanged methylene blue content was measured. Observations had previously been made which demonstrated that the color of irradiated aqueous solutions of methylene blue remained stable for a t least 5 hours after irradiation.
6 MAXIMUM R)R RANGE R AVERAGE FOR RANGE R
NlMUM FOR RANGER
EXPERIMENTAL
The materials used in this research included methylene blue chloride (National Aniline), resazurin, glycerol, and methanol (absolute) of Mallinckrodt Analytical Reagent Grade, 2,3,5-triphenylazolium chloride (Arapahoe Chemical Co.), and Bactoagar in water. a W
Figure 2. Ionization in Depth for Maximum, Minimum, and Average Doses of 3-1M.e.v. Cathode Rays
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To obtain an indication of any structural changes caused by the radiations ultraviolet absorption spectra of diluted solutions of methylene blue were determined with a Beckman Model D U ultraviolet spectrophotometer. When resazurin or 2,3,5-triphenylazolium chloride was used in the tests, 8 ml. of a 14-mg. dye solution was added to a liter of molten agar (20 grams of agar per liter of distilled water). The agar-dye mixture was then poured into sanitary cans of various sizes and allowed t o gel.
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RESULTS
Figure 1. Penetration of 2- and 3-M.e.v. Cathode Rays in Sardine Cans Filled with Agar Gel and Resazurin Dye
QUALITATIVE STUDIES. The use of agar gels containing dyes that are changed in color by ionizing radiations has been found to be a satisfactory means of quickly determining visually the extent of penetration of a beam of ionizing radiations into matter of unit density. For example, in its reduction resazurin changes in color from blue to pink to colorlesfi.
Two types of radiation units were used-a Van de Graaff electron accelerator capable of producing mono-energetic cathode rays in the voltage range of 1 to 4 m.e.v. and a linear electron accelerator capable of producing mono-energetic cathode rays up to 16 m.e.v.
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Figure 1 illustrates thi: type of results obtained whcn oblong aardine can8 filled with resazurin dissolved in agar were irradiated with 2 - or 3-m.e.v. cathode rays. The cross-hatched section of each diagram represents thitt portion of ihe can's content8 in which the dye was unaffected by the cathode rays, the solid black section that portion where the dye w a ~changed to a pink color (indicative of level8 of irmdiation sufficient to cause partial ateriliziltion), and t,he blank section that portion where complete d i e coloration of tho dye (indicative of levels capable of causing complftt. si.criliaation) had occurred.
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When the linear accelerator K ~ uscd S its the S O U ~ C of~ the cathode rays and one end o f a .To. 2 can, having a diameter of 3 '/,. inches and a height o f 4°/,s ineheq filled with a mixture of agar gel and 2,3,5-triphenylasolium chloride was exposed to the radiations, the meximum depth of penetration of the 16-m.e.v. rays w86 8.5 cm. The distrihut,ion of the untreated cathode ray beam in t~heNo. 2 can thus exposed is illustrated in Figure 4. Tho dye, which is normally colorless, was changed to a dark red in that portion penetrated by the eat,hode rays. With this high voltage equipment it is possible to sterilize completely the contents oi a No. 2 can by exposing both ends of tho can to the radiations. Qualitative field distribution studiea may be readily made by placing agar-dye gel8 in the radiation beam and observing the relat,ivo changc in color after the gel is swtioned. For this purpose cryatallising dishes arc convenient for liargosized fields, whereas ram may bc used for fields oi small diameter. The change8 in color of dyes dissolved in agar have been found to be impermanent and are reversible. Moreover, limitations are preserrt,ed by diffusion of the colored substances through the gels over R pcriod of time, which blur an init.ially nliarp picture of dose diubribution. Iferrce, ohrcrvstions must be begun immediately after irmdist,ion.
PENETRATION (cm)IN MATERIAL OF UNIT DENSITY
figure 3.
Ionization in Depth in Steel Sardine Cans Filled with mnt~sialof unit drnxity Cross-Bred by 3.1-m.e.r. eaihode rays
T h e extent of ponetrdiori of the cathode raya when directed at, the top of t.he sardine C ~ (16 S mm. in depth) is shown by thc two di&grnmaio the l e f t half of Figure 1. It is qqxircnt that ncithor t.he 2- nor tho 3-m.e.v. cathode rays p&etrated the can conpletely.
The exteiit of penetration when the sardine can8 were exposed to cathode rays from hoth sides through the short dimension-that is, were crossfired-ia indicated by the diagrams in the right half 01 Figure 1. When the Can was crossfired with 2kn.e.v. electrons, it WILL not completely penetrated by t h o mya. There still remained a bimd of resasorin-agar in the eentcr of the c m that was blue (cross-liutr:lied), that is, eomplet~elyuntouclied by the cathodc rays. However, when 3-1n.e.v. electrons WPK used in the same msnner, the can WBB completely penetrated with tho required radiation for stcrility. Thia may be understood when Figures 2 and 3 arz eonsidered. Figure 2 represents tho ionization in depth of 3-m.e.v. cathode rays. The inaxirnuni range of penetration oi 2-m.e.v. electrons is 1.0 cm. and that of 3-m.e.v. electrons, 1.5 cm. The pink band of resasurin-gel (solid hlack section in the diagrams in Figure 1) represents the lower end of the ionization curve shown in Figure 2. Ifore the ionization i 8 less-that is, the dose is smsller than that in the firat three fifths of the range of penetration of the electrons. The total dose is represented hy the area under the curve. The relative dose at m y level heneath the mrface (heneath the curve) is the ionization n t t h n t depth of penetration. The maximum dose occurs at one third of the maximurn range (Rmsx.)or st 0.5 em. for 3 -".e.". cathode ntys. At threofiiths Itmsx.the dose is eqnnl to that at thc top of the can (60%) and at R,,,. thc dose I& oif to zero. The solid black sections of {.hediagranw in Figure 1 SIP represontstivo of the lower done betmeon thrcofifths R,,,. and RmtLx. When an oblong sardine can filled with remauriii-gel is exposed to 3.1hii.e.v. cnthode rays Eroiri both sides, thc C U N ~ R in Figure 3 art. oht:iind In thifi cnse the penehtion is suifieient, for conp1et.e st.erilizal.ion. This OCIDUIII,as may k seen iri Figure 3, by tho additivit,y of both parts oi the lmver ends of the ioaimtion curves represented hy the pink re win-gel, with the result that tile percentage of iouization at the center oi the r m is equivalent to that at tho top and bottom surfaces nnd complete steriiiinttion owum. This iu repreaentcd in Figure 1 by the diagmm in the 10ww rietit corner Isbeled coiorlsss.
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h m ~ .Qunnt.itative ponetrzation studies may be mi~deby plating viab conbrrining aqueous solutions of methylene t h e in YLENE:
the piith of the elect.ron beam, the vials being staakod in depth, and measuring the unchanged mnt.hylene blue remaining after irradiation. Figure 5 represents such a study with the linear aceelerator. In another serirs of quantitative penetration studies, five bets of vials containing aqueous solutions of methylane blue of five differmt ioncentrations (weieht to volnrne)-namelv. " _96.5.. !l4.0. 88.0, 45.75, and 8.60 micrograms per ml.'-were irradiabed by doses of cathode mys varying from 5000 to 2,W,WO roentgeneiluivnlents-physicrtl. The doses given in thia psgm itre avwage doses ealculated nccordine to the method of Trunro. Wrieht. and
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(r.e.p.), tho encrgy converrion factor of 1 r.e.p. being consCdeFed to bo 83 ergs per gram o f tismo. No attempt W B S madc to rPmOVe the ossecn from the mnnoules in which the soliitionx were irradi-
The data indic.ate that thc methylenc blue in aqueous suitition by the ionizing radiations. In no instance did t,he blue color return to tho solutions in t,ho vials trentrd with hydrogen peroxide. The curves Ear ultrmiolet absorption ~pcctrnlikewise indicated disruption of thc nwthylene blue molecule, for on W~LSdestro,yed
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TABLE I. EFFECTO F SUPERVOLTAGE CATHODE RAYSO N AQUEOUS SOLUTIONS OF METHYLENE BLUE Ihactivation Concn. Ori ins1 Methylene Dose, DO", and Dose, Bfue Remaining R.E.P. R.E.P. Unchanged, % ' 96.5 pg./ml. 100.0 Control 318;OOO 85.5 50,000 69.9 280,000 100,000 61.7 310,000 150,000 53,l 314,000 200,000 44.1 305,000 250,000 41.4 340,000 300,000 32.4 357,000 400 000 20.2 313,000 500:000 94 pg./ml. 100.0 Control 3801000 97.4 10,000 156,000 85.2 25,000 242,000 81.3 50.000 248,000 73.9 75,000 260,000 68.0 100,000 222,000 56.9 125,000 326,000 63.1 150,000 253 000 50.0 175,000 270 000 47.6 200,000 271 ,000 33.0 300,000 24.4 (355,000) 500,000 (420,000) 9.2 1 000 000 (505 000) 5.1 1 :500:000 (673:OOO) 5.1 2,000,000 88 g/ml. &oAtrol 50,OW' 100,000 150,000 200,000 250,000 300,000 500,000 c45.75 pg./ml 100.0 Control 113;000 63.9 50,000 131,000 46.7 100,000 148,000 36.3 150,000 145.000 25.1 200,000 153,000 19.5 250 000 166,000 16.4 300'000 191,000 12.3 400 000 222,000 10.3 500,000 8.601pg./mI. 100.0 Control 2i:eoo 83.6 5,000 35,600 75.8 10,000 31,600 62.2 15,000 33,700 55.3 20,000 0 Values in parentheses were not used in calculating the average inactivation doses. D o , recorded in Table 11.
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the spectra occurred. The conclusion is that the methylene blue in aqueous solutions, in the presence of oxygen, was irreversibly destroyed by the supervoltage *cathode rays, with probable disr u p t i o n of t h e ring structure. Similar results in this respect have b e e n o b t a i n e d with other aromatic 80 '' ring-type compounds ( 1 , 48). % The average inactivation doses, Do, and 40 the specific inactiva\ 20 tion doses, D,/C, cal\ culated for these stud0 ies are summarized 0 2 4 6 8 1 0 in TableII. DISTANCE FROM BEAM T h e inactivation dose, D,, is defined as the amount of radiaORIGIN OF tion t o which a solute BE P M is exposed in Figure 5. Ionization in Depth s o l u t i o n w h i c h will in Vials result in 63% destrucVials oontained aqueous solutions of m e t h y l e n e blue tion of the solute or, Irradiated w i t h 16-m.e.v. cathode c o n v e r s e l y , 37% rerays
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tention. It may be calculated for any single determination by the equation
n/no = e - D / D o where nln, is the amount of solute remaining unchanged when it is exposed t o a dose D (r.e.p.), Dois the inactivation dose, and e is a natural logarithm (4). The average inactivation dose, Do,is the average of the individual determinations for any one concentration when irradiated over a range of doses. (See Tables I and 11.)
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Figure 6. Ultraviolet Absorption Spectra of Aqueous Solutions of Methylene Blue after Irradiation by High Energy Cathode Rays The fact that the average inactivation dose is proportional to, or dependent on, the concentration is indicative of an indirect action of the cathode rays (4). Moreover, the absolute magnitude of the average inactivation dose is practically incompatible with a direct action. The deviations from the first-order reaction a t the higher irradiation doses noted in certain instances may in all probability have been due t o the molecular fragments resulting from the cathode ray bombardment, fragments that competed for the free radicals produced by the radiations.
TABLE11. AVER.4GE INACTIVATION DOSES AND SPECIFIC INACTIVATION DOSESFOR AQUEOUS SOLUTIONS OF METHYLENE BLUEOF DIFFERENT CONCENTRATIONS Ori inal Concn. of; Solution, Micrograms/Ml. 96.50 94.00 88.00 45.75 8.60
Average Inactivation Dose, Do, R.E.P. 317,000 263,000 318,000 159,000 32,200
Specific Inactivation Dose, Do/(?, R.E.P./G./Ml. 3.29 X 100 2.80 X 100 3.61 X 109 3.48 X 100 3.74 x 109
QUANTITATIVE STUDIES WITH NONAQUEOUS SOLUTIONSOF METHYLENE BLUE. I n these studies an attempt was made first t o irradiate methylene blue in glycerol. Because glycerol is viscous, quantitative transfers of the solutions by pipet were difficult. Solutions of 50 micrograms per ml. were irradiated by relatively high doses of 1.0 X lo6and 1.5 X lo8r.e.p. of cathode rays. Examination of the vials immediately after irradiation showed that the solutions were almost completely discolorized. However, when the irradiated solutions R'ere left a t room temperature for approximately 1hour, over 90% of the original color returned.
INDUSTRIAL AND ENGINEERING CHEMISTRY
February 1952
DOSAGE (rap a lo5)
Figure 7. Effect o f High Energy Cathode Rays on Retention of Color of Nonaqueous (Methanol) Solution of Methylene Blue
The writers have not studied the effect of dissolved oxygen on this reaction. However, it has been suggested t o them (6)t h a t *the low initial yield in the irradiation of methanol solutions of methylene blue might well be due t o dissolved oxygen and t h a t the gradual return of the color which is noted is due at least partially t o the diffusion of oxygen. Moreover, while the samples were being returned from the irradiation room to the laboratory, i t was observed t h a t the solution t h a t had been irradiated with 500,000 r.e.p. changed from being almost colorless immediately after irradiation t o a definitely blue color again. With t h e methanol solutions, therefore, the cathode ray bombardment caused some reversible reduction. However, the destruction curve (Figure 7 and Table 111)did not appear t o be exponential. PERCENT OF MAXIMUM DOSAGE
Although quantitative data could not be obtained because some of the glycerol-methylene blue solutions may have adhered t o the pipets, the fact t h a t there was a 90% return of the color shows that methylene blue, when exposed in glycerol t o cathode ray irradiation does have a remarkable reversibility. T o obtain more quantitative results with a nonaqueous solvent, methylene blue (92.5 micrograms per ml.) was irradiated in absolute methanol. T h e results obtained (Table I11 and Figures 7 and 8 ) were of the same nature, although not nearly so marked, as those when glycerol was t h e solvent. T h e irreversible destruction of the dye (column 2 of Table 111) was much lesa than t h a t of the dye of corresponding concentration irradiated in aqueous solution.
TABLE111. EFFECTOF SUPERVOLTAGE CATHODERAYS ON METHYLENE BLUEDISSOLVED I N ABSOLUTE METHANOL Concentration, 92.50 Micrograms per Ml. Original Methylene Inactivation Blue Remaining Dose. Do, R.E.P. Unchanged, % Dose Control 100.0 100.0 50,000 3 666'000 100 000 97.3 91.8 2'440' 000 200:000 81 .a 1 :428:OOO 300,000 600,000 58.2 924,000 Range of epecific inactivation dose, D o / C , 3.98 X 1010 to 9.99 X 1 0 9 r.e.p per g. per ml.
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