BIODEGRADATION RADIOACTIVE
TESTING WASTE
FORMS
OF *
ROBERT D. ROGERS and JOHN W. M c C O N N E L L , Jr. Biotechnology and Waste Management Groups, INEL, P.O. Box 1625, Idaho Falls, 119 83415, U.S.A.
(Received February 1988) Abstract. Biodegradation tests were conducted on solidified waste forms containing ion exchange resins
contaminated with high levels of radioactive nuclides. These tests were part of a program to test waste forms in accordance with the U.S. NRC Branch Technical Position on Waste Forms. Small waste forms were manufactured using two different solidification agents, Portland Type I-II cement and vinyl ester-styrene (VES). Ion exchange material was taken from a filter system which had been used to remove radionuclides from highly contaminated water. As specified by NRC, the waste forms were evaluated for their resistance to biological degradation using the G21 and G22 procedures of the American Society for Testing Materials (ASTM). Results showed that microbial growth can be supported by the VES waste forms. The particular organisms used in the tests did not grow in the presence of the cement waste forms. It is also shown that the ASTM tests specified in the Technical Position are not suitable for the use intended. A different testing methodology is recommended which would provide direct verification of waste form integrity.
1. Introduction
Filters e x p e n d e d in the c l e a n - u p o f liquid wastes at the T h r e e Mile I s l a n d U n i t 2 ( T M I - 2 ) n u c l e a r p o w e r r e a c t o r c o n t a i n o r g a n i c a n d i n o r g a n i c ion exchange resins which are c o n t a m i n a t e d with g a m m a - a n d b e t a - p r o d u c i n g r a d i o n u c l i d e s . T h e I d a h o N a t i o n a l E n g i n e e r i n g L a b o r a t o r y ( I N E L ) used these ion e x c h a n g e resins to o b t a i n i n f o r m a t i o n on s u r v i v a b i l i t y o f waste f o r m s c o m p o s e d o f the resins solidified in m a t r i c e s o f P o r t l a n d T y p e I - I I c e m e n t a n d vinyl ester-styrene (VES). These waste f o r m s were tested to d e t e r m i n e if they m e t the r e q u i r e m e n t s o f U.S. N u c l e a r R e g u l a t o r y C o m m i s s i o n ( N R C ) r e g u l a t i o n 10 C F R P a r t 61, ' L i c e n s i n g R e q u i r e m e n t s f o r L a n d D i s p o s a l o f R a d i o a c t i v e W a s t e ' [1]. E m p h a s i s was p l a c e d o n a p p l y i n g r e g u l a t i o n 10 C F R P a r t 61 using m e t h o d s specified in the B r a n c h T e c h n i c a l P o s i t i o n o n W a s t e F o r m [2] (TP) o f the N R C O f f i c e o f N u c l e a r M a t e r i a l S a f e t y a n d Safeguards. O n e o f the s t r u c t u r a l stability r e q u i r e m e n t s for waste f o r m s in 10 C F R P a r t 61 is resistance to b i o d e g r a d a t i o n . This p a p e r p r o v i d e s results o b t a i n e d f r o m testing o f solidified resins using the initial b i o d e g r a d a t i o n m e t h o d s specified b y the T P .
* Work supported by the U.S. Nuclear Regulatory Commission, Office of Nuclear Regulatory Research, under DOE Contract No. DE-AC07-76ID01570. Environmental Monitoring and Assessment 11 (1988) 89-100. 9 1988 by Kluwer Academic Publishers.
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ROBERT D. ROGERS A N D J O H N W. MCCONNELL JR.
2. Materials and Methods 2.1. DESCRIPTION OF WASTE FORMS Waste forms used in the biodegradation test were composed of solidified filter ion exchange resin wastes. Two resin waste mixtures were solidified. Type A was a mixture o f synthetic organic ion exchange resins (phenolic cation, strong acid cation, and strong base anion resins), while Type B was a mixture o f synthetic organic ion exchange resins (strong acid cation and strong base anion resins) and inorganic zeolite. Portland Type I-II cement and VES were Used to solidify both types o f resin wastes. VES is a proprietary solidification agent developed and supplied by the Dow Chemical Company * and is composed of a binder (styrene monomer and vinyl ester resin), a catalyst, and a promoter. Individual waste forms were manufactured by allowing a mixture of solidification agent and resin waste to solidify in 4.8 cm diameter by 10.2 cm high polyethylene molds. Enough of the mixture was added to each vial to produce waste forms with average dimensions of 4.8 cm diameter by 7.6 cm length (137.5 cm3). A complete description of waste form manufacture is given in Reference [3]. 2.2. PROCEDURES SPECIFIED FOR DETERMINING BIODEGRADATION The NRC T P specifies that procedures from the American Society for Testing Materials (ASTM) be used to determine the resistance of solidified waste forms to fungi and bacteria. Those procedures can be found in ASTM G21, 'Standard Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi' [4], and ASTM G22, 'Standard Practice for Determining Resistance of Plastics to Bacteria' [5]. If either o f those tests indicate that a waste form is capable o f supporting microbial growth, the T P states that a more conclusive procedure like that described by Bartha and Prame [6] be initiated to determine if biodegradation is occurring. Both ASTM procedures are described in detail as referenced and will only be highlighted here. Five of the fungal cultures specified for the ASTM G21 procedure were obtained from the American Type Culture Collection (ATCC), Rockville, MD, and the sixth, designated ILF-I, was isolated in the laboratory from a VES waste form. The ATCC fungal strains were Aspergillus niger (9642), Penicillium funiculosum (9644), Chaetomium globosum (6205), Gliocladium virens (9645), and Auerobasidium pullulans (9348). Cultures of these fungi were received freeze-dried and were revived following ATCC procedures. Stock cultures were maintained on potato dextrose agar (PDA) plates and recultured as necessary. Stock cultures were kept in a refrigerator at 5 ~ cultures used to produce inoculum were maintained at 28 ~ Pseudomonas aeruginosa (ATCC 13388) as specified by ASTM G22 was used for bacterial testing. Freeze-dried cultures were revived and cultured on nutrient agar * Mention of specific products and/or manufactures in this document implies neither endorsement of preference nor disapproval by the U.S. Government, any of its agencies, or EG + G Idaho, Inc., of the use of a specific product for any purpose.
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91
salts. Stock cultures were refrigerated at 5 ~ while the culture used for inoculum was maintained at 28 ~ All procedures used to culture and maintain organisms, prepare inoculum, as well as the actual testing procedures, were as specified by ASTM G21 for the fungi or ASTM G22 for the bacteria. Agar used for the waste form testing was the ASTM 'nutrient salts' (NS) formulation. This media contained inorganic nutrients but no organic carbon sources and was intended to supplement those organisms which could utilize the waste forms as a carbon source. In addition to the controls mandated by the ASTM Procedures, each test also had controls consisting only of inoculated NS media. These controls were used to assure that any microbiological growth on or near the waste forms was not being supported by the NS media. Also, to assure that the waste forms did not come in contact with other sources of usable carbon, they were stored either in sealed 'whirl-pak' bags or glass beakers and handled only with tongs. All work was conducted with 100 x 15 mm petri dishes except that with the full size waste forms which required 100 x 80 mm culture dishes. The diameter and height of the larger dishes were such that they could each accommodate a waste form laid on its side. After a waste form was placed in a dish, 106 mL of molten agar, cooled to 55 ~ was poured in, thus submerging all but the upper one-fifth of the form. A number of ASTM G21 preliminary investigations were performed on VES and VES mixed with nonradioactive exchange resin before the radioactive waste forms were evaluated. Sequentially, though not necessarily chronologically, work with nonradioactive VES was conducted on the individual components usedto make the VES polymer (binder, catalyst, promoter), powder made from a waste form, wafers cut from a waste form, and a whole waste form. Work with the polymer components was conducted with both NS agar and NS liquid. A quantity of 0.2 mL of each of those VES components was placed on a separate section of the agar in each of six petri dishes. Each of the agar surfaces was inoculated with one of the fungal species used in the ASTM G21 test. After 20 days' of incubation, the agar surface was examined to detect growth of the fungi. For the liquid study, 1 mL of each of the polymer components was added to 20 mL of sterilized NS liquid contained in a 50 mL Erlenmeyer flask. Each of the three flasks was then inoculated with 0.2 mL of the fungal spore suspensions and sealed with a sterile rubber stopper. For the studies using the solidified waste form, VES containing exchange resin was ground to pass a 35 mesh sieve (0.42 ram) and the powder was sprinkled onto the surface of NS agar inoculated with fungal spores. In this case, duplicate plates with VES and an inoculated control were used. Wafers were obtained from dry cutting 0.30 cm sections. A whole nonradioactive waste form was also used. Radionuclide contents of radioactive waste forms used in this study are shown in Table I. Because of the high radiation doses emanating from the waste forms, three separate biodegradation test setups were required at different times to accommodate the waste forms with the available shielding. In the first evaluation, waste forms C1A-28, C1A-29, D1-8, and D1-9 were tested under the ASTM G21 procedure, while D I - l l , Dl-12, C1A-31, and C1A-32 were used with ASTM G22. In addition, DI-10
92
ROBERTD. ROGERSAND JOHN W. MCCONNELLJR. TABLE I Contact gamma dose for waste form used in biodegradation study
Waste Form ID
Contact Gamma Dosea (R/h)
WasteForm ID
Contact Gamme Dosea (R/h)
CIA-28b C1A-29 C1A-30 CIA-31 C1A-32 C2A-22 C2A-23 C2A-25 C2A-31 C2A-32
2.2 2.2 2.1 2.1 2.0 11.0 11.0 11.0 11.0 11.0
DI-8 DI-9 DI-10 DI-ll DI-12 D2A-3 D2A-4 D2A-5 D2A-6 D2A-7
4.0 3.7 3.6 3.4 3.1 16.0 14.5 13.5 13.5 11.5
a Waste forms have the following curie contents: 134C8 137Cs
9~
Total
DI 4.38x 10-3 66.22 x 10-3 3.92x 10-3 74.52 x 10-3 D2A 18.22 x 10-3 275.45 x 10-3 0.64 x 10-3 294.31 x 10-3 C1A 2.95 x 10-3 44.58 x 10-3 2.64x 10-3 50.17 • 10-3 C2A 13.53 x 10-3 204.59 x 10-3 0.47 x 10-3 218.59 x 10_3 b 'C' indicates Portland cement; 'D' indicates vinyl ester-styrene; ' 1' indicates Type A waste; '2' indicates Type B waste; and 'A' indicates the first of two batches.
a n d C1A-30 were i n c u b a t e d u n d e r A S T M G21 c o n d i t i o n s except that the A S T M NS agar was replaced with the c a r b o n - r i c h P D A . The p u r p o s e o f using P D A was to provide controls to determine if there were i n h e r e n t properties o f the waste forms (i.e., chemical or r a d i a t i o n ) which w o u l d prevent or reduce growth o f test organisms. The second set o f tests used waste forms D2A-3 a n d D2A-4 for A S T M G21 tests, D2A-5 a n d D2A-6 for A S T M G22 tests, a n d D2A-7 for the c a r b o n - r i c h P D A control. T h e third a n d final test used waste forms C2A-22 a n d C2A-23 for A S T M G21, C2A-25 a n d C2A-31 for A S T M G22, a n d C2A-32 for the c a r b o n - r i c h control. F o r each testing period as specified by A S T M G21, sterile paper strips were placed o n NS agar (in the absence o f waste forms) a n d sprayed with the fungal i n o c u l u m . I n a d d i t i o n , the surface o f a n NS agar plate was sprayed with i n o c u l u m to d e t e r m i n e if either the spore s u s p e n s i o n or the NS agar could s u p p o r t f u n g a l growth. A f t e r 20 days o f i n c u b a t i o n , the waste forms a n d associated agar were inspected as detailed in A S T M G21 or G22. W h e n dealing with the radioactive forms, a close, p r o l o n g e d i n s p e c t i o n was n o t possible; d e t e r m i n a t i o n o f the extent of the growth had to be ascertained f r o m color p h o t o g r a p h s o f each waste f o r m in its agar matrix.
53. Results and Discussion 3.1. RESULTS OF A S T M G22 BACTERIA TESTING N o n r a d i o a c t i v e specimens of VES a n d cement were f o u n d n o t to s u p p o r t the test
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
93
bacteria Psudornonas aeruginosa under ASTM G22 conditions. The same was also true for those tests conducted on eight radioactive waste forms (four VES and four Portland cement). These data do not support the earlier work of Piciulo et al. [7], who reported that various mixtures of VES and simulated waste (including a deionized water control) supported the bacteria. However, because the bacterial growth in the referenced study was determined by an indirect method not specified by the ASTM G22 procedure, the results have been considered inconclusive [8]3.2. RESULTS OF ASTM G21 FUNGAL TESTING Results of the initial investigations using nonradioactive material will be presented first, followed by those on the radioactive waste forms. In those tests involving VES components, all of the NS agar areas to which VES components had been added supported at least one of the fungal species in addition to fungi which were able to grow at the component agar interface (Table II). The inoculated control plate which consisted of NS agar without a carbon source had no traces of fungal growth, thus showing that neither the NS agar nor the spore suspension contained usable carbon. After a 30-day period of incubation of VES components in liquid NS media, the contents of the flasks were examined for the presence of fungal growth. Growth was noted visually (appearance of hyphal strands) in all cases, with the most apparent growth in the NS/catalyst mixture and the least in the NS/promoter mixture. Even after 18 months, growth associated with the catalyst and binder remained viable. This was shown when samples of fungal hyphae from these mixtures produced abundant growth on nutrient agar plates. These findings support the work of others [9, 10] who have found that styrene and styrene dimers can be biodegraded. Within 30 days of incubation, sporadic heavy growth could be seen on both duplicate inoculated plates hosting powdered VES (Figure 1) but not on the NS control. An enlargement (10 x ) of one of the areas of growth (Figure 2) shows that the fungi were growing on the VES. Growth of fungi at the NS agar wafers interface with and without exchange resins was noted within 30 days. Close examiT A B L E 11 Support o f fungal growth by individual components of Dow polymer (VES) Component Fungus
Binder
Catalyst
Promoter
ILF-I A.
_a
+b
_
+
+
+
G. virens P. funiculosum
+c
+_
+
A. pullulans
+_
+
+
niger
a No fungal growth noted. b Fungal growth on c o m p o n e n t (usually an ASTM G21 rating o f 1 or 2). c Fungal growth surrounding component.
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ROBERT D. ROGERS AND JOHN W. MCCONNELL JR.
Fig. 1.
Fig. 2.
Fungal growth on a powdered nonradioactive VES waste form.
Close-up view (10 x ) o f fungal growth on a powdered nonradioactive VES waste form.
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
Fig. 3.
Fig. 4.
95
Funga/growth on water cu~ from aoaradioacZJve wasze form after 18 months.
Ciose-ttp view (10 x ) of fungai growth on wafer cut from nonradioactive VES waste form after 18 months.
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ROBERT D. ROGERS AND J O H N W. MCCONNELL JR.
nation after 60 days showed that fungi were growing on the edges of both types of wafers. After 18 months, more extensive growth (ASTM rating of three) was seen on the edge and surface of the wafer with resins (Figures 3 and 4). During the development of a method to implement the ASTM testing of full-size waste forms, an unidentified fungus (later given the designation of ILF-1) was found growing on the agar surface directly above a buried, unirradiated VES waste form. The source of the fungus was apparently the waste form, since the agar was not inoculated. Within 30 days, the fungus covered the exposed NS agar surface (Figure 5). The supposed carbon source for the fungus was some soluble component of the waste form. This fungus was cultured on PDA and, as allowed by the ASTM G21 protocol, was included as a stock culture for that test. After the initial work using the nonradioactive VES was complete, ASTM G21 testing of the irradiated EPICOR-II waste forms began. Cement waste forms in NS agar failed to support fungal growth. The cement waste form which was used as a control in the carbon nutrient PDA did allow for growth of fungi over the complete surface of the agar. So, while the waste form itself was not able to support the fungi, growth was supported by a supplemental carbon source. Results of radioactive VES testing showed that these waste forms could support the growth of fungi (Figure 6). Growth can clearly be seen at the agar/waste form interface. Because of the mottled appearance of the waste form surface coupled with the inability for close examination, it is not clear whether it is supporting fungal growth. No growth rating based on the ASTM scale was assigned. The PDA surface containing a VES waste form had a heavy covering of fungi. The NS agar control with no carbon source had no fungal growth, while NS agar with paper strips (ASTM control) had good growth on the paper. Following the NRC TP procedures, those waste forms which had supported fungal growth (VES) were prepared for retesting while the others (cement) were used for compression testing. Preparation for retesting was accomplished by first rinsing the specimens in methanol [7] and then allowing them to air dry for 30 days. The waste forms which had supported fungal growth initially were again subjected to the G21 procedure. After the 20 day period of incubation, fungi were again seen to be growing at the/agar waste form interface of each specimen. The control of NS agar remained free from fungal growth. 9.3. DISCUSSIONOF ASTM G21 RESULTS It is obvious that work with cement waste forms should have been conducted with microorganisms known to degrade cement. (Thiobacilli [ 11], nitrobacter [12]). While fungal growth was not seen on the NS agar, growth was noted in the cement PDA control. Piciulo et al. [7], noted that fungi did not grow during the ASTM G21 test with cement and attributed this to pH or ionic strength changes created by the cement. While cement probably does cause such changes, it still appears that an available carbon source will promote growth near the waste form. Using the results from Piciulo et al. [7], Bowerman et al. [8], suggested that the cement waste forms
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
Fig. 5.
Fig. 6.
Fungal growth on NS agar covering nonradioactive VES waste form.
Fungal growth associated with radioactive VES waste form DI-9 (first ASTM test).
97
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ROBERT D. ROGERS AND J O H N W. MCCONNELL JR.
might cause a biocidal or inhibitory effect in a burial environment. It now appears that this effect may not be as pronounced as first reported. Work with VES has demonstrated that it can promote fungal growth, although the carbon source has not been determined. Evidence from this study indicates that the polymer or components (particularly the catalyst and binder) could be contributors. In addition, the study shows that once organisms have established themselves with these materials they can remain viable for several months. The growth of fungi away from a VES waste form suggests that some carbon source is being leached from the VES. Since this pattern o f growth continued in a second testing even after the waste form surface was rinsed, it can be assumed that the source of the soluble carbon was internal. The application o f the Bartha-Pramer [6] test method would provide information which could be used to determine the extent of the carbon source. Because it was not possible to closely examine the radioactive VES waste forms, it was not clear whether the fungi were actually growing on the VES surface. However, results from two of the initial studies with nonradioactive material suggest that fungi could indeed have been growing on the surface. It was shown that fungi would grow on a powdered VES waste form (Figure 2) as well as wafers cut from waste forms with and without ion exchange resins. With time, this growth can become heavy (Figure 4). No data are available on whether the fungi are penetrating the waste form surface. This work was not intended to determine if VES polymer bonds were being biocatalyzed. It is a commonly held theory that chain branching in polymers has an inhibitory effect on the biodegradation of VES [13]. However, when VES samples (mixed with distilled water) were evaluated in a Bartha-Pramer test, CO2 production was maintained at a continuous rate during a 6 month test [7]. Those data suggest that a carbon source in excess o f any free monomer, promoter, or catalyst was being utilized. A study of the data from compression testing of those cement waste forms exposed to biodegradation testing reveals that compression testing is unlikely to detect any biologically induced physical changes in a waste form. This is because of the insensitivity o f compression testing to detect those slight structural variations which could occur over the brief period (21 days) of the biotests with relatively bulky samples. Finally, there have been some suggestions that the biodegradation of radioactive waste forms is moot, since the radiation associated with this material would kill any microorganisms. This does not appear to be the case as was evidenced by the results from waste forms tested in this study which contains high radionuclide loadings and exhibited radiation doses of 2-16 R / h at contact (Table I). In those cases where a readily available carbon source was supplied (PDA), the VES as well as the cement waste forms had copious quantities of fungai growth. It is expected that microbes can exist in an exposure range o f 2 - 1 0 0 ( O rads [14]. This would appear to be well within the range produced by commercial-sized radioactive waste forms.
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99
4. Conclusions and Recommendations As a result of this study, the following conclusions can be drawn: (1) VES with or without irradiated ion exchange resins promotes microbial growth. (2) The carbon source for growth could be any or all unreacted components used to produce VES or it could be the VES itself. (3) Cement waste forms containing radioactive ion exchange resins do not support microbial growth of the types specified by ASTM G21 and G22; neither do they completely inhibit growth when a carbon source is provided. (4) Neither VES or cement waste forms promote the growth of the bacterium P. aeruginosa. (5) Radiation at the levels found in the EPICOR-II radioactive waste forms used in this study does not inhibit microbial growth. (6) The ASTM G21 and G22 tests are not suitable for determining the extent of waste form biodegradability or for establishing physical effects due to biodegradation. While this study and others [8, 15] have provided evidence that VES and cement can support the growth of microbes, it was concluded that the fundamental requirements of the T P have not been satisfied because the extent of degradation and any effect it could have on the physical characteristics of the waste form were not and could not be determined with the ASTM methods. Because of the unlikelihood that the present methodology can be modified in a way that will completely satisfy the demands for an accelerated biodegradation test, it is recommended that the emphasis of the test be changed from the evaluation of waste forms to an evaluation of materials which will be used as solidification agents. By doing this, the question of biodegradation of a solidification agent and its consequences could be answered by one in-depth investigation conducted by personnel familiar with biodegradation testing rather than recurring limited tests conducted at numerous waste generation sites using individuals with varying training, experience, and equipment to conduct the testing. This concept is based on the conclusion that the greatest possibility for structural failure o f a waste form rests with the solidifying agent. If the solidified matrix retains its integrity, then there is reason to assume that there will be no structural failure even if the waste material is biodegradable. However, some testing with standard waste forms containing various known wastes would be required to substantiate this position. If this suggestion were to be adopted, then the responsible agency could maintain complete control of verifying the integrity of waste forms simply by certifying the solidification agent. Such certification would come from a source other than the manufacturer and would involve a very conservative (i.e., rigorous) test protocol. The justification for such rigorous testing is that it needs to occur only once for each solidification agent. It may also be necessary to verify that the producer of the waste form is adhering to the specifications for making the solidification agent. The proposed testing would be of the type which Bowerman et al. [8] summarized as inherent biodegradability testing. Such a method would include: exposure to a
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ROBERT D. ROGERS AND JOHN W. MCCONNELL JR.
wide range of microbial species (especially to those known to affect the solidification agent); appropriately sized specimens for testing; possible use of alternative carbon (energy) sources to provide opportunity for co-metabolism of a specimen; and extension of the time period allowed for testing (up to 6 months). Degradative effects would be determined directly by measuring metabolic activity, such as CO2 production or oxygen consumption, or some physical parameters like specimen weight loss and visual deterioration. Notice This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, or any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use, or the results of such use, of any information, apparatus, product or process disclosed in this report, or represents that its use by such third party would not infringe privately owned rights. The views expressed in this report are not necessarily those of the U.S. Nuclear Regulatory Commission.
References [1] United States Nuclear Regulatory Commission, 'Licensing Requirements for Land Disposal of Radioactive Waste', Title 10 Code of Federal Regulations Part 61, U.S. Federal Register, Vol. 46, No. 142, 24 July 1987. [2] United States Nuclear Regulatory Commission, 'Branch Technical Position on Waste Form', 11 May 1983. [3] Neilson, R M. and McConnell, J. W.: Solidification of EPICOR-II Resin Waste Forms, GENDINF-055, August 1984. [4] American Society for Testing Materials, 'Standard Practice for Determining Resistance of Synthetic Polymeric Materials for Fungi', ASTM G21-70, 1981 Annual Book of Standards, Part 35. [5] American Society for Testing Materials, 'Standard Practice for Determining Resistance of Plastic to Bacteria', ASTM G22-76, 1981 Annual Book of Standards, Part 35. [6] Bartha, R. and Pramer, D.: 1965, 'Features of a Flask and Methods for Measuring the Persistence and Biological Effects of Pesticides in Soil', Soil Science 100, 68-70. [7] Piciulo, P. L., Shea, C. E., and Barletta, R. E.: Biodegradation Testing of Solidified Low-Level Waste Streams, NUREG/CR-4200, May 1985. [8] Bowerman, B. S., et al., An Evaluation of the Stability Test Recommended in the Branch Technical Position on Waste Forms and Container Materials, NUREG/CR-3829, March 1985. [9] Sielicki, M., Focht, D. D., and Martin, J. P.: 1978, 'Microbial Transformations of Styrene and [14C] Styrene in Soil and Enrichment Cultures', Appl. Environ. Microbiol. 35, 124-138. [10] Higashimura, T., Sawamoto, M., Hiza, T., Karaiwa, M., Tsuchii, R., and Suzuki, T.: 1983, 'Effect of Methyl Substitution on Microbial Degradation of Linear Styrene Dimers by Two Soil Bacteria', Appl. Environ. Microbiol. 46, 386-391. [11] Sand, W., Bock, E., and White, D. C.: 1987, 'Biotest Systems for Rapid Evaluation of Concrete Resistance to Sulfuroxidizing Bacteria', Mater. Perform. 26, 14-17. [12] Bock, E.: 1987, 'Biologisch Induzierte Korrosion von Natustein-Starker Befall mit Nitrifikanten', Bautenschutz. Bausanierung 10, 24-27. [13] Potts, J. E.: 'Biodegradation', in H. H. G. Jellinek (ed.), Aspects of Degradation and Stabilization of Polymers, Elsevier Scientific Publishing Co., New York, NY, 1978. [14] Choppin, G. R. and Rydberg, J.: Nuclear Chemistry: Theory and Application, Pergamon Press, Oxford, 1980. [15] Zajie, J. E.: Microbial Biogemochemistry, Academic Press, NY 1969, p. 345.
TESTING WASTE
FORMS
OF *
ROBERT D. ROGERS and JOHN W. M c C O N N E L L , Jr. Biotechnology and Waste Management Groups, INEL, P.O. Box 1625, Idaho Falls, 119 83415, U.S.A.
(Received February 1988) Abstract. Biodegradation tests were conducted on solidified waste forms containing ion exchange resins
contaminated with high levels of radioactive nuclides. These tests were part of a program to test waste forms in accordance with the U.S. NRC Branch Technical Position on Waste Forms. Small waste forms were manufactured using two different solidification agents, Portland Type I-II cement and vinyl ester-styrene (VES). Ion exchange material was taken from a filter system which had been used to remove radionuclides from highly contaminated water. As specified by NRC, the waste forms were evaluated for their resistance to biological degradation using the G21 and G22 procedures of the American Society for Testing Materials (ASTM). Results showed that microbial growth can be supported by the VES waste forms. The particular organisms used in the tests did not grow in the presence of the cement waste forms. It is also shown that the ASTM tests specified in the Technical Position are not suitable for the use intended. A different testing methodology is recommended which would provide direct verification of waste form integrity.
1. Introduction
Filters e x p e n d e d in the c l e a n - u p o f liquid wastes at the T h r e e Mile I s l a n d U n i t 2 ( T M I - 2 ) n u c l e a r p o w e r r e a c t o r c o n t a i n o r g a n i c a n d i n o r g a n i c ion exchange resins which are c o n t a m i n a t e d with g a m m a - a n d b e t a - p r o d u c i n g r a d i o n u c l i d e s . T h e I d a h o N a t i o n a l E n g i n e e r i n g L a b o r a t o r y ( I N E L ) used these ion e x c h a n g e resins to o b t a i n i n f o r m a t i o n on s u r v i v a b i l i t y o f waste f o r m s c o m p o s e d o f the resins solidified in m a t r i c e s o f P o r t l a n d T y p e I - I I c e m e n t a n d vinyl ester-styrene (VES). These waste f o r m s were tested to d e t e r m i n e if they m e t the r e q u i r e m e n t s o f U.S. N u c l e a r R e g u l a t o r y C o m m i s s i o n ( N R C ) r e g u l a t i o n 10 C F R P a r t 61, ' L i c e n s i n g R e q u i r e m e n t s f o r L a n d D i s p o s a l o f R a d i o a c t i v e W a s t e ' [1]. E m p h a s i s was p l a c e d o n a p p l y i n g r e g u l a t i o n 10 C F R P a r t 61 using m e t h o d s specified in the B r a n c h T e c h n i c a l P o s i t i o n o n W a s t e F o r m [2] (TP) o f the N R C O f f i c e o f N u c l e a r M a t e r i a l S a f e t y a n d Safeguards. O n e o f the s t r u c t u r a l stability r e q u i r e m e n t s for waste f o r m s in 10 C F R P a r t 61 is resistance to b i o d e g r a d a t i o n . This p a p e r p r o v i d e s results o b t a i n e d f r o m testing o f solidified resins using the initial b i o d e g r a d a t i o n m e t h o d s specified b y the T P .
* Work supported by the U.S. Nuclear Regulatory Commission, Office of Nuclear Regulatory Research, under DOE Contract No. DE-AC07-76ID01570. Environmental Monitoring and Assessment 11 (1988) 89-100. 9 1988 by Kluwer Academic Publishers.
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ROBERT D. ROGERS A N D J O H N W. MCCONNELL JR.
2. Materials and Methods 2.1. DESCRIPTION OF WASTE FORMS Waste forms used in the biodegradation test were composed of solidified filter ion exchange resin wastes. Two resin waste mixtures were solidified. Type A was a mixture o f synthetic organic ion exchange resins (phenolic cation, strong acid cation, and strong base anion resins), while Type B was a mixture o f synthetic organic ion exchange resins (strong acid cation and strong base anion resins) and inorganic zeolite. Portland Type I-II cement and VES were Used to solidify both types o f resin wastes. VES is a proprietary solidification agent developed and supplied by the Dow Chemical Company * and is composed of a binder (styrene monomer and vinyl ester resin), a catalyst, and a promoter. Individual waste forms were manufactured by allowing a mixture of solidification agent and resin waste to solidify in 4.8 cm diameter by 10.2 cm high polyethylene molds. Enough of the mixture was added to each vial to produce waste forms with average dimensions of 4.8 cm diameter by 7.6 cm length (137.5 cm3). A complete description of waste form manufacture is given in Reference [3]. 2.2. PROCEDURES SPECIFIED FOR DETERMINING BIODEGRADATION The NRC T P specifies that procedures from the American Society for Testing Materials (ASTM) be used to determine the resistance of solidified waste forms to fungi and bacteria. Those procedures can be found in ASTM G21, 'Standard Practice for Determining Resistance of Synthetic Polymeric Materials to Fungi' [4], and ASTM G22, 'Standard Practice for Determining Resistance of Plastics to Bacteria' [5]. If either o f those tests indicate that a waste form is capable o f supporting microbial growth, the T P states that a more conclusive procedure like that described by Bartha and Prame [6] be initiated to determine if biodegradation is occurring. Both ASTM procedures are described in detail as referenced and will only be highlighted here. Five of the fungal cultures specified for the ASTM G21 procedure were obtained from the American Type Culture Collection (ATCC), Rockville, MD, and the sixth, designated ILF-I, was isolated in the laboratory from a VES waste form. The ATCC fungal strains were Aspergillus niger (9642), Penicillium funiculosum (9644), Chaetomium globosum (6205), Gliocladium virens (9645), and Auerobasidium pullulans (9348). Cultures of these fungi were received freeze-dried and were revived following ATCC procedures. Stock cultures were maintained on potato dextrose agar (PDA) plates and recultured as necessary. Stock cultures were kept in a refrigerator at 5 ~ cultures used to produce inoculum were maintained at 28 ~ Pseudomonas aeruginosa (ATCC 13388) as specified by ASTM G22 was used for bacterial testing. Freeze-dried cultures were revived and cultured on nutrient agar * Mention of specific products and/or manufactures in this document implies neither endorsement of preference nor disapproval by the U.S. Government, any of its agencies, or EG + G Idaho, Inc., of the use of a specific product for any purpose.
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91
salts. Stock cultures were refrigerated at 5 ~ while the culture used for inoculum was maintained at 28 ~ All procedures used to culture and maintain organisms, prepare inoculum, as well as the actual testing procedures, were as specified by ASTM G21 for the fungi or ASTM G22 for the bacteria. Agar used for the waste form testing was the ASTM 'nutrient salts' (NS) formulation. This media contained inorganic nutrients but no organic carbon sources and was intended to supplement those organisms which could utilize the waste forms as a carbon source. In addition to the controls mandated by the ASTM Procedures, each test also had controls consisting only of inoculated NS media. These controls were used to assure that any microbiological growth on or near the waste forms was not being supported by the NS media. Also, to assure that the waste forms did not come in contact with other sources of usable carbon, they were stored either in sealed 'whirl-pak' bags or glass beakers and handled only with tongs. All work was conducted with 100 x 15 mm petri dishes except that with the full size waste forms which required 100 x 80 mm culture dishes. The diameter and height of the larger dishes were such that they could each accommodate a waste form laid on its side. After a waste form was placed in a dish, 106 mL of molten agar, cooled to 55 ~ was poured in, thus submerging all but the upper one-fifth of the form. A number of ASTM G21 preliminary investigations were performed on VES and VES mixed with nonradioactive exchange resin before the radioactive waste forms were evaluated. Sequentially, though not necessarily chronologically, work with nonradioactive VES was conducted on the individual components usedto make the VES polymer (binder, catalyst, promoter), powder made from a waste form, wafers cut from a waste form, and a whole waste form. Work with the polymer components was conducted with both NS agar and NS liquid. A quantity of 0.2 mL of each of those VES components was placed on a separate section of the agar in each of six petri dishes. Each of the agar surfaces was inoculated with one of the fungal species used in the ASTM G21 test. After 20 days' of incubation, the agar surface was examined to detect growth of the fungi. For the liquid study, 1 mL of each of the polymer components was added to 20 mL of sterilized NS liquid contained in a 50 mL Erlenmeyer flask. Each of the three flasks was then inoculated with 0.2 mL of the fungal spore suspensions and sealed with a sterile rubber stopper. For the studies using the solidified waste form, VES containing exchange resin was ground to pass a 35 mesh sieve (0.42 ram) and the powder was sprinkled onto the surface of NS agar inoculated with fungal spores. In this case, duplicate plates with VES and an inoculated control were used. Wafers were obtained from dry cutting 0.30 cm sections. A whole nonradioactive waste form was also used. Radionuclide contents of radioactive waste forms used in this study are shown in Table I. Because of the high radiation doses emanating from the waste forms, three separate biodegradation test setups were required at different times to accommodate the waste forms with the available shielding. In the first evaluation, waste forms C1A-28, C1A-29, D1-8, and D1-9 were tested under the ASTM G21 procedure, while D I - l l , Dl-12, C1A-31, and C1A-32 were used with ASTM G22. In addition, DI-10
92
ROBERTD. ROGERSAND JOHN W. MCCONNELLJR. TABLE I Contact gamma dose for waste form used in biodegradation study
Waste Form ID
Contact Gamma Dosea (R/h)
WasteForm ID
Contact Gamme Dosea (R/h)
CIA-28b C1A-29 C1A-30 CIA-31 C1A-32 C2A-22 C2A-23 C2A-25 C2A-31 C2A-32
2.2 2.2 2.1 2.1 2.0 11.0 11.0 11.0 11.0 11.0
DI-8 DI-9 DI-10 DI-ll DI-12 D2A-3 D2A-4 D2A-5 D2A-6 D2A-7
4.0 3.7 3.6 3.4 3.1 16.0 14.5 13.5 13.5 11.5
a Waste forms have the following curie contents: 134C8 137Cs
9~
Total
DI 4.38x 10-3 66.22 x 10-3 3.92x 10-3 74.52 x 10-3 D2A 18.22 x 10-3 275.45 x 10-3 0.64 x 10-3 294.31 x 10-3 C1A 2.95 x 10-3 44.58 x 10-3 2.64x 10-3 50.17 • 10-3 C2A 13.53 x 10-3 204.59 x 10-3 0.47 x 10-3 218.59 x 10_3 b 'C' indicates Portland cement; 'D' indicates vinyl ester-styrene; ' 1' indicates Type A waste; '2' indicates Type B waste; and 'A' indicates the first of two batches.
a n d C1A-30 were i n c u b a t e d u n d e r A S T M G21 c o n d i t i o n s except that the A S T M NS agar was replaced with the c a r b o n - r i c h P D A . The p u r p o s e o f using P D A was to provide controls to determine if there were i n h e r e n t properties o f the waste forms (i.e., chemical or r a d i a t i o n ) which w o u l d prevent or reduce growth o f test organisms. The second set o f tests used waste forms D2A-3 a n d D2A-4 for A S T M G21 tests, D2A-5 a n d D2A-6 for A S T M G22 tests, a n d D2A-7 for the c a r b o n - r i c h P D A control. T h e third a n d final test used waste forms C2A-22 a n d C2A-23 for A S T M G21, C2A-25 a n d C2A-31 for A S T M G22, a n d C2A-32 for the c a r b o n - r i c h control. F o r each testing period as specified by A S T M G21, sterile paper strips were placed o n NS agar (in the absence o f waste forms) a n d sprayed with the fungal i n o c u l u m . I n a d d i t i o n , the surface o f a n NS agar plate was sprayed with i n o c u l u m to d e t e r m i n e if either the spore s u s p e n s i o n or the NS agar could s u p p o r t f u n g a l growth. A f t e r 20 days o f i n c u b a t i o n , the waste forms a n d associated agar were inspected as detailed in A S T M G21 or G22. W h e n dealing with the radioactive forms, a close, p r o l o n g e d i n s p e c t i o n was n o t possible; d e t e r m i n a t i o n o f the extent of the growth had to be ascertained f r o m color p h o t o g r a p h s o f each waste f o r m in its agar matrix.
53. Results and Discussion 3.1. RESULTS OF A S T M G22 BACTERIA TESTING N o n r a d i o a c t i v e specimens of VES a n d cement were f o u n d n o t to s u p p o r t the test
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
93
bacteria Psudornonas aeruginosa under ASTM G22 conditions. The same was also true for those tests conducted on eight radioactive waste forms (four VES and four Portland cement). These data do not support the earlier work of Piciulo et al. [7], who reported that various mixtures of VES and simulated waste (including a deionized water control) supported the bacteria. However, because the bacterial growth in the referenced study was determined by an indirect method not specified by the ASTM G22 procedure, the results have been considered inconclusive [8]3.2. RESULTS OF ASTM G21 FUNGAL TESTING Results of the initial investigations using nonradioactive material will be presented first, followed by those on the radioactive waste forms. In those tests involving VES components, all of the NS agar areas to which VES components had been added supported at least one of the fungal species in addition to fungi which were able to grow at the component agar interface (Table II). The inoculated control plate which consisted of NS agar without a carbon source had no traces of fungal growth, thus showing that neither the NS agar nor the spore suspension contained usable carbon. After a 30-day period of incubation of VES components in liquid NS media, the contents of the flasks were examined for the presence of fungal growth. Growth was noted visually (appearance of hyphal strands) in all cases, with the most apparent growth in the NS/catalyst mixture and the least in the NS/promoter mixture. Even after 18 months, growth associated with the catalyst and binder remained viable. This was shown when samples of fungal hyphae from these mixtures produced abundant growth on nutrient agar plates. These findings support the work of others [9, 10] who have found that styrene and styrene dimers can be biodegraded. Within 30 days of incubation, sporadic heavy growth could be seen on both duplicate inoculated plates hosting powdered VES (Figure 1) but not on the NS control. An enlargement (10 x ) of one of the areas of growth (Figure 2) shows that the fungi were growing on the VES. Growth of fungi at the NS agar wafers interface with and without exchange resins was noted within 30 days. Close examiT A B L E 11 Support o f fungal growth by individual components of Dow polymer (VES) Component Fungus
Binder
Catalyst
Promoter
ILF-I A.
_a
+b
_
+
+
+
G. virens P. funiculosum
+c
+_
+
A. pullulans
+_
+
+
niger
a No fungal growth noted. b Fungal growth on c o m p o n e n t (usually an ASTM G21 rating o f 1 or 2). c Fungal growth surrounding component.
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ROBERT D. ROGERS AND JOHN W. MCCONNELL JR.
Fig. 1.
Fig. 2.
Fungal growth on a powdered nonradioactive VES waste form.
Close-up view (10 x ) o f fungal growth on a powdered nonradioactive VES waste form.
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
Fig. 3.
Fig. 4.
95
Funga/growth on water cu~ from aoaradioacZJve wasze form after 18 months.
Ciose-ttp view (10 x ) of fungai growth on wafer cut from nonradioactive VES waste form after 18 months.
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ROBERT D. ROGERS AND J O H N W. MCCONNELL JR.
nation after 60 days showed that fungi were growing on the edges of both types of wafers. After 18 months, more extensive growth (ASTM rating of three) was seen on the edge and surface of the wafer with resins (Figures 3 and 4). During the development of a method to implement the ASTM testing of full-size waste forms, an unidentified fungus (later given the designation of ILF-1) was found growing on the agar surface directly above a buried, unirradiated VES waste form. The source of the fungus was apparently the waste form, since the agar was not inoculated. Within 30 days, the fungus covered the exposed NS agar surface (Figure 5). The supposed carbon source for the fungus was some soluble component of the waste form. This fungus was cultured on PDA and, as allowed by the ASTM G21 protocol, was included as a stock culture for that test. After the initial work using the nonradioactive VES was complete, ASTM G21 testing of the irradiated EPICOR-II waste forms began. Cement waste forms in NS agar failed to support fungal growth. The cement waste form which was used as a control in the carbon nutrient PDA did allow for growth of fungi over the complete surface of the agar. So, while the waste form itself was not able to support the fungi, growth was supported by a supplemental carbon source. Results of radioactive VES testing showed that these waste forms could support the growth of fungi (Figure 6). Growth can clearly be seen at the agar/waste form interface. Because of the mottled appearance of the waste form surface coupled with the inability for close examination, it is not clear whether it is supporting fungal growth. No growth rating based on the ASTM scale was assigned. The PDA surface containing a VES waste form had a heavy covering of fungi. The NS agar control with no carbon source had no fungal growth, while NS agar with paper strips (ASTM control) had good growth on the paper. Following the NRC TP procedures, those waste forms which had supported fungal growth (VES) were prepared for retesting while the others (cement) were used for compression testing. Preparation for retesting was accomplished by first rinsing the specimens in methanol [7] and then allowing them to air dry for 30 days. The waste forms which had supported fungal growth initially were again subjected to the G21 procedure. After the 20 day period of incubation, fungi were again seen to be growing at the/agar waste form interface of each specimen. The control of NS agar remained free from fungal growth. 9.3. DISCUSSIONOF ASTM G21 RESULTS It is obvious that work with cement waste forms should have been conducted with microorganisms known to degrade cement. (Thiobacilli [ 11], nitrobacter [12]). While fungal growth was not seen on the NS agar, growth was noted in the cement PDA control. Piciulo et al. [7], noted that fungi did not grow during the ASTM G21 test with cement and attributed this to pH or ionic strength changes created by the cement. While cement probably does cause such changes, it still appears that an available carbon source will promote growth near the waste form. Using the results from Piciulo et al. [7], Bowerman et al. [8], suggested that the cement waste forms
BIODEGRADATION TESTING OF RADIOACTIVE WASTE FORMS
Fig. 5.
Fig. 6.
Fungal growth on NS agar covering nonradioactive VES waste form.
Fungal growth associated with radioactive VES waste form DI-9 (first ASTM test).
97
98
ROBERT D. ROGERS AND J O H N W. MCCONNELL JR.
might cause a biocidal or inhibitory effect in a burial environment. It now appears that this effect may not be as pronounced as first reported. Work with VES has demonstrated that it can promote fungal growth, although the carbon source has not been determined. Evidence from this study indicates that the polymer or components (particularly the catalyst and binder) could be contributors. In addition, the study shows that once organisms have established themselves with these materials they can remain viable for several months. The growth of fungi away from a VES waste form suggests that some carbon source is being leached from the VES. Since this pattern o f growth continued in a second testing even after the waste form surface was rinsed, it can be assumed that the source of the soluble carbon was internal. The application o f the Bartha-Pramer [6] test method would provide information which could be used to determine the extent of the carbon source. Because it was not possible to closely examine the radioactive VES waste forms, it was not clear whether the fungi were actually growing on the VES surface. However, results from two of the initial studies with nonradioactive material suggest that fungi could indeed have been growing on the surface. It was shown that fungi would grow on a powdered VES waste form (Figure 2) as well as wafers cut from waste forms with and without ion exchange resins. With time, this growth can become heavy (Figure 4). No data are available on whether the fungi are penetrating the waste form surface. This work was not intended to determine if VES polymer bonds were being biocatalyzed. It is a commonly held theory that chain branching in polymers has an inhibitory effect on the biodegradation of VES [13]. However, when VES samples (mixed with distilled water) were evaluated in a Bartha-Pramer test, CO2 production was maintained at a continuous rate during a 6 month test [7]. Those data suggest that a carbon source in excess o f any free monomer, promoter, or catalyst was being utilized. A study of the data from compression testing of those cement waste forms exposed to biodegradation testing reveals that compression testing is unlikely to detect any biologically induced physical changes in a waste form. This is because of the insensitivity o f compression testing to detect those slight structural variations which could occur over the brief period (21 days) of the biotests with relatively bulky samples. Finally, there have been some suggestions that the biodegradation of radioactive waste forms is moot, since the radiation associated with this material would kill any microorganisms. This does not appear to be the case as was evidenced by the results from waste forms tested in this study which contains high radionuclide loadings and exhibited radiation doses of 2-16 R / h at contact (Table I). In those cases where a readily available carbon source was supplied (PDA), the VES as well as the cement waste forms had copious quantities of fungai growth. It is expected that microbes can exist in an exposure range o f 2 - 1 0 0 ( O rads [14]. This would appear to be well within the range produced by commercial-sized radioactive waste forms.
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99
4. Conclusions and Recommendations As a result of this study, the following conclusions can be drawn: (1) VES with or without irradiated ion exchange resins promotes microbial growth. (2) The carbon source for growth could be any or all unreacted components used to produce VES or it could be the VES itself. (3) Cement waste forms containing radioactive ion exchange resins do not support microbial growth of the types specified by ASTM G21 and G22; neither do they completely inhibit growth when a carbon source is provided. (4) Neither VES or cement waste forms promote the growth of the bacterium P. aeruginosa. (5) Radiation at the levels found in the EPICOR-II radioactive waste forms used in this study does not inhibit microbial growth. (6) The ASTM G21 and G22 tests are not suitable for determining the extent of waste form biodegradability or for establishing physical effects due to biodegradation. While this study and others [8, 15] have provided evidence that VES and cement can support the growth of microbes, it was concluded that the fundamental requirements of the T P have not been satisfied because the extent of degradation and any effect it could have on the physical characteristics of the waste form were not and could not be determined with the ASTM methods. Because of the unlikelihood that the present methodology can be modified in a way that will completely satisfy the demands for an accelerated biodegradation test, it is recommended that the emphasis of the test be changed from the evaluation of waste forms to an evaluation of materials which will be used as solidification agents. By doing this, the question of biodegradation of a solidification agent and its consequences could be answered by one in-depth investigation conducted by personnel familiar with biodegradation testing rather than recurring limited tests conducted at numerous waste generation sites using individuals with varying training, experience, and equipment to conduct the testing. This concept is based on the conclusion that the greatest possibility for structural failure o f a waste form rests with the solidifying agent. If the solidified matrix retains its integrity, then there is reason to assume that there will be no structural failure even if the waste material is biodegradable. However, some testing with standard waste forms containing various known wastes would be required to substantiate this position. If this suggestion were to be adopted, then the responsible agency could maintain complete control of verifying the integrity of waste forms simply by certifying the solidification agent. Such certification would come from a source other than the manufacturer and would involve a very conservative (i.e., rigorous) test protocol. The justification for such rigorous testing is that it needs to occur only once for each solidification agent. It may also be necessary to verify that the producer of the waste form is adhering to the specifications for making the solidification agent. The proposed testing would be of the type which Bowerman et al. [8] summarized as inherent biodegradability testing. Such a method would include: exposure to a
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ROBERT D. ROGERS AND JOHN W. MCCONNELL JR.
wide range of microbial species (especially to those known to affect the solidification agent); appropriately sized specimens for testing; possible use of alternative carbon (energy) sources to provide opportunity for co-metabolism of a specimen; and extension of the time period allowed for testing (up to 6 months). Degradative effects would be determined directly by measuring metabolic activity, such as CO2 production or oxygen consumption, or some physical parameters like specimen weight loss and visual deterioration. Notice This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, or any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party's use, or the results of such use, of any information, apparatus, product or process disclosed in this report, or represents that its use by such third party would not infringe privately owned rights. The views expressed in this report are not necessarily those of the U.S. Nuclear Regulatory Commission.
References [1] United States Nuclear Regulatory Commission, 'Licensing Requirements for Land Disposal of Radioactive Waste', Title 10 Code of Federal Regulations Part 61, U.S. Federal Register, Vol. 46, No. 142, 24 July 1987. [2] United States Nuclear Regulatory Commission, 'Branch Technical Position on Waste Form', 11 May 1983. [3] Neilson, R M. and McConnell, J. W.: Solidification of EPICOR-II Resin Waste Forms, GENDINF-055, August 1984. [4] American Society for Testing Materials, 'Standard Practice for Determining Resistance of Synthetic Polymeric Materials for Fungi', ASTM G21-70, 1981 Annual Book of Standards, Part 35. [5] American Society for Testing Materials, 'Standard Practice for Determining Resistance of Plastic to Bacteria', ASTM G22-76, 1981 Annual Book of Standards, Part 35. [6] Bartha, R. and Pramer, D.: 1965, 'Features of a Flask and Methods for Measuring the Persistence and Biological Effects of Pesticides in Soil', Soil Science 100, 68-70. [7] Piciulo, P. L., Shea, C. E., and Barletta, R. E.: Biodegradation Testing of Solidified Low-Level Waste Streams, NUREG/CR-4200, May 1985. [8] Bowerman, B. S., et al., An Evaluation of the Stability Test Recommended in the Branch Technical Position on Waste Forms and Container Materials, NUREG/CR-3829, March 1985. [9] Sielicki, M., Focht, D. D., and Martin, J. P.: 1978, 'Microbial Transformations of Styrene and [14C] Styrene in Soil and Enrichment Cultures', Appl. Environ. Microbiol. 35, 124-138. [10] Higashimura, T., Sawamoto, M., Hiza, T., Karaiwa, M., Tsuchii, R., and Suzuki, T.: 1983, 'Effect of Methyl Substitution on Microbial Degradation of Linear Styrene Dimers by Two Soil Bacteria', Appl. Environ. Microbiol. 46, 386-391. [11] Sand, W., Bock, E., and White, D. C.: 1987, 'Biotest Systems for Rapid Evaluation of Concrete Resistance to Sulfuroxidizing Bacteria', Mater. Perform. 26, 14-17. [12] Bock, E.: 1987, 'Biologisch Induzierte Korrosion von Natustein-Starker Befall mit Nitrifikanten', Bautenschutz. Bausanierung 10, 24-27. [13] Potts, J. E.: 'Biodegradation', in H. H. G. Jellinek (ed.), Aspects of Degradation and Stabilization of Polymers, Elsevier Scientific Publishing Co., New York, NY, 1978. [14] Choppin, G. R. and Rydberg, J.: Nuclear Chemistry: Theory and Application, Pergamon Press, Oxford, 1980. [15] Zajie, J. E.: Microbial Biogemochemistry, Academic Press, NY 1969, p. 345.
