Infectivity Studies of Both Ash and Air Emissions ... - ACS Publications

The 263K strain of hamster-adapted scrapie was chosen because the concentration of infectivity in brain tissue of terminally ill animals is as high or...
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Environ. Sci. Technol. 2004, 38, 6155-6160

Infectivity Studies of Both Ash and Air Emissions from Simulated Incineration of ScrapieContaminated Tissues P A U L B R O W N , * ,† E D W A R D H . R A U , ‡ PAUL LEMIEUX,§ BRUCE K. JOHNSON,† ALFRED E. BACOTE,† AND D. CARLETON GAJDUSEK| Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, and Division of Environmental Protection, Office of Research Facilities Development and Operations, National Institutes of Health, United States Department of Health and Human Services, Bethesda, Maryland 20892, National Homeland Security Research Center, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711, and Institut Alfred Fessard, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette, France

We investigated the effectiveness of 15 min exposures to 600 and 1000 °C in continuous flow normal and starvedair incineration-like conditions to inactivate samples of pooled brain macerates from hamsters infected with the 263K strain of hamster-adapted scrapie with an infectivity titer in excess of 109 mean lethal doses (LD50) per g. Bioassays of the ash, outflow tubing residues, and vented emissions from heating 1 g of tissue samples yielded a total of two transmissions among 21 inoculated animals from the ash of a single specimen burned in normal air at 600 °C. No other ash, residue, or emission from samples heated at either 600 or 1000 °C, under either normal or starved-air conditions, transmitted disease. We conclude that at temperatures approaching 1000 °C under the air conditions and combustion times used in these experiments, contaminated tissues can be completely inactivated, with no release of infectivity into the environment from emissions. The extent to which this result can be realized in actual incinerators and other combustion devices will depend on equipment design and operating conditions during the heating process.

Introduction Safe disposal of medical wastes, carcasses of cattle with bovine spongiform encephalopathy (BSE), cervids with chronic wasting disease (CWD), sheep with scrapie, and more generally, any human or animal tissue infected or potentially infected with one of the agents that cause transmissible spongiform encephalopathy (TSE) continues to be an issue * Corresponding author phone: (301)652.5940; fax: (301)652-4312; e-mail: [email protected]. † Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health. ‡ Division of Environmental Protection, National Institutes of Health. § National Homeland Security Research Center. | Centre National de la Recherche Scientifique. 10.1021/es040301z CCC: $27.50 Published on Web 10/19/2004

 2004 American Chemical Society

of concern. High temperature incineration has been the method of choice for treatment of medical and veterinary wastes by virtue of its proven ability to inactivate all types of conventional pathogens, high throughput capacity, and significant volume reduction. However, TSE agents are uniquely resistant to most physical and chemical methods of disinfection, including dry heat (1-3). In a previous series of experiments (4), we showed that transmission could occur even after ashing infected tissue in a covered crucible at 600 °C: the ash from one sample of fresh brain tissue heated for 15 min transmitted to five of 18 animals (another sample heated for 5 min did not transmit to any of the 15 animals), and one formalin-fixed sample heated for 5 min transmitted to one of 24 animals. As no transmissions occurred from any sample heated to 1000 °C, the infectivity extinction point was somewhere between 600 and 1000 °C, most probably very close to 600 °C, approaching the operating temperature of some incineration units. Because of concerns about the reproducibility of these unprecedented results, and about the possibility that some infectivity might be entrained in stack gases vented during incineration, we designed an experimental apparatus to produce conditions that reflect more closely actual incineration conditions, in which gases flowed across a heated open crucible containing contaminated tissue, oxidizing or pyrolyzing the tissue, and partially entraining some of the ash; we also performed infectivity bioassay measurements of both ash and emissions. The 263K strain of hamster-adapted scrapie was chosen because the concentration of infectivity in brain tissue of terminally ill animals is as high or higher than in any other TSE, natural or experimental, and thus allows the maximum measure of reduction, and because this strain shows resistance to heat that is comparable to that of BSE and superior to other tested TSE strains (refs 5-8 and personal communication from Dr. David Taylor, Edinburgh, Scotland). We here report that once again, despite the nearly total destruction of over 109 LD50, and individual bioassay animal caging to avoid any possibility of cross-contamination, an ashed sample of scrapie-infected tissue transmitted disease after having been exposed to 600 °C for 15 min, and once again, we found no survival after exposure to 1000 °C. We also show that no infectivity escaped into air emissions from 15 min test burns at either 600 or 1000 °C. Whatever the mechanism of this minimal level of survival in extreme heatswhether a result of incomplete combustion, the existence of a mineralized template for replication, or some other unimagined phenomenonsit may be concluded that the exposure under carefully controlled laboratory conditions of a small sample of contaminated tissue to 1000 °C, under either an oxidizing or reducing atmosphere, will ensure complete sterilization of the ash and emissions. Exposure at 600 °C allows a minimal level of infectivity to persist in the ash but generates air emission products that are noninfective.

Experimental Procedures Tissue Samples. Brains from 20 terminally ill hamsters infected with the 263K strain of hamster-adapted scrapie were pooled, homogenized, and distributed into 1 g aliquots. The same procedure was used for a small pool of uninfected control brains. Samples were frozen until the test burns were initiated. Simulated Incineration. The incineration simulation apparatus was constructed, and test burns were performed at the U.S. Environmental Protection Agency’s National Risk VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic view of incineration simulation apparatus showing, from left to right, the gas inlet, Lindberg furnace surrounding a removable combustion chamber (quartz reactor tube), quartz exhaust tube, emission impingers (ice water bath followed by dry ice bath), and exhaust through filter into fume hood.

FIGURE 2. Photograph of the incineration simulation apparatus. Management Research Laboratory located at Research Triangle Park, NC. Tissue samples were heated in a 2.54 cm (1 in.) diameter quartz reactor (Prism Research Glass, Research Triangle Park, NC) placed inside a Lindberg furnace (Blue M Model 542 32-V). Three separate, nearly identical quartz reactors were used. One reactor was only used for the 1000 °C tests, and the other two were alternated for the 600 °C tests. Gas flow through the experimental system was controlled with a rotameter (Gilmont Instruments, a Barnant Company, Barrington, IL). A two-stage impinger was used to collect emissions from the gas stream exiting the reactor: the first stage discharged 6156

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the gas through deionized water in a tube held in an ice bath; gas exhausted from the first impinger trap flowed into a second trap suspended in dry ice within a polystyrene foam container. The apparatus is shown schematically in Figure 1 and is photographed in Figure 2. All components of the impinger system were made of quartz glass and connected with Teflon couplings. Metal components were avoided because of their tendency to bind amyloid protein (9). The entire apparatus was located in a fume hood. The experimental matrix included testing of normal and infected tissues at both 600 and 1000 °C. Experiments were performed under oxidative (combustion) and reducing

(pyrolytic) conditions: oxidative conditions utilized humidified air, and reducing conditions utilized humidified nitrogen (N2) as the reactor inlet gas. These parameters were selected to simulate conditions within incinerators commonly used for destruction of medical waste. Before each test, the reactor was immersed for 30 min in a 1:1 aqueous solution of freshly prepared sodium hypochlorite (Clorox bleach) and then extensively rinsed with deionized water and allowed to dry. The clean, dry reactor was then placed into the Lindberg furnace, and the furnace temperature controls were adjusted to the desired setting based on calibrations that were performed prior to the experiments. Each 1 g tissue sample was thawed and placed into a new quartz crucible. Before beginning the experiments, a type-K thermocouple (Omega, Model No. KQXL-18G-12) was used to measure the gas temperature at the axial location of the reactor where the crucible would be inserted. The temperature was allowed to equilibrate until no significant temperature change occurred. The furnace temperature was then logged, and the thermocouple was removed. After removal of the thermocouple, the impinger train (Prism Research Glass, Research Triangle Park, NC) was installed onto the outlet of the reactor, the inlet gas system was connected to the reactor, and the temperature of the impinger train was logged. A bubble meter was used to perform a leak check by setting the gas rotameter to the desired flow rate and checking the impinger train outlet flow rate. After the leakage rate was determined for each test and airflow was found to be within the acceptable range (50-70 mL/min), the tissue sample contained in the quartz crucible was placed into the crucible holder and inserted into the reactor, and a clip was placed around the joint between the reactor and the crucible holder. All samples were heated for 15 min. Collection and Processing of Ash and Emission Samples. At the end of each test burn, the clip holding the crucible holder to the reactor was removed, and the crucible holder with crucible inside was removed and immediately cooled. The crucible with its contained ash was then placed in a labeled sample vial. It was noted that the reactor walls and crucible were coated with opaque, glasslike surface deposits after the 1000 °C tests. When found in the reactor, these deposits were dislodged using a stainless steel spatula, collected from the reactor as thoroughly as possible, and placed in the labeled sample vial along with the ash from each crucible. The collected ash and deposits were then transferred to a Tenbroeck tissue grinder and homogenized in 1 mL of distilled water. Following each test burn, the impinger train was removed, labeled, and sealed. The entire impinger train was placed at 4 °C for storage and later transported to the National Institutes of Health in Bethesda, Maryland for bioassay of the impinged emission materials that were recovered separately from the glass tubing leading from the burner to the first impinger and from the traps. Visible deposits from the tubing were assiduously scraped, rinsed into a tissue grinder, and homogenized in 1 mL of distilled water. Water from the first trap was allowed to evaporate inside a laminar flow hood to a volume of approximately 1 mL, which was transferred together with all associated tube residues from both traps to a tissue grinder and homogenized. Each test burn yielded three samples: (1) residue collected from the crucible and deposits from the inside of the heated zone of the reactor (ash); (2) residue from the exhaust zone of the reactor tube to the first impinger trap (exit tube residue); and (3) commingled water and residues from the two impinger traps (air emission samples). Bioassays. The total volume of each sample was inoculated undiluted into groups of healthy female weanling hamsters (0.05 mL per animal by the intracerebral route;

TABLE 1. Bioassay Results for Combustion Products from Heated Infected Hamster Brain Tissue Macerates and Controlsa test conditions

bioassay specimen

tissue

gas

°C

crucible

exit tube

traps

normal normal normal normal normal infected infected infected infected infected

air air N2 air N2 air air N2 air N2

ambient 600 603 1015 1000 ambient 612 598 996 997

NA 0/20 0/21 0/23 0/20 NA 2/21 0/20 0/15 0/23

NA NT 0/18 NT 0/18 NA 0/22 0/19 0/26 0/18

NA NT NT NA 0/24 0/26 0/23 0/23

a For each test group, fractions represent number of PrPres-positive animals over total number of inoculated animals. Residues from the exit tubes and emissions from the impinger traps were combined for bioassays of the uninfected control samples subjected to 600 and 1000 °C under N2. NA ) not applicable; NT ) not tested.

approximately 20 animals per sample). Twenty uninoculated sentinel animals were randomly positioned among the inoculated bioassay animals, all of which were individually caged, to avoid fighting and any possibility of crosscontamination. Animals were observed for a period of 12 months for clinical signs of scrapie, at which point the survivors were euthanized. The brains of all animals, whether dying during the observation period, or surviving to its conclusion, were examined for the presence of proteinaseresistant protein (PrPres) by Western blot immunoassays. The 12 month observation period was mandated by considerations of cost and space associated with prolonged care of the large number of animals (∼450) needed to conduct this study. The occurrence of rare transmissions after longer incubation periods in rodents inoculated intracerebrally with low dose infectious material has been documented (10, 11), but this possibility was mitigated in our experiment by the examination of all brains for the presence of PrPres, which is visible well before the onset of symptomatic disease (12, 13). Western Blot Immunoassays. Approximately 0.1 g of brain tissue was extracted per sample by the phosphotungstic acid method described by Safar et al. (14) and blotted using the monoclonal anti-hamster PrP antibody 3F4 at a dilution of 1:2000. Samples giving a questionable positive result were reextracted using the purification/concentration method of Xi et al. (15): all six such samples were found to be clearly negative on retesting.

Results and Discussion Bioassay results for each tested sample are summarized in Table 1. It is important to note that the all material recovered from each test burnsapproximately 1 mL volumes of resuspended ash, residues, or emissionsswas inoculated to avoid any sampling error that can be significant when dealing with very low levels of infectivity. Two unheated 263K brain tissue samples were assayed, yielding levels of infectivity of 109.2 and 109.7 LD50/g of tissue macerate. Incubation periods in the lowest dilution (10-1) groups were between 50 and 60 days; incubation periods in the highest positive dilution groups (titration end point) ranged from 120 to 180 days. The residual ash from the 1 g sample of 263K brain macerate heated at 600 °C in normal air transmitted disease to two of 21 inoculated animals after incubation periods of 261 and 303 days, and their brains were positive for PrPres. The clinical signs and PrPres patterns in both hamsters were VOL. 38, NO. 22, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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indistinguishable from those of the positive control animals that received unheated inocula. No other heated samples were infectious, based on the absence of symptomatic disease and brain PrPres, including reactor exit tube residues and emission samples from tissues heated to 600 °C; ash, exit tube residues, and emissions from tissues heated to 1000 °C; and normal brain tissue heated to 600 °C (bioassays were not done on normal tissue heated to 1000 °C). In particular, no clinically healthy animal surviving to the observation end point was found to have PrPres in the brain (i.e., no preclinical or subclinical infections were detected 12 months after inoculation). All uninoculated sentinel animals also remained asymptomatic and PrPresnegative. Comparison of Experimental and Actual Incineration Conditions. The question as to whether medical waste incinerators and other types of combustion units used for disposal of contaminated materials provide the conditions necessary for inactivation of TSE cannot be completely answered by laboratory experimentation. It is acknowledged that experiments such as these cannot duplicate the dynamic operating conditions and complex rheology of incinerators and the myriad of interactions with other waste constituents that occur in a combustion environment. However, smallscale simulations can provide valuable qualitative information regarding the behavior of materials in a high temperature combustion environment under tightly controlled conditions. With this limitation in mind, we offer the following comparisons of our experimental conditions with those expected in actual incinerators and comment on the implications of our data for potential environmental releases of infectivity from combustion processes. Types of Incinerators and Operating Temperatures. In the U.S., three types of incinerators are typically used for disposal of medical wastes: controlled-air two-stage modular systems, excess air batch systems, and rotary kilns (16, 17). Of these, the controlled-air (also referred to as starved-air) systems are the most widely used today (18). In these units, combustion of wastes occurs in two stages. In the first stage, waste is fed into the primary chamber, which is operated with less than the stoichiometric amount of air required for combustion. Air enters from both above and below the burning bed of waste, which is dried, volatilized, pyrolized, and partially combusted. In this chamber, the air temperature above the waste is typically 760-980 °C. In the second stage, air is added to the gases produced in the first chamber to complete combustion, and the gas temperature is higher, typically 980-1095 °C. The partial combustion of the waste in the first stage yields a gas with sufficient heating value to operate the combustion process in the second stage without the need for additional fuel. Gas temperatures in each chamber of controlled-air incinerators are thus higher than the temperatures observed in our experiments to achieve, respectively, near-total and total inactivation of the agent. Excess air medical waste incinerators are typically small modular units, usually designed with two chambers and provisions for manual loading of waste into the primary chamber and removal of ash. Burners are ignited to bring the secondary chamber to an operating temperature of 870980 °C. When this temperature is reached, the burner in the primary chamber is ignited. The unit is operated with levels of air that are approximately 100% higher than the stoichiometric amount of air required for combustion. The operating temperatures of modern medical waste incinerators, which typically operate well above 600 °C, should reduce TSE infectivity concentrations to levels at or very close to extinction. However, it should be noted that these temperatures are usually measured in the gases above the bed of burning waste, not in the bed itself. The maximum temperatures achieved in the bed may be as much as 100 °C 6158

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lower than the gases, depending on the bed depth, composition of the input material, and other factors. Accordingly, incinerators used to dispose of TSEs should not be operated at lower temperatures. Indeed, studies by the EPA have shown that incinerators operated at air temperatures of about 600 °C may not even inactivate conventional pathogens that are much less resistant to thermal inactivation than TSEcontaminated materials (35). Large-volume, nonmedical waste streams that may contain TSE-contaminated livestock and wildlife carcasses or meat and bone meal (MBM) have been disposed of by various methods. In the U.K., all carcasses are incinerated in dual chamber facilities with primary and secondary chamber temperatures of approximately 850 and 1000 °C, respectively. These incinerators typically operate at a carcass input batch rate ranging between 100 and 1000 kg/h (average 450 kg/h), with a solid-phase residence time of 1 h (personal communication, Dr. Stephen Wyllie, Department for Environment, Food and Rural Affairs, U.K.). MBM may also be incinerated, may be subjected to other thermal technologies including rotary kilns, fluidized beds, and cement plants, or co-fired in power plants with fuels such as coal, lignite, and other wastes (19). If these nonincineration systems operate under conditions similar to incinerators, they may be expected to provide a similar level of inactivation. Evaluations by the German Federal Institute for Viral Illnesses in Animals and the Institute for Biological Safety indicate that the incinerators used in Germany for disposal of MBN can achieve a temperature of 600 °C for 15 min in the waste if specific operating conditions are met (20, 21). Incinerators used in the U.S. for disposal of municipal waste operate at temperatures above 1000 °C (22). Concentration of the Infectious Agent. In these experiments, the waste load consisted of pure brain tissue with an extremely high concentration of infectivity (>109LD50/g). In actual incinerators, the concentration of infectivity in the waste load will be much lower than that in our experiments because the brain infectivity concentration in hamsters infected with the 263K strain of scrapie is at least 2-3 logs higher than in livestock infected with either BSE or scrapie (CWD brain has not been titered) and also because high infectivity central nervous system tissues are diluted in the mix of peripheral carcass (or MBM) tissues that contain little or no infectivity. In medical waste incinerators, mixing of TSE tissues and contaminated items with other materials also dilutes the concentration of the agent in the waste load. Tissues usually comprise only a small percentage of the total volume of most hospital waste streams; almost all of the mass of material that is classified as medical waste is comprised of noninfectious materials such as paper and plastic (23); and medical wastes are often burned together with noninfectious, nonmedical waste. Dilution of the infectious agent in a much larger volume of noninfectious material is theoretically advantageous because it reduces the probability of the agent being in localized areas of the incinerator, which may have less than optimal conditions for inactivation. An example of such an area is the zone near the incinerator walls, which may be cooler than the rest of the chamber. Conversely, mixing TSEcontaminated materials with other wastes could adversely impact inactivation by insulating the agent and decreasing its total time of exposure to inactivation temperatures. Combustion Gases. In some of these experiments, pure nitrogen gas was used to simulate the combustion gas in the primary chamber of a controlled-air incinerator. Oxygen in the small volume of air that entered the reactor during the few seconds when the crucible holding the tissue sample was inserted would have been rapidly purged from the system, probably before the sample was dried out and heated

to the target temperature. Thus, virtually all of the test burns using nitrogen were carried out under anoxic conditions. This differs somewhat from actual starved-air incineration conditions where limited amounts of air enter the chamber throughout the combustion cycle, and partial oxidation of waste constituents occurs. Volatilized organic and particulate materials from the primary chamber enter the secondary chamber where excess air is added to complete oxidization of these materials. The ash from controlled-air incinerators has a relatively high carbon content, typically from 3 to 6% and values as high as 30% are common (17). The high carbon content is of concern because there is some evidence (24) that the presence of carbon may protect TSE infectivity, and some of the residues observed in the reactor exit tube and impinger traps in these experiments were similar in color and form to carbon black. Although the nitrogen used as a reactor carrier gas did not contain any oxygen, as would be present in actual controlled-air incinerators, the results yielded information relevant to inactivation mechanisms at higher temperatures. No transmissions were detected in ash or emissions from infected issues in the test burns performed in nitrogen, confirming that the presence of oxygen is not required to inactivate the agent and that any carbon formed was not sufficiently protective to prevent its inactivation. The lack of transmission from test burns in anoxic conditions also suggests that denaturation or some inactivation mechanism other than chemical oxidation may be operative at incineration temperatures. These results may have potential application in selection of waste processing technologies, particularly for high-volume waste streams such as MBM and animal carcasses. High-temperature, anoxic waste pyrolysis systems that can yield biofuels and other useful byproducts could be considered as alternatives for incineration, which is usually a strictly destructive process. Secondary Chamber. Another aspect of these experiments that differed from actual incineration conditions was that the vented gases from the reactor tube, which is functionally similar to the primary chamber of an incinerator, were exhausted directly into a cold impinger train. In actual incinerators, the vented emissions from the primary chamber typically enter a secondary chamber, which is usually operated at a temperature higher than the primary chamber, providing additional opportunity for the inactivation of any pathogens carried in the gas phase. Depending on the system design, gases exiting the secondary chamber may then be cooled and passed through scrubbers or other types of air pollution control equipment before they are released to the environment. In our experiments, no infectivity was detected in emission deposits collected directly from the exhaust end of the reactor tube. This suggests that the agent would be inactivated or retained in the ash in the first chamber of an incinerator and that the potential for contamination of residues, wastewater, and other effluents generated by gas cooling and air pollution control systems is minimal.

Conclusion Any thermal treatment processswhether incineration or alternative technologysthat ensures exposure of TSE wastes to temperatures of 1000 °C for at least 15 min should result in sterile output products, as the minimum temperature required to achieve sterility is probably only marginally above 600 °C. Treatment at 600 °C may thus produce an ash that is either sterile or contains a level of residual infectivity well within regulatory requirements for reductions of conventional pathogens in sterile products. In our experiments, over one billion LD50 of scrapie infectivity were reduced to less than a single LD50 (two transmissions among 21 inoculated animals) by a 15 min exposure to dry heat at 600 °C. Although

it may be objected that even this degree of reduction does not achieve zero risk, it is approximately 10-fold greater than the most stringent process validation guidelines issued by the FDA to ensure the safety of biological products (up to 8 log virus removal) (25) or than the standard used by the EPA for registration of sterilants (no growth of Bacillis subtilis in 720 carriers each having at least 2 × 105 spore counts) (26). For TSE inactivation conditions to be met, incinerators and other thermal treatment systems must be properly selected and operated. In actual incinerators, inactivation conditions can be adversely affected by an array of operational factors, such as overloading, cold start-ups and shut-downs, inadequate control of air flow, insufficient or excessive turbulence, and loss of partially burned material through grates. Under these failure mode conditions, inactivation may be incomplete. Given a hypothetical potential for survival of trace amounts of TSE infectivity in the combustion products of incinerators operated under suboptimal conditions, the likelihood of disease transmission via environmental media is minimized by several factors including the following: dilution; hydrophobic properties of agents that would be expected to reduce their mobility in water and soils; containment provided by ash landfill design and operations; biological degradation; species barriers; and the inefficiency of likely routes of exposure (27-32). It should also be noted that neither humans nor animals appear to be susceptible to air-borne TSE infections, further diminishing any potential risk from incinerator emissions. Prior to this study, data on inactivation of TSE-infected tissues in incinerator emissions were not available for risk assessment, and probabilistic approaches were used to assess the risks of combustion processes, including the burning of carcasses in open pyres. These approaches led to conclusions that the risk of transmission to humans was extremely low (33, 34). Our study provides actual data on inactivation under incineration conditions and offers further reassurance that TSE materials can be safely disposed of via incineration.

Acknowledgments The authors thank Mr. David Liles of ARCADIS G&M, who set up and operated the reactor and impinger train, and Mr. George Nelson, who conscientiously scraped all tubing and traps for emission residues. The combustion test portion of this work was performed under Interagency Agreement RW75938614 between the National Institutes of Health and the U.S. Environmental Protection Agency.

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Received for review January 2, 2004. Accepted June 14, 2004. ES040301Z