Due to the varying risk of biological agents, the facilities that handle these agents need to be designed and classified according to specific special wastewater treatment specifications.

Issue: 11/02

(Editor's Note: Due to difficulties with the graphic representation of the figures referenced in this article, please see the print edition to view the figures.)

West Nile Virus and Anthrax deaths, along with the incidents of mad cow disease and terrorist threats in the wake of September 11, have brought the subject of biosafety to the forefront. Funding for laboratories that deal with exotic and infectious diseases has increased, as has the scheduling of related seminars, conferences and trade shows.

At the heart of biosafety is the containment of hazardous agents through multiple levels of barriers. Primary barriers pertain to equipment such as gloves, gowns, masks, biosafety cabinets, respiratory protection and positive-pressure ventilation suits, as well as the use of good laboratory techniques. Secondary barriers are addressed through facility design with air-tight rooms, air handling and filtration, air locks, showers, laundry, sewage treatment, waste disposal, sterilizers, redundant services and equipment and material finishes. Tertiary barriers deal with the physical operation with items such as walls, fences, security and animal exclusion zones. Due to the varying risk of biological agents, the facilities that handle these agents need to be designed and classified accordingly.

Liquid Wastes

Often, biosafety facilities will use experimental animals for researching vaccines and other countermeasures. Invariably, work with animals will involve the disposal of liquid wastes. Consideration must be given to the need for wastewater treatment, and the attributes of the treatment system. Certain issues should be considered when selecting technologies for treating liquid animal wastes, such as waste type and waste quantity. Types of liquid animal wastes may include liquid manure, urine, blood and other necropsy wastes and water from wash-down procedures. Other issues include the type of infectious agents, as many are resistant to certain chemical disinfectants, and the solids content of the liquid waste. Solids in wastewater can interfere with the effectiveness of both chemical and heat sterilization processes. Solids can react with and "consume" chemical disinfectants and reduce their ability to attack pathogens. Settling and scaling in treatment tanks can also interfere with heat transfer. Large bulk solids such as bedding can plug piping and strainers, interfering with the operation of pumps and valves. Hard, finer solids making their way through strainers can cause abrasion and wearing of pump seals. Decomposition of organic solids in storage tanks may lead to the corrosion of ferrous components.


Sterilization is the use of a physical or chemical procedure to destroy all microorganisms, including large numbers of bacterial spores. Heat treatment is the most appropriate method for the sterilization of wastewater that contains high levels of solids. These systems usually consist of one or more tanks in which batches of wastewater are "cooked" under temperatures and pressures typical of autoclaves. Autoclaves may be used for treating small quantities of liquid wastes generated in laboratories or small animal rooms. Large animal facilities and facilities with high numbers of small animals may require centralized wastewater treatment systems where wastewater is treated in batches of hundreds or even thousands of gallons at a time. Heat treatment systems can also feature grinders or other particle size reduction equipment, storage tanks and heat recovery systems.

Chemical treatment systems are not as effective in treating wastewater from animal facilities because of the potential for interference with chemical disinfection by solids in the wastewater. However, chemical treatment systems can be used successfully in some animal applications. Chemical treatment systems can range from something as simple as keeping drain traps filled with disinfectant, to complex centralized treatment systems with treatment tanks fitted with agitators, chemical feed equipment and computerized controls.

Where animal facilities are served by municipal sanitary sewage collection and treatment systems (or comparable onsite systems), treating wastewater before it is discharged to the sanitary sewer is sometimes unnecessary. These sewage treatment systems include solids removal, organic load reduction and disinfectant processes that are all monitored closely under state or federal permits. If the type and concentration of infectious agents in the wastewater are comparable to sanitary sewage or the wastewater generated by a hospital, additional treatment at the animal facility may not be required.(1)

System Design

There are common elements to the design and operation of wastewater treatment systems for biosafety level 3 facilities where small animals such as rodents, rabbits and monkeys would be used (see "Biosafety Levels" sidebar below). The following description outlines the features of one system that was designed for a large non-profit research organization located in the Midwest. The project included adding on to an animal research facility that tripled the number of animal holding rooms and laboratories at the location.

All plumbing fixtures in the facility except the water closets for the staff were connected to the sterilized waste system. These fixtures included restroom lavatories, sinks located in the laboratories and the anterooms, and the floor drains located in the animal holding rooms. The facility addition did not include necropsy, autoclaves or rack wash areas, where large solids would need to be handled. Waste for the most part was in liquid form, with a trace amount of rodentia droppings. Any large solids that were generated would need to be collected and disposed of before they entered the wastewater system. The under-slab waste piping drained by gravity to two 580-gallon sterilization tanks. After the sterilization process, the tanks were pumped out to an after-cooler that lowered the temperature below 140 degrees F before discharging to the sanitary system.

System Components

Commercial water heaters can be customized for the specialized application of wastewater sterilization. This project used vertical, stainless steel tank-type water heaters with all stainless steel steam tube bundles, circulation pumps and associated piping and specialties. The heaters were skid mounted and factory insulated with a protective metal jacket. All steam components not in contact with the wastewater, including piping, steam traps, control valves and strainers, were carbon steel. The steam tube bundles were mounted low in the tank, allowing flexibility in system operation, as the tank could be heated when at low wastewater levels. This design was an improvement over an installation in the main facility, as it consisted of only one sterilizer configured horizontally, with the tube bundle mounted high in the tank. When this tank was full, there was no place to divert the incoming waste, and the process could not begin until the tube bundle was sufficiently covered with wastewater. Additionally, a horizontal tank configuration did not lend itself to probe-type sensor control, as the spacing of levels tended to be too close.

The system was designed with the sterilizers being supplied with saturated steam at 60 psig, which corresponds to 307 degrees F and allows the tanks to be quickly heated to 250 degrees F. The tank is held at this temperature for one hour. High-pressure condensate from this process is discharged through float and thermostatic traps to a flash tank, then to a condensate return unit (see Fig. 1).

The sterilizer circulating pumps serve a dual role in promoting heat transfer across the immersed steam bundles and in delivering wastewater to the test panel located on the floor above. The sterilizers are pumped out at the end of the batch cycle with multi-staged stainless steel pumps to a blowdown separator that served as an after-cooler.

Sequence of Operation

Although the system was not strictly automated, it did have a control panel (see Figure 2) that allowed for easy opening and closing of valves and turning pumps on and off by flipping toggle switches. The sterilizer tanks are normally vented through a central HEPA-filtered exhaust system, although they can be vented to the outdoors by operation of manual isolation valves.

The gravity filling of the primary tank over the course of the day displaces the air in the tank through this vent line. When the tank is half full, operating personnel are notified through the building automation system (BAS). When the wastewater level in the tank reaches the 5/6 mark, the BAS again notifies personnel. At this point, valve VB-4 is opened by positioning of a toggle switch at the sterilization control panel, to allow effluent to enter the standby tank (see Figure 3). Valve VA-4 is then closed off on the operating tank to keep wastewater from entering. If the tank is filled beyond the 5/6 level, the safety relief valve could open during the sterilization procedure due to thermal expansion of the liquid. The quantity of air at the top of the tank acts as a cushion, as in an expansion tank, to prevent this from happening. Discharge of this wastewater through the relief valve before the end of the sterilization process would necessitate an extensive decontamination operation at the sterilizer pit level.

If the wastewater level reaches the full mark, the BAS will send an alarm to the operator workstation. At this point, wastewater can be transferred to the standby tank through the chemical feeder with the pump-out pump P-14 until the operating tank again reaches the 5/6-full mark. Before the tank heating system is activated, vent valve VA-3 is closed to provide an airtight pressure vessel. Toggle switches are then positioned to activate the heating circuit and the tank circulating pump P-16. Controls furnished with the sterilizer modulate the steam control valve to maintain the tank temperature at 250 degrees F. The tank pressure is allowed to rise above atmospheric, as it is desirable to keep the wastewater in liquid form during the procedure. The regulating authorities, such as the EPA, prefer to see circular charts. Therefore, chart recorders, as well as the BMS, keep a record of the tank temperature. After an hour at 250 degrees F, the heating circuit and the circulating pump can be toggled to the "off" position. At this point, the vent valve VA-3 can then be opened, causing the hot liquid to boil and to flash steam up the vent line as it drops to atmospheric pressure.

The pump-out pump P-14 can now be activated by a toggle switch to transfer the wastewater to the after-cooler. The pump-out pumps were selected to accommodate hot liquid with a low vapor pressure to prevent cavitation. The pumps were also placed at a sub-pit level 4 1/2 feet below the level of the tanks to ensure positive suction head. The original installation in the main facility had the pump at the same level as the tanks, which would not permit the tank to be fully pumped out. In addition, cold water had to be introduced to the tank's wastewater to prevent cavitation. The fully insulated tank would not be sufficiently cool even after 24 hours of sitting idle to allow pumping without cavitation. A level sensor at the base of the tank will automatically shut down the pump-out pump P-14 to prevent an empty tank condition and cavitation from occurring. This feature can be over-ridden manually to permit fully evacuating the tank.

The after-cooler that is employed is simply a boiler blowdown separator. It is vented and has a cold water connection with a temperature regulating valve. This ensures that the wastewater enters the main sanitary system at no greater than 140 degrees F.

During the sterilization procedure, the circulating pump P-16 distributes wastewater to the test panel where it is cooled for sampling purposes by heat exchanger HX-8. The shell side of this heat exchanger uses cold water for cooling. The potable water source is protected by backflow prevention, and for an added measure of safety, the heat exchanger is of the double-wall design to ensure a leak will not permit contact between the two fluids. Chlorine and ph analyzers are mounted on the test panel used by operating personnel to take readings for their safety records. The chlorine analyzer does its work at atmospheric pressure so that if Tank A is being analyzed, Valve VB-6 to Tank B needs to be open to permit the small sampling volume to be sent there.

Any part of the system that does not come in contact with the hot sterilized wastewater must be decontaminated when maintenance is to be performed. This would include the gravity drain components ahead of the sterilizer. One of these components is the basket strainer that requires periodic cleaning. This is accomplished by closing the isolation valve on the upstream side of the strainer and opening the vent valve to the tank being serviced. The drain valve on the bottom of the strainer can then be opened to drain to either tank. Only enough contaminated water needs to be drained from the strainer to permit the required amount of reagent to be admitted. Chlorine in the form of household bleach is often used and is injected through the decontamination ports on either side of the strainer. Because of the use of decontamination chemicals, the gravity piping in the vicinity of the tank, the valves and the basket strainers are all stainless steel. After a time, the top of the strainer can be safely unbolted, and the strainer basket can be removed and cleaned. The entire tank can be decontaminated without the use of heat by using the chemical feeder and the pump-out pumps to circulate through the tanks with valves VA-5 and VB-5.

Other Considerations

Systems such as the one described above may need to be registered with the state Environmental Protection Agency, and consequently, a Permit To Install (PTI) would need to be obtained. Licensing for treatment of infectious wastes may be considered a new technology and could create long delays in obtaining approvals. If this is the case, an alternative strategy might be to modify the system to operate as an autoclave, which may not require a PTI. This approach might require that the effectiveness of the process be verified on a monthly basis. This could be accomplished by installing sample tubes in the sterilizer tanks that would allow spore samples to be inserted at the center of the load. The monthly samples would be tested after the process for inactivation of the spores.

Certain applications may see larger solids enter the wastewater system. These solids could damage pumps and other mechanical equipment. If this is a possibility, modifications would need to be made to the system described previously. Grinder pumps or process pumps would be employed. Grinder pumps typically cannot tolerate water temperatures above the 160-180 degrees F range. Process pumps can be selected for much higher temperatures, but the low vapor pressure of the hot wastewater at atmospheric pressure could cause cavitation. For this reason, the cooling of the wastewater would need to occur between the sterilizer outlet and the pump inlet instead of just prior to discharge to the sanitary system.

The design of a wasterwater sterilization system for a biosafety facility can be a challenging task and requires an intimate understanding of how the operators will perform their various tasks. The system described in this article can serve as a starting point in the dialogue with the laboratory staff.

SIDEBAR: Biosafety Levels

Biosafety in Microbiological and Biomedical Laboratories, published by the U.S. Department of Health and Human Services, contains recommendations for working with infectious agents in laboratory settings. It outlines various combinations of equipment, practices and building guidelines that constitute four biosafety levels. These are as follows:

Biosafety Level 1 involves secondary educational training and teaching laboratories and other laboratory settings where work is done with strains of microorganisms not known to consistently cause disease in healthy adult humans. This type of facility would rely on containment based upon standard microbiological practices with no special primary or secondary barriers other than a sink for hand washing.

Biosafety Level 2 applies to clinical, diagnostic, teaching and other laboratories where work is done with moderate-risk agents that are present in the community and associated with human disease. The agents dealt with can be used safely in activities on an open bench, granted that the potential for producing splashes and aerosols is low. Hepatitis B virus, HIV and salmonellae are representative of the organisms assigned to this containment level. Primary barriers include biological safety cabinets, gowns, gloves and face protection. Secondary barriers include hand washing sinks and waste decontamination facilities.

Biosafety Level 3 facilities include clinical, diagnostic, teaching, research and production operations where work is done with indigenous or exotic agents with a potential for respiratory transmission and which may cause serious and potentially lethal infection. Tuberculosis and St. Louis encephalitis virus are representative of the microorganisms assigned to this containment level. More emphasis is placed on primary and secondary barriers to protect the community and the environment, as well as the laboratory personnel. All laboratory manipulations should be performed in a biological safety cabinet or other closed equipment. Secondary barriers include controlled access to the space and ventilation requirements that minimize the release of infectious aerosols from the laboratory.

Biosafety Level 4 involves work with dangerous and exotic agents that pose a high individual risk of life-threatening disease, which may be transmitted by aerosol and for which there is no available vaccine or therapy. Viruses such as Congo-Crimean hemorrhagic fever are manipulated at this biosafety level. The primary hazard to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane or broken skin exposure to infectious droplets, and auto-inoculation. Work with these agents poses a high risk of exposure and infection to laboratory personnel, the community and the environment. Worker isolation from aerosols is accomplished by working in Class III biological safety cabinets or in a full-body, air-supplied positive-pressure personnel suit. The Biosafety Level 4 facility is generally a separate building or completely isolated zone with complex, specialized ventilation requirements and waste management systems to prevent release of viable agents to the environment.(1)

(1) U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Institutes of Health, "Biosafety in Microbiological and Biomedical Laboratories," Fourth Edition, May 1999.