TABLE 7. Floor plan of the USDA Foreign Disease and Weed Science Research Unit BLP-3 Containment Facility in Frederick, Maryland¹⁸

Note that major changes to the layout can necessitate further structural modifications, such as the addition of partitions and/or hallways within a previously undivided greenhouse. These changes may in turn call for revamping environmental control schemes, utilities, ventilators, and primary structural components as well.

Entry doors and locks Standard lockable hinged doors can be used for exterior and corridor entrances. Sliding doors are acceptable at BL1-P and BL2-P but do not seal tightly enough for higher containment levels. Both styles of doors can be fitted with locks to limit access. For security reasons, the distribution of greenhouse keys should be carefully controlled and monitored. Greenhouse rooms dedicated to transgenic research could be re-keyed to assure access is limited to authorized personnel only. It is also advisable to restrict the total number of keys issued to a practical minimum and to strictly limit the number of master or sub-master keys made. Doors should fit tightly against the jamb and have a sweep at the threshold. The most commonly used standard door sweep consists of a neoprene or rubber strip or a short plastic brush attached to an aluminum holder that can be fastened to any relatively flat surface (Fig. 8). Although sweeps cannot restrict all small insects that are intent on entering or exiting a space, they can easily exclude rodents, birds, and larger flying insects. Figure 8. Neoprene door sweep The NIH Guidelines stipulate a double set of self-closing and locking doors for BL3-P and BL4-P containment. Building codes prescribe the presence and placement of emergency exits regardless of containment needs. Therefore local officials must be consulted before amending or creating entrances and exits. Glazing The condition of the glazing and bedding putty should be carefully evaluated before conducting transgenic research. Properly installed glazing provides low infiltration and generally affords a high degree of containment. Bedding putty for standard lapped glass greenhouses, however, wears out long before the glass, a condition that may precipitate glass breakage and cracking. If a glass greenhouse needs new bedding putty, which is a very labor intensive job, it may be economically advantageous to consider reglazing at the same time with sheet materials, new styles of glass, or inflated films. Simultaneously replacing bedding putty and glazing would provide tighter containment, better environmental control, and energy cost savings. Standard greenhouse glazing material will satisfy the requirements for BL1-P and BL2-P. Glass glazing is the most enduring material and provides the greatest amount of natural light. Laminated and heat-strengthened glass is preferred or, depending on building codes, may be required. Standard tempered glass is more prone to spontaneous breakage and shattering which can both breach containment and create a hazard. Glass can be manufactured in lengths that extend from the eaves to the ridge, though lengths over eight feet become impractical. Sheets of rigid plastics such as Lexan® polycarbonate or Exolite® acrylic also are commonly used for glazing. Polycarbonate costs less and is more fire resistant than acrylic; acrylic glazing, however, lasts longer and permits better light transmission. Double-walled sheets of rigid plastic glazing shift significantly within their framing with temperature fluctuations; therefore, inspections should be made seasonally for openings in these materials. Various types of film plastic glazing are commonly available, e.g., p1olyester, polyethylene, polyvinyl chloride, and so on. Double-layer plastics rely on a fan to inflate the space between the sheets, and require regular inspections to detect loose holddown clamps and tears. Film plastics also have a relatively short life (less than four years on average), become brittle with age, and are easily penetrated, accidentally or intentionally. Therefore they are not the preferred choice for a containment greenhouse. Reglazing with the new generation of film plastics may be a viable option; project managers are advised to consult contractors and institutional officials. Films such as Hostaflon® can be installed in three layers and still transmit light as efficiently as glass. Longevity with some of the new films has increased from four to 20 years, and they can resist hail damage better than most rigid materials. Standards for BL3-P and BL4-P require windows to be closed, sealed, and resistant to breakage. This requirement can be met by using double-paned sealed glass or rigid, double-walled plastic panels. Examples are Sedo®, a brand of double-paned glass that contains an inert gas between the panes, and two readily available sheet materials, Lexan® and Exolite®, as noted above. Reglazing with doublepaned sealed glass is likely to require extensive structural renovations to bear the additional weight. The National Greenhouse Manufacturers Association published glazing standards that allow manufacturers to run standard tests on their products19. Test results allow consumers to make comparisons between various glazing products on the market. Screening Screening is an especially important consideration when retrofitting an existing structure to attain a higher containment level. Screening should be carefully installed on all ventilation intake vents. Figure 9 demonstrates a method of screening around moving vent arms. Figure 9. Screen panels over ridge (Reprinted with permission of Agritechnove, Inc., St. Anselme, Quebec, Canada.) For containment purposes, screening side vents is recommended for BL1-P and required for BL2-P. If evaporative cooling pads made of aspen fiber or corrugated cellulose are used on the intake side vents, screening is still useful since insects can find their way through these materials. Screen mesh size should be gauged relative to the size and shape of the organisms to be contained or excluded. A comparison of commercial screening materials20 indicates that in some instances screens with a larger hole size may have exclusion efficiencies similar to those with smaller holes. This is because holes are not always perfectly square in commercially-made screens, a factor that may or may not favor insect exclusion, depending on hole shape. Further, thread diameter and mesh material also influence exclusion properties. Relatively rigid stainless steel mesh may offer better exclusion than softer mesh with a similar hole size. Fine mesh screen requires high maintenance; therefore consideration should be given to ease of replacement and cleaning. Screen size can greatly affect airflow, cooling efficiency, CO2 retention, humidity level, and light transmission. Proper sizing of screen to the ventilation system is critical, regardless of the type of cooling systems installed—passive, fan only, fan and pad, or mechanical (air-conditioned). A piece of 64-mesh screen with a thread thickness of 0.008" has a total of only 23.8% open space. Dust accumulation on screens can also affect their efficiency—as the screen opening size decreases, the need to keep the screens clean by washing or vacuuming increases. Regardless of where screening is placed, airflow considerations are paramount because of temperature changes associated with reduced air movement. Airflow, cooling, and fan performance are significantly affected by the installation of any screen, especially when using the finer mesh sizes. One solution to the airflow restriction problem is to build a "screen box" outside the cooling pad frame (Fig. 10) to provide adequate surface area for airflow though the cooling pads. Figure 10. Typical insect screen installation shown on intake vent end of greenhouse (Book of Standards. National Greenhouse Manufacturers Association. Reprinted with permission.) Ventilation, Cooling and Heating Motorized and/or manual hinged vents, located at the roof ridge and/or sidewall, are a common feature of most greenhouse cooling and ventilating systems. The passive ventilation afforded by vents can be activated with the addition of exhaust fans and evaporative cooling systems. Air intake screening (but never air outlet) and motorized or gravity-driven exhaust fan louvers are recommended for BL1-P and required for BL2-P. Motorized louvers should be interlocked so they open and close with fan startup and shutdown. Gravity-operated louvers are also adequate. The vent operator arms or racks that pass through screen are generally fitted with brushes or flexible barriers to prevent rodents and other large pests from entering the greenhouse. Fog cooling systems, if suitable for the structure and climate, may offer a better and more convenient alternative to evaporative cooling pad systems. Fog cooling should always be used in conjunction with a good control system to insure precise relative humidity measurement and proper fog delivery. Screens should be made to fit all vent openings, fans sized accordingly, and the system installed inside the greenhouse as specified by the manufacturer. Recirculating fans and curtain systems are also used to help control temperature. The use of mechanical cooling, i.e., air conditioning, is the only option for higher levels of containment. Construction and operation costs are very high due to the enormous heat load of a greenhouse. Typical greenhouse heating systems include hot water radiation, steam radiation, infrared electric, solar, and forced air. All are adequate for every containment level. Benching Standard greenhouse benches are adequate for most GMO research projects though wood is not recommended. Benching made of expanded galvanized steel or aluminum is preferred since these materials are resistant to water and most chemicals. In addition, such benches are readily available, meet higher containment standards, and allow for thorough cleaning, which contributes positively to a pest control program regardless of the research protocol. In some cases, benches with solid tops have adequate framing to allow replacement with expanded metal. In cases where the IBC, as recommended by the NIH Guidelines, stipulates collection of runoff water, a solid bench may be installed that drains runoff into a holding tank for treatment with chemicals or heat before being released to the sewer or ground. A bench that collects water for recirculation, also called an Ebb and Flow bench (Fig. 11), could also be modified to collect runoff for subsequent treatment, or simply desiccation, if that renders the propagules in question inactive. Figure 11. Ebb and flow bench At BL3-P, other provisions may be needed to collect and treat runoff water. These may involve collection from the bench and consequent treatment but would more likely involve whole room collection using a sewer. Higher levels of containment also require seamless bench tops and other work surfaces that are impervious to water and chemicals and can withstand mild heat. These requirements may make retrofitting for high levels of containment cost prohibitive. Floors and Drains Requirements for greenhouse floors vary according to the biosafety level indicated (Table 7). Floors and drains may need to be renovated to meet containment standards for transgenic greenhouses. Gravel and soil beds can be used under benches in BL1-P greenhouses only if experimental material cannot travel through these beds and leave the greenhouse; concrete walkways are preferred. A BL2-P greenhouse must have an impervious floor surface. Retrofitting a greenhouse with concrete floors and walkways can substantially improve containment and sanitation practices. Coatings can be applied to concrete surfaces to make them easier to clean and disinfect. If a new floor is to be installed, it may be advantageous to install floor drains designed to collect all runoff. This is particularly true if research projects that use genetically engineered microbes are underway or expected. Retrofitting with a biowaste collection and treatment system can be prohibitively expensive if the existing concrete slab and underground piping must first be removed and reinstalled. Such renovations could easily push the cost of retrofitting an existing facility to exceed that of new construction. Control Systems Standard digital, analog, pneumatic, or mechanical greenhouse control systems are suitable for GMO research at BL1-P and BL2-P. Computer or other control systems that incorporate alarms and interact with headhouse systems are recommended for BL3-P and BL4-P. Sensors, usually under computer control, are also required for high containment facilities to monitor differential air pressures. Sensor technology has become prevalent and could be employed in any modern research greenhouse. Control systems can be easily upgraded in most situations. Greenhouse control systems technology has become highly advanced, reliable, and cost effective. It is strongly recommended that any control system used in the greenhouse itself be designed and manufactured exclusively for greenhouses, in contrast to building control systems, which cannot meet the exacting specifications for a research greenhouse. Piping systems Heating, watering, and fertilizing systems are typically piped into and throughout the greenhouse. Automatic watering and fertilizing systems are advantageous because they reduce the amount of traffic into the greenhouse, thus decreasing the opportunity to spread transgenic pollen, seed, and other propagative materials. The relative ease and affordable cost of installing these systems makes them an attractive option. However, for containment reasons, new piping should be installed with a minimal number of intrusions. All new and existing intrusions should be sealed with a durable material to help ensure containment (see Fig. 1). SCREENHOUSES Screenhouses are acceptable for GMO research only when they meet the requirements for BL1-P or BL2-P level greenhouses, including floors, and contain organisms that would have minimal impact on the environment, if released. Though they have limited utility for research, screenhouses may offer a low cost alternative to greenhouses when sited in an appropriate climate. Retrofitting screenhouses involves many of the same measures listed for greenhouses. Upgrades could include the addition of concrete floors, well-fitting, lockable doors, individual compartments, sealed joints (Fig. 12) and utility intrusions, and specialized screening. BL3-P experiments would likely not be approved for screenhouse containment. Figure 12. Sealed framing joints in a containment screenhouse GROWTH CHAMBERS If the quantity of plant material is not large, use of a growth chamber or growth room may be the best option for containment at the higher levels. A growth chamber modified to meet BL3-P requirements is shown in Fig. 13. The two main retrofits to the chamber are a HEPA filter and a system for collecting runoff water. If large quantities of plant material are produced, then renovation of existing facilities may be as cost effective as retrofitting the growth chamber. Figure 13. Growth chamber with HEPA filtration (Reprinted with permission of Conviron, Inc., Winnipeg, Manitoba, Canada.)