Access

Access to greenhouses housing transgenic research materials is restricted, regardless of the biosafety level. Such restrictions are intended to minimize the spread of transgenic pollen, seed, or other propagative material that could be carried by people moving between rooms or facilities. At BL1-P, access is limited or restricted at the discretion of the greenhouse manager or PI when experiments are in progress. At BL2-P, the manager is required to limit greenhouse access to individuals directly involved with the experiments, and at BL3-P, the manager, in consultation with the PI, should determine access authorization on an individual basis. Discretionary access is generally reserved for maintenance personnel and accompanied visitors who have a special interest in the research.

If the greenhouse consists of one large room as opposed to individual compartments, access to the entire facility may need to be restricted; all authorized personnel should have access to a key to enter. Signs must be posted at the entries to the greenhouse indicating that access is restricted for the experiment in progress. These signs may also contain access instructions. An entry and exit logbook is required for BL4-P greenhouses only. However, when exotic infectious agents are present in the research facility, APHIS recommends keeping a record of the personnel who regularly work there, visitor, and service personnel visits. The log should include the names, dates, and times of everyone entering and exiting the facility.

Apparel and Hygiene

Personnel entering BL1-P and BL2-P facilities may wear their usual street or lab clothing. For entry into BL3-P greenhouses, disposable lab gowns or the equivalent may be required at the discretion of the greenhouse manager. If special clothing is required, it must be removed before leaving the facility and decontaminated (usually by autoclaving) before washing or disposal. Users are also required to wash their hands before leaving BL3-P restricted areas.

BL4-P facilities maintain strict apparel and hygiene protocols. All users are required to enter only through the dressing/shower rooms and must shower when leaving the facility. Users are also required to remove all street clothing and don protective clothing before entering. Likewise, personnel leaving the facility must remove protective clothing before showering and exiting. The clothing must be stored in the inner change room and autoclaved before laundering. Showering before entering is required only when there is concern that organisms will be brought into the containment area from the outside.

Signage

No special signs are required for BL1-P containment greenhouses. Entryways into BL2-P and higher facilities should be posted with signs indicating that access is limited to authorized personnel only. If the experiment uses organisms that pose a risk to the local ecosystem or agriculture, a sign so stating must be placed on the access doors to the greenhouse. A description of the potential risk may be posted on the restricted access sign as long as this is not confidential information. The sign should state the name and telephone number of the responsible individual, the plants in use, and any special requirements for using the area. It may include contact information for the greenhouse manager and others to be called in case of emergency.

Transgenic material in a greenhouse room must be marked to distinguish it from non-transgenic organisms such as plants serving as experimental controls or those not involved with the experiment. Individual pots, bench sections, or entire benches can be marked with stakes or signs that identify the plant and the primary genetic modification, for example, "Soybeans with viral coat protein gene" (Fig. 6). All organisms in the room must be treated in accordance with the highest level of containment standards required by any experimental material present.

Seed Storage

Transgenic seed should be stored in a locked cabinet located preferably in the greenhouse room so as to minimize handling in unconfined spaces. When stored or handled outside the area of confinement, such as in a cabinet or on a potting bench in a headhouse corridor, the seed should be in a spillproof container. The transgenic seed should be clearly identified and labeled to distinguish it from other stored seeds or materials in the cabinet. Greenhouse personnel should take ordinary precautions to prevent seed germination in unwanted locations.

Transfer of Materials

The NIH Guidelines specify requirements for transporting experimental materials to and from a greenhouse for levels BL2-4P. For BL2-P and higher facilities, transgenic material in the form of seeds or propagules, potted plants, trays of seedlings, etc. are to be transferred in a closed non-breakable container. For BL3-P and BL4-P containment, the guidelines require an additional sealed secondary container for movement of experimental materials. The exterior surface of the secondary chamber should be decontaminated either chemically or in a fumigation chamber if the same plant, host, or vector is present within the effective dissemination distance of the propagules of the experimental organism.

Termination and Disposal

To prevent the unintended survival of GMOs outside the greenhouse environment, all experimental materials must be rendered biologically inactive (devitalized) before disposal. Termination procedures for the safe disposal of soil and plant material should be part of the experimental plan for a research project. The IBC may institute a policy that outlines acceptable disposal procedures for GM research materials, taking into consideration the biosafety level of the experiment and the volume of material to be handled. Devitalization of plant material and soil should be completed before it leaves a greenhouse or laboratory and goes to a landfill.

Plants and associated organisms can be inactivated though steam or chemical sterilization procedures. Steam forced into special carts or boxes has traditionally been used in greenhouses for treating growing beds, pasteurizing or sterilizing media, and disinfecting containers, thus it is likely to be available. Materials from smaller experiments can be inactivated by autoclaving all plants, plant parts, containers, and potting media. For larger volumes, composting is an acceptable treatment for experimental plant and soil materials that pose no recognized harm to the environment. Plants can be devitalized through desiccation simply by withholding water or they can be chopped or minced to pieces unable to grow independently under natural conditions. Incineration may also be used to destroy easily combustible, dry plant material; however, incineration must be used with caution since not all seeds are easily burned, e.g., cottonseed. At higher containment levels, it is recommended that all materials leaving the greenhouse be sterilized in an autoclave.

Disposing of very small transgenic seeds requires special care. Fine mesh bags can be secured around flower heads prior to disposal; a sheet of dampened white paper such as BenchKoteª placed on the work surface facilitates recovery of easily scattered seeds. The gravel under benches in BL2-P facilities should be decontaminated by, for example, treatment with a sodium hypochlorite (household bleach) solution. Catching liquid in a large open pan and allowing it to evaporate is a simple alternative.

Abandoned or forgotten experimental materials are not an infrequent problem for greenhouse managers. An IBC policy stipulating that GMO material is the responsibility of the PI would clarify authority in disposing of neglected or abandoned materials. This policy would preclude a source of gene escape that may occur when a PI leaves transgenic material in the greenhouse due to death, resignation, or simple oversight.

Pest Control

The NIH Guidelines call for a pest control program for all biosafety levels when working with transgenic organisms in a greenhouse setting. Rodents and birds can transport transgenic seed outside the facility; insects and other organisms can transfer pollen to receptive plants located within or outside the containment area. A stringent pest control program, using physical, chemical, or biological control measures, alone or in combination, should be implemented and monitored for effectiveness. Screens are recommended for BL1-P and required for BL2-P to exclude pollinating insects and birds; BL2-P facilities must have louvers fitted on exhaust fans that are open only when fans are running. The perimeters of greenhouses of every containment level should be sealed to prevent rodents and other large pests from entering. Fumigation can be used to control certain insect pests such as whiteflies. Biological control measures may involve the introduction of predators, parasites, and parasitoids to control pests.

Greenhouse research commonly uses insect pests as part of the experimental protocol, such as in testing plants for disease or insect resistance. In these cases, selective control measures are needed to eliminate the unwanted pest without killing the required pest organism. When insect vectors are used to transmit genetically modified viruses, particular care should be taken to eliminate the vector once the transmission has been accomplished.

Training and Reference Manuals

Personnel instruction is an important component of good management practices. A reference manual should be prepared containing directives covering all safety considerations pertaining to the transgenic research being conducted. Staff are required to read, understand, and follow the instructions provided in the manual before entering the greenhouse. Personnel training is best accomplished through interactive sessions that include the PI, greenhouse manager, or other safety-management staff.

For BL2-P and higher facilities, emergency and contingency plans, as well as documents pertaining to routine operations, are required to be included in the reference manual. It is not necessary to include experimental protocols in the manual, however researchers and greenhouse staff may find that a copy of the experimental protocol aids compliance with containment procedures. Conversely, relevant portions of the manual may be included in the project documents submitted for IBC approval.

Monitoring Containment Effectiveness

Escaped organisms may be detected by placing susceptible host plants, insect traps, or spore/pollencatching devices both inside and outside the containment area. Traps and bioindicator plants can be used to detect unintended virus transmission, insect migration, and pollen or spore spread. For example, if an experiment involves a caged insect-vectored plant disease system, uninfected plants placed in the same greenhouse but not in the caged area can be monitored for evidence of disease transmission. Light traps placed in corridors and operated at night are useful to indicate the presence of insects that have escaped the greenhouse rooms.

Procedures for Loss of Containment

The integrity of containment measures is susceptible to equipment malfunctions, acts of nature such as fire, flood, and storm damage, and human error. A loss of BL1-P containment due to any of these factors would likely have only minor environmental consequences, if any, and would not require any response. At BL2-P or higher, such events may present larger concerns.

Facilities operated above BL1-P should be equipped with an alarm system designed to alert someone when mechanical or weather-related events causing a loss of containment occur. Greenhouse systems that monitor automated environmental controls should have built-in local and remote alarms. Instances of human error, such as a door left open or ordinary disposal of unlabeled transgenic materials, is actually a more common cause of containment loss than facility malfunctions or structural damage. Designated people who are promptly alerted to problems can make timely decisions in regards to contacting or dispatching appropriate response personnel.

The NIH Guidelines require BL2-P and higher facilities to have contingency plans for handling emergency situations that also apply in cases of theft or vandalism. These plans, drawn up by the BSO and/or IBC in consultation with the PI, must include measures to contain the breach, a personnel notification sequence, and decontamination procedures. In addition, the plans should include names and contact information for repair personnel, researchers, relevant authorities, and greenhouse staff.

Records

The extent of record keeping required for research using transgenic organisms is commensurate with the level of biosafety. Records of experiments in progress must be kept for all biosafety levels. At BL2-P and higher, additional records must be kept of all plants and plant-associated organisms entering or leaving the greenhouse. A record of the dates and times of personnel visits must be kept for BL4-P facilities.

Although the NIH Guidelines do not specify who should keep records, the PI is the logical choice as he/she is responsible for tracking experimental material. It is also appropriate that someone stationed in the facility (e.g., the greenhouse manager or equivalent) has responsibility for entry and exit logs when required.

Inspections

Greenhouses should be inspected periodically to ensure that containment measures appropriate for the transgenic plants and other organisms held inside are being rigorously followed. Inspections should be conducted on a regular schedule and whenever new types of experimental materials are brought into the facility. Inspectors may include the greenhouse manager, BSO, IBC representative, or state agriculture officials. Officials from USDA/APHIS may, upon request, visit a facility to observe containment features. However, USDA does not certify or otherwise designate a greenhouse's suitability for research materials requiring a specific biosafety level(s) unless there is present a plant pest requiring a permit from APHIS-PPQ.

Inspection checklists help ensure that a greenhouse complies with all necessary physical, biological, and managerial requirements for a given Biosafety Level. Inspection checklists facilitate IBC approval, provide an outline for internal monitoring, and serve as documentation of compliance. A sample of an APHIS "Facility Inspection Checklist for Containment of Genetically Engineered Organisms" is included as an Appendix. Public and private sector research organizations usually develop their own inhouse checklists. Checklists may be customized by combining items from the APHIS checklist, other lists, and the list below. Where several levels of containment are provided by different rooms within a single facility, checklists tailored to each level simplify the inspections.

For each room or research project, an inspection checklist may ask:

• Who is the responsible party? Is their contact information posted on the door?
• What is the nature of the GMO and how is it identified?
• What is the prescribed level of containment? Do the physical facilities meet this level?
• What specific physical and biological measures are being used to achieve that level of containment?
• Are prescribed practices being followed?
• Is there any evidence of deficiencies with regard to containment?
• How is the area secured? What security is required?
• Is there a written plan for responding to loss of containment? What is the most likely containment breach?

If GMOs under APHIS permit are in a greenhouse with the same species of non-GMOs, APHIS recommends that the two groups (or more) be well separated to avoid inadvertent cross pollination. Also, it is recommended that the GMOs have some designated boundary on the bench such as colorcoded markers. Additionally, Plant Protection and Quarantine Officers of APHIS may conduct unannounced re-visits to facilities housing GMOs under federal permit. The unannounced inspections occur during normal business hours and are a Standard Permit Condition.

Periodic reinspections of the greenhouse should be conducted. The presence of light, heat, and water within a facility promotes gradual deterioration of equipment and structural features over time. Additionally, an inspection serves as an opportunity to review any special practices that may be required, as staff adherence to non-standard procedures may tend to relax over time.

A Note about Vandalism

Vandalism is an increasing concern for greenhouse managers. Some individuals and organizations opposed to recombinant DNA research have targeted greenhouse and field trial research projects, causing substantial damage. Determined individuals gain entry either by force, by defeating security hardware, or they may be admitted inadvertently by authorized personnel—self-closing doors may be propped open, rooms and entries left unlocked, and strangers not always confronted. Facility users should be advised that they share responsibility for maintaining security.

When the threat of vandalism is politically motivated, a situation termed "domestic terrorism" by the US Federal Bureau of Investigation, an institution may wish to create a response team. This group typically is composed of a high level administrator, a public information officer, the facility manager, legal counsel, and relevant others whose job it is to review physical deterrents and develop public relations strategies. Because political actions generally are designed to garner sympathy for a cause via the news media, it is important that an institution have an opportunity to respond quickly and clearly to threats or acts of vandalism.