SAFETY PROTOCOLS for TOXICITY BIOASSAYS with Pfiesteria

SAFETY PROTOCOLS FOR PFIESTIERIA TOXICITY BIOASSAYS

In The

AQUATIC BIOMONITORING FACILITY

October 15, 1999

Reviewed by:

Principal Investigator Associate Director Environmental Sciences Safety and Environmental Programs VIMS VIMS

Department Chair, Director of Research & Advisory Services Environmental Sciences

Dean/Director VIMS CONTENTS

Safety Features of the Facility 1

Emergency Procedures 1

Pfiesteria Toxicity Issues 2

Decontamination 2

Entry 3

Preparation Room and Laboratory 4

Primary Exposure Room 5

Emergency Response to Spills 6

Decontamination/Undressing 6

Secondary Exposure Room and Water Maintenance Area 8

Maintenance Schedule 8

Figures

Figure 1. Schematic of Aquatic Biomonitoring Facility

Appendices

CDC: Recommended Guidelines for Biosafety Level 3 i-v

CDC: Guidelines for Work with Toxins of Biological Origin vi-viii

Example methods for experimental exposures ix

Training checklist for work in facility x

SAFETY FEATURES OF THE FACILITY The facility is engineered for unidirectional air flow to pass through a dual, three-tiered filter system (Figure 1). These ventilation features lie at the heart of the safe use of the facility for exposure and toxicity studies. The filter system consists of a fiber prefilter for large particles, an activated charcoal/carbon filter to remove potential toxins, and a standard HEPA filter to further reduce aerosol contaminants. Other safety features include a self-contained, restricted access work environment, separation of laboratory and aquaria space into 3 separate rooms, a safety shower, floor drains in the Exposure Rooms, a small biosafety cabinet (level 1 with HEPA filter), and splash-containment tubs. Additional safety procedures include the use of personal protective equipment (PPE) including full-face respirators (with P-100 cartridges) in exposed areas, rigid protocols for handling and observing toxic and suspect toxic fluids, limiting the generation of aerosols, and regular maintenance of the ventilation system.

By definition a Biosafety Level 3 facility has strict access control and a specialized ventilation system. Safety procedures and equipment are designed to inhibit aerosol infection or contamination of the researchers, community, and environment (Centers for Diseases Control 1984). Level 3 practices include standard microbiological techniques, decontamination of wastes, limited and controlled access, protective gloves, biohazard warning signs, and special laboratory clothing (as deemed appropriate to the agent). Level 3 safety equipment includes partial containment equipment for all manipulations, and mechanical barriers and procedural means to limit aerosol exposure.

There are numerous variations on what constitutes a BSL3 facility. The CDC (1999) published the following statement: "It is recognized that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g., access zone, sealed penetrations, and directional air flow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g., diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in laboratories where facility features satisfy Biosafety Level 2 recommendations provided the recommended "Standard Microbiological Practices," "Special Practices," and "Containment Equipment" for Biosafety Level 3 are rigorously followed." The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director" (CDC, 1984).

EMERGENCY PROCEDURES Toxic Exposure Personal protective equipment (gloves, boots, aprons, etc.) will limit contact with toxic materials. An emergency shower, however, will be used in case of contact with toxic culture media. Soap and water washes are prescribed for contact with toxic media in field conditions, and will be adapted for use in the laboratory (see below). Full-face respirators (with P-100 cartridges) will limit inhalation of toxic materials. A doctor will be sought immediately in case of inhalation or if an exposure results in skin lesions or unusual behaviors.

 Level 1 biosafety cabinets are designed to protect personnel from exposure to the contents of the hood. Such cabinets do not protect the contents from potential microbial contamination. That is not an issue for the planned work as the bioassays are not sterile.

1 Fire The building is to be exited in case of fire. A fire extinguisher is located inside the entry door to the facility. All personnel working in the facility must be trained to use a fire extinguisher prior to their working therein.

Injury Call console attendant at x7000/7001 in case of life threatening injuries. The attendant will call 9-911 and notify the Office of Safety & Environmental Programs. Non-life threatening injuries should be immediately reported to the Worker's Compensation Coordinator for a referral to a doctor treating work-related injuries.

PFIESTERIA TOXICITY ISSUES "The Centers for Disease Control (CDC) set of exposure criteria and clinical signs and symptoms for Pfiesteria are defined as exposure to estuarine water characterized by one of the following: (1) fish with lesions consistent with Pfiesteria piscicida or morphologically related organisms (MRO) toxicity (20 percent of at least 50 fish of one species having lesions); (2) a fish kill with fish having lesions consistent with Pfiesteria or MRO toxicity; or (3) a fish kill involving fish without lesions, in the presence of Pfiesteria or MRO, without an alternative reason for the fish kill. The clinical features include the following signs and symptoms: (1) memory loss, (2) confusion, (3) acute skin burning (upon direct contact with water), or (4) three or more of the following: a. headaches; b. skin rash; c. eye irritation; d. upper respiratory irritation; e. muscle cramps; f. nausea/vomiting/diarrhea/abdominal cramps" (CDC, 1998).

Toxic blooms of P. piscicida can occur at relatively low cell densities of 250-300 cells/ml (Burkholder et al., 1992; 1995). Sublethal toxicity in fish may arise from as few as 100 cells/ml. Human health is apparently affected at densities over 1000 cells/ml; but exposure levels remain to be determined (Glasgow et al. 1995). Subcutaneous injections of water samples with toxins caused memory and learning disorders in rats (Levin et al., 1997). The toxins appear to have two components: a water-soluble, and a lipid soluble fraction (McClellan et al., 1997). The water- soluble fraction has been tested against human and rat cell cultures with good results (McClellan et al., 1997, Burkholder & Glasgow, 1997. Cytotoxic effects were noted with blebbing of the cytoplasm, and degradation and eventual lysis of the cell. The toxins reportedly have a low LC50 of 1x10-17 g/ml (McClellan et al., 1997; Burkholder & Glasgow, 1997). The potential toxicity of the Pfiesteria or Pfiesteria-like organisms has led to safety concerns regarding lab cultures of the toxic forms. At present, “toxic cultures” require isolation, and suitable ventilation (with HEPA and activated charcoal filters); thus limiting the number of facilities capable of working with the toxin-producing stages.

DECONTAMINATION The toxins produced by Pfiesteria are not well understood, but they apparently have a short half life (minutes to hours) in estuarine waters (Burkholder et al., 1995). For laboratory situations, decontamination will follow general BSL 2 guidelines using dilute (1.0%) bleach to decontaminate surfaces and disposables. In addition, and for field samples, we will follow the procedures outlined in a general memo on Pfiesteria and fish kill safety procedures (see attached, Hobbs, June 6, 1998). Briefly, contaminated surfaces and gear are washed in soap and water and thoroughly rinsed. Cleaned gear is then allowed to air dry. Respirators are cleaned and

2 decontaminated as per manufacturer's instructions, and if necessary as above. Filter cartridges are replaced weekly or when wet.

Aquaria with live cultures are decontaminated with bleach. Bleach is added directly to the media to make an approximate 5-10% solution, incubated for 30-60 min, and neutralized with sodium thiosulfate. The media is then flushed down the sink to a connecting sewer line to the Hampton Roads Sewage Treatment plant.

ENTRY Entry to the trailer is limited to qualified personnel who have read this safety document. When toxic cultures are present, no admittance will be allowed to those who do not meet the below standards.

Entry Criteria Those actively working in the trailer must (a) pass a general physical examination administered by a qualified medical doctor, (b) be trained in the use of and possess a custom-fitted full-face respirator with HEPA filter cartridges, (c) must sign a memorandum of understanding attesting to their having read and understood this document, (d) otherwise practice and adhere to the safety standards and protocols detailed herein, and (e) have the written approval of the Principal Investigator, and the Office of Safety and Environmental Programs. As per NIH biosafety guidelines (CDC, 1984, 1999), a certification checklist (for microbiological and hazardous materials training) will be maintained and updated annually for all personnel working in the facility (see Appendices).

To practice good safety procedures, two people must be present in the facility when toxic or suspect toxic cultures are present.

Once established, the review and practice of these safety protocols will be done quarterly by all personnel. Each quarter personnel must sign a memo stating that they have read the protocols.

The trailer is designated as a Biohazard Laboratory, and is marked accordingly. No food or drink is allowed inside the facility.

Entry Requirements Only authorized individuals as above may enter and work within the facility. For safety purposes and for proper documentation, an Entry Logbook will be maintained. Those entering the facility must sign in and out, noting the date, times of entry and exit, and activities in the logbook. A list of authorized personnel will be maintained at the entryway.

Dressing Area The dressing area is used for changing into the necessary protective gear to work in the Exposure Rooms. No contaminated or exposed gear is allowed in the area. New gloves, Tyvek clothing, and filter cartridges are kept in the storage compartment.

3 PREPARATION ROOM AND LABORATORY The Prep Room and Laboratory will not be directly or indirectly exposed to toxins or toxic byproducts. Respirators will not need to be worn in this part of the facility, but they must be available in case of emergency. The sink-mounted safety drench serves as an emergency eye wash and shower if needed.

Note that sealed containers of toxic cultures will be examined in the Prep Room, and sealed waste containers must be transported out of the facility through it. As a standard precaution the following personal protective equipment is to be worn while working in the Prep Room:

 gloves  lab coats  eye protection  fully covered shoes  pants

Exposed or contaminated gloves, boots, aprons, and clothing are not to be brought into the Prep Room unless decontaminated by washing with soap and water (see Decontamination above and Undressing below).

Handling sealed materials Toxic and suspect toxic cultures must be assessed for cell densities, morphologies, etc. Within the Exposure Rooms, aliquots of toxic cultures will be pipetted into tissue culture flasks, and sealed tightly with a lid. The sealed flasks will be taken to the Prep Area and examined with an inverted microscope. In certain circumstances (subsampling for fixations, density manipulations, etc.), sealed flasks will be opened but only within the biosafety cabinet. A relatively low volume of contaminated waste will be generated. Pipettes and other materials will be placed in a small Biohazard bag within the cabinet for later incineration. Waste materials (pipettes, plasticware, paper towels, liners, etc.) from the Exposure Rooms will pass through the Prep Area. Such waste materials will be "double-bagged" in Biohazard bags for transportation.

Biological Safety Cabinet The biosafety cabinet (Class I) is lined with an absorbent paper lining to catch any potential spills. The HEPA filter unit will be monitored weekly and serviced as needed. The biosafety hood will be certified annually. Alcohol swipes will be applied prior to and after use to keep the inside of the cabinet clean.

4 PRIMARY EXPOSURE ROOM Before entry the following personal protective equipment will be worn for splash protection:

 standard latex or nitrile gloves  shoulder length nylon/rubber gloves  One piece Tyvek jumpsuits with hoods  full-length rubber apron  knee-high rubber boots  full-face respirator with P-100 filter cartridge  two qualified personnel in the trailer  respirator fit check

While toxic or suspect toxic cultures are present, at least two qualified personnel must be in the trailer for anyone to enter the Exposure Rooms. Prior to entry into the Primary Exposure Room, a respirator fit check is required to insure a proper fit. One qualified person shall remain in the Preparation Room at all times when the Primary or Secondary Exposure Rooms are occupied.

The Primary Exposure Room is divided into two sections (Figure 1). The anterior section contains the entry portal, shower, and one bank of aquaria. These aquaria are for control, nontoxic cultures of fish only. The posterior half of the room contains two banks of aquaria that will be used for cultures of toxic dinoflagellates. The Secondary Exposure Room also contains a bank of aquaria for toxic exposures (see below).

Contact Aquarium maintenance (feeding, cleaning, checking airlines, etc.) and collection of water samples will necessitate frequent contact with toxic and suspect toxic cultures. Much of this contact will be minimized by the use of long handled nets, scrapers, pipettes and siphons. In certain circumstances personal protective equipment (gloves, boots, and aprons) will become exposed. Decontamination is outlined below. In case of contact with skin, a decontamination shower shall be performed using the safety shower in the Primary Exposure Room.

Limiting Aerosol Exposure Four safety features help limit the generation and spread of aerosols in the Exposure Rooms.

(1) Whisper filters are used instead of box filters. Aeration will be used for menhaden studies but the aquaria will be covered and located "downwind" in the air flow. (2) Plastic shielding is installed around the shelving and over aquaria as needed. (3) The unidirectional air flow will collect possible aerosols downwind of unexposed areas toward and through the HEPA filter system. (4) We have demarcated the entry area, and an adjacent area within the Primary Exposure Room as UNEXPOSED areas. Only aquaria serving as controls will be placed in these areas.

5 EMERGENCY RESPONSE TO SPILLS Staff will undergo quarterly spill response exercises to drill for possible accidents involving toxins. In the event that there is a large spill, breakage of aquaria, or other toxin-involved accidents we will practice the following acryonym: SWIM.

Minor spills In the event that there is a large spill, breakage of aquaria, or other toxin-involved accidents we will practice the following measures (acryonym: SWIM):

Stop other activities. (Seek emergency help immediately in case of injury. Evacuate if necessary.) Warn your coworkers that there has been a toxic spill. Isolate the damage to prevent further contamination if possible. Minimize by diluting the toxin if in the containment tubs, and flush through the disposal system with soap and water. Use towels to clean up, double bag for incineration or autoclave. Clear broken glass to a box, double bag for autoclave. Evaluate the spill to be sure clean up is complete, and before leaving the area.

Lastly, provide timely notification of spill to Principal Investigator, and Safety Officer immediately after clean up.

Major spill with evacuation In the event that there is a large spill with injury or spill of catastrophic proportions we will practice the following safety measures (acryonym: SWIM)

Stop other activities. (Seek emergency help immediately in case of injury. Evacuate if necessary.) Warn your coworkers that there has been a toxic spill. Notify first link in phone chain (Safety Officer or Principal Investigator). Evacuate if damage is extensive. Isolate the damage to prevent further contamination if possible. Minimize by diluting the toxin if in the containment tubs, and flush through the disposal system with soap and water. Use towels to clean up, double bag for incineration or autoclave. Clear broken glass to a box, double bag for autoclave. Evaluate the spill to be sure clean up is complete, and before leaving the area.

Safety shower for 15 minutes followed if necessary by an emergency shower under the guidance of the Safety Office. Timely notification to Director of Research and Advisory Services, and Dean/Director of VIMS.

STANDARD DECONTAMINATION/SPILL Appropriate procedures for handling toxic cultures of Pfiesteria have not been well established. We have adopted procedures from the Centers for Disease Control and Prevention (http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ai.htm). In addition, we will follow procedures recommend for decontamination of field equipment (Hobbs memo), and standard practices used in BSL 2 and 3 containment. For field gear and large equipment items, contaminated surfaces

6 and gear are washed in soap and water (using a deck-mounted shower) and thoroughly rinsed. Cleaned gear is toweled off, then allowed to air dry in the rack space adjacent to the shower. Gloves, boots, aprons, and other non-permeable gear will be dipped in 1% bleach and air dried. Semi-permeable protective clothing (Tyvek suits) will be bagged in the Exposure Room, double- bagged in the Preparation Room, and moved to the holding room (CBH B30) prior to incineration. Dead fish will be handled as above, but placed in the freezer in CBH B30 prior to incineration. Respirators will be cleaned and decontaminated as per manufacturer's instructions, and, if necessary, as above. Filter cartridges will be replaced biweekly or when wet.

Aquaria with live cultures are decontaminated with bleach. Bleach is added directly to the media to make an approximate 5-10% solution, incubated for 30-60 min, and neutralized with sodium thiosulfate. The media is then flushed down the sink to a sewer line.

Toxic Exposure Personal protective equipment should limit skin and inhalation contact with toxic materials. If contact with the skin is made with a toxic or suspect toxic culture, the exposed person must shower immediately and for at least 15 minutes. Soap and water washes will be used for decontamination. Respirators will limit inhalation of toxic materials. A doctor will be sought immediately in case of inhalation, or if a toxic exposure results in skin lesions or unusual behaviors. The Safety Office, Director of Research and Advisory Services, and Dean/Director of VIMS will be notified in case of toxic exposure.

Waste materials A relatively low volume of solid toxic waste will be generated. Solid waste consisting of plasticware, toweling, dead fish and shellfish, etc. will be double-bagged in Biohazard-labeled trash bags for incineration or autoclaving. Tissue waste will be "double-bagged" as above, and transported to a storage freezer in CBH B30 until removed for commercial incineration.

Undressing To maintain a safe work area, a rigid protocol must be adhered to when leaving the Primary Exposure Room to the Preparation Area. (1) Visual check of safety gear for moisture. (2) Any wet gear must be washed with soap and water in the shower area before removal. (3) While in the shower area, the gear is blotted dry with cloth towels. (4) Step out of shower area, into changing area, remove boots, etc. but leave respirator and booties on. (5) Cleaned gear is placed in the rack adjacent to the shower area to dry. (6) Tyvek clothing is discarded into an adjacent Biohazard bag. (7) Exit while wearing respirator. [Note that the unidirectional air flow will ensure that the rack area remains uncontaminated.] (8) Remove respirator after exiting.

7 SECONDARY EXPOSURE ROOM AND WATER MAINTENANCE AREA This room has the greatest risk of exposure as the air flow from the entire facility is directed here. All of the above safety standards apply to entry into this room.

The Secondary Exposure Room contains two large (175 gal.) reservoir water tanks for the artificial sea water, pump, an ISCO Water Sampler, the HEPA filter for the facility, and one bank of aquaria (in a containment tub). Artificial sea water is mixed in the large tanks, and used as needed in the aquaria. A small pump is used to dispense the sea water. Aerators are available for use, but only in the large tanks. Covers over the tanks limit potential contamination. The ISCO water sampler may be used for timed sampling of toxic cultures. It will be decontaminated using bleach washes as described above.

SCHEDULE FOR MAINTENANCE AND REVIEW OF SAFETY PROCEDURES

Item Schedule

Check magnahelic gauge (for HEPA filters) in facility daily

Change prefilters in facility monthly

Change HEPA filters in facility annually as needed

Certification of biosafety cabinet annually

Check air flow in biosafety cabinet weekly

Check and cycle emergency generator weekly

Check building air flow for maintenance of one-way flow monthly

Change respirator cartridges weekly or after 8 hrs

Plan and undertake mock emergency exercises monthly

Checklist for training annually

Full review of safety procedures and protocols 3 months

8 Literature cited Burkholder J.M., and H.B. Glasgow, Jr. 1997. Pfiesteria piscicida: a new and expanding problem in the Eastern US – Biology, life cycle, and general toxicity. Research on Toxic Algae: Pfiesteria-like Organisms Burkholder, J.M., E.J. Noga, C.W. Hobbs, H.B. Glasgow Jr. and S.A. Smith. 1992. New "phantom" dinoflagellate is the causative agent of major estuarine fish kills. Nature 358:407-410; 360:768.

Burkholder, J.M., H.B. Glasgow Jr., and C.W. Hobbs. 1995. Fish kills linked to a toxic ambush- predator dinoflagellate: distribution and environmental conditions. Mar. Ecol. Prog. Ser. 124: 43-61.

CDC, 1998, Federal Register, 63 (13): 3139; from a CDC Workshop, 1997, http://www.cdc.gov/nceh/press/1997/971003pf.htm. CDC, 1999, Biosafety in microbiological and biomedical laboratories. HHS. http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm CDC, 1999, guidelines for work with toxins of biological origin, (http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ai.htm Glasgow Jr H.B., J.M.Burkholder, D.E. Schmechel, P.A. Tester, P.A. Rublee. 1995. Insidious effects of a toxic estuarine dinoflagellate on fish survival and human health. J Toxicol Environ Health 46:101-122.

Levin, E.D., Schmechel, D.E., Burkholder, J.M., Glasgow, H.B., Jr., Deamer-Melia, N.J., Moser, V.C., Harry, G.J. 1997. Pfiesteria piscicida exposure in rats results in persisting learning deficits. Research on Toxic Algae: Pfiesteria-like Organisms. McClellan-Green, P.D., Noga, E.J., Baden, D., Jaykus, L.A., Green, D.P. 1997. Morphological and cytological effects of Pfiesteria piscicida toxin(s). Research on Toxic Algae: Pfiesteria-like Organisms.

9 APPENDICES

BIOSAFETY LEVEL 3 Excerpted from http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

BIOSAFETY LEVEL 3 is applicable to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents which may cause serious or potentially lethal disease as a result of exposure by the inhalation route. Laboratory personnel have specific training in handling pathogenic and potentially lethal agents, and are supervised by competent scientists who are experienced in working with these agents.

All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets or other physical containment devices, or by personnel wearing appropriate personal protective clothing and equipment. The laboratory has special engineering and design features.

It is recognized, however, that many existing facilities may not have all the facility safeguards recommended for Biosafety Level 3 (e.g. access zone, sealed penetrations, and directional airflow, etc.). In these circumstances, acceptable safety may be achieved for routine or repetitive operations (e.g. diagnostic procedures involving the propagation of an agent for identification, typing, and susceptibility testing) in Biosafety Level 2 facilities. However, the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 must be rigorously followed. The decision to implement this modification of Biosafety Level 3 recommendations should be made only by the laboratory director.

The following standard and special safety practices, equipment and facilities apply to agents assigned to Biosafety Level 3:

A. Standard Microbiological Practices 1. Access to the laboratory is limited or restricted at the discretion of the laboratory director when experiments are in progress.

2. Persons wash their hands after handling infectious materials and animals, after removing gloves, and when they leave the laboratory.

3. Eating, drinking, smoking, handling contact lenses, and applying cosmetics are not permitted in the laboratory. Persons who wear contact lenses in laboratories should also wear goggles or a face shield. Food is stored outside the work area in cabinets or refrigerators designated for this purpose only.

4. Mouth pipetting is prohibited; mechanical pipetting devices are used.

5. All procedures are performed carefully to minimize the creation of aerosols.

6. Work surfaces are decontaminated at least once a day and after any spill of viable material.

7. All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are to be placed in a durable, leakproof container and closed for transport from

i the laboratory. Materials to be decontaminated at off-site from the laboratory are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility.

8. An insect and rodent control program is in effect.

B. Special Practices 1. Laboratory doors are kept closed when experiments are in progress.

2. The laboratory director controls access to the laboratory and restricts access to persons whose presence is required for program or support purposes. For example, persons who are immunocompromised or immunosuppressed may be at risk of acquiring infections. Persons who are at increased risk of acquiring infection or for whom infection may be unusually hazardous are not allowed in the laboratory or animal rooms. The director has the final responsibility for assessing each circumstance and determining who may enter or work in the laboratory.

3. The laboratory director establishes policies and procedures whereby only persons who have been advised of the potential biohazard, who meet any specific entry requirements (e.g., immunization), and who comply with all entry and exit procedures, enter the laboratory or animal rooms.

4. When infectious materials or infected animals are present in the laboratory or containment module, a hazard warning sign, incorporating the universal biohazard symbol, is posted on all laboratory and animal room access doors. The hazard warning sign identifies the agent, lists the name and telephone number of the laboratory director or other responsible person(s), and indicates any special requirements for entering the laboratory, such as the need for immunizations, respirators, or other personal protective measures.

5. Laboratory personnel receive the appropriate immunizations or tests for the agents handled or potentially present in the laboratory (e.g., hepatitis B vaccine or TB skin testing).

6. Baseline serum samples are collected and stored for all laboratory and other at-risk personnel. Additional serum specimens may be collected periodically, depending on the agents handled or the function of the laboratory.

7. A biosafety manual is prepared or adopted. Personnel are advised of special hazards and are required to read and to follow instructions on practices and procedures.

8. Laboratory personnel receive appropriate training on the potential hazards associated with the work involved, the necessary precautions to prevent exposures, and the exposure evaluation procedures. Personnel receive annual updates, or additional training as necessary for procedural changes.

9. The laboratory director is responsible for insuring that, before working with organisms at Biosafety Level 3, all personnel demonstrate proficiency in standard microbiological practices and techniques, and in the practices and operations specific to the laboratory facility. This might include prior experience in handling human pathogens or cell cultures, or a specific training program provided by the laboratory director or other competent scientist proficient in safe microbiological practices and techniques.

ii 10. A high degree of precaution must always be taken with any contaminated sharp items, including needles and syringes, slides, pipettes, capillary tubes, and scalpels. Needles and syringes or other sharp instruments should be restricted in the laboratory for use only when there is no alternative, such as parenteral injection, phlebotomy, or aspiration of fluids from laboratory animals and diaphragm bottles. Plasticware should be substituted for glassware whenever possible.

a. Only needle-locking syringes or disposable syringe-needle units (i.e., needle is integral to the syringe) are used for injection or aspiration of infectious materials. Used disposable needles must not be bent, sheared, broken, recapped, removed from disposable syringes, or otherwise manipulated by hand before disposal; rather, they must be carefully placed in conveniently located puncture-resistant containers used for sharps disposal. Non-disposable sharps must be placed in a hard-walled container for transport to a processing area for decontamination, preferably by autoclaving.

b. Syringes which re-sheathe the needle, needle-less systems, and other safe devices should be used when appropriate.

c. Broken glassware must not be handled directly by hand, but must be removed by mechanical means such as a brush and dustpan, tongs, or forceps. Containers of contaminated needles, sharp equipment, and broken glass should be decontaminated before disposal, according to any local, state, or federal regulations.

11. All manipulations involving infectious materials are conducted in biological safety cabinets or other physical containment devices within the containment module. No work in open vessels is conducted on the open bench.

12. Laboratory equipment and work surfaces should be decontaminated with an appropriate disinfectant on a routine basis, after work with infectious materials is finished, and especially after overt spills, splashes, or other contamination with infectious materials. Contaminated equipment should also be decontaminated before it is sent for repair or maintenance or package for transport in accordance with applicable local, state, or federal regulations, before removal from the facility. Plastic-backed paper toweling used on non-perforated work surfaces within biological safety cabinets facilitates clean-up.

13. Cultures, tissues, or specimens of body fluids are placed in a container that prevents leakage during collection, handling, processing, storage, transport, or shipping.

14. All potentially contaminated waste materials (e.g., gloves, lab coats, etc.) from laboratories or animal rooms are decontaminated before disposal or reuse.

15. Spills of infectious materials are decontaminated, contained and cleaned up by appropriate professional staff, or others properly trained and equipped to work with concentrated infectious material.

iii 16. Spills and accidents which result in overt or potential exposures to infectious materials are immediately reported to the laboratory director. Appropriate medical evaluation, surveillance, and treatment are provided and written records are maintained.

17. Animals and plants not related to the work being conducted are not permitted in the laboratory.

C. Safety Equipment (Primary Barriers) 1. Properly maintained biological safety cabinets are used for all manipulation of infectious materials.

2. Outside of a BSC, appropriate combinations of personal protective equipment are used (e.g., special protective clothing, masks, gloves, face protection, or respirators), in combination with physical containment devices (e.g., centrifuge safety cups, sealed centrifuge rotors, or containment caging for animals).

3. This equipment must be used for manipulations of cultures and of those clinical or environmental materials which may be a source of infectious aerosols; the aerosol challenge of experimental animals; harvesting of tissues or fluids from infected animals and embryonated eggs, and necropsy of infected animals.

4. Face protection (goggles and mask, or faceshield) is worn for manipulations of infectious materials outside of a biological safety cabinet.

5. Respiratory protection is worn when aerosols cannot be safely contained (i.e., outside of a biological safety cabinet), and in rooms containing infected animals.

6. Protective laboratory clothing such as solid-front or wrap-around gowns, scrub suits, or coveralls must be worn in, and not worn outside, the laboratory. Reusable laboratory clothing is to be decontaminated before being laundered.

7. Gloves must be worn when handling infected animals and when hands may contact infectious materials and contaminated surfaces or equipment. Disposable gloves should be discarded when contaminated, and never washed for reuse.

D. Laboratory Facilities (Secondary Barriers) 1. The laboratory is separated from areas which are open to unrestricted traffic flow within the building. Passage through two sets of self-closing doors is the basic requirement for entry into the laboratory from access corridors or other contiguous areas. A clothes change room (shower optional) may be included in the passage way.

2. Each laboratory contains a sink for handwashing. The sink is foot, elbow, or automatically operated and is located near the laboratory exit door.

3. The interior surfaces of walls, floors, and ceilings are water resistant so that they can be easily cleaned. Penetrations in these surfaces are sealed or capable of being sealed to facilitate decontamination.

iv 4. Bench tops are impervious to water and resistant to acids, alkalis, organic solvents, and moderate heat.

5. Laboratory furniture is sturdy, and spaces between benches, cabinets, and equipment are accessible for cleaning.

6. Windows in the laboratory are closed and sealed.

7. A method for decontaminating all laboratory wastes is available, preferably within the laboratory (i.e., autoclave, chemical disinfection, incineration, or other approved decontamination method).

8. A ducted exhaust air ventilation system is provided. This system creates directional airflow that draws air from "clean" areas into the laboratory toward "contaminated" areas. The exhaust air is not recirculated to any other area of the building, and is discharged to the outside with filtration and other treatment optional. The outside exhaust must be dispersed away from occupied areas and air intakes. Laboratory personnel must verify that the direction of the airflow (into the laboratory) is proper.

9. The High Efficiency Particulate Air (HEPA)-filtered exhaust air from Class II or Class III biological safety cabinets is discharged directly to the outside or through the building exhaust system. If the HEPA-filtered exhaust air from Class II or III biological safety cabinets is to be discharged to the outside through the building exhaust air system, it is connected to this system in a manner (e.g., thimble unit connection)136 that avoids any interference with the air balance of the cabinets or building exhaust system. Exhaust air from Class II biological safety cabinets may be recirculated within the laboratory if the cabinet is tested and certified at least every twelve months.

10. Continuous flow centrifuges or other equipment that may produce aerosols are contained in devices that exhaust air through HEPA filters before discharge into the laboratory.

11. Vacuum lines are protected with liquid disinfectant traps and HEPA filters, or their equivalent, which are routinely maintained and replaced as needed.

12. An eyewash facility is readily available.

v BMBL Appendix I (http://www.cdc.gov/od/ohs/biosfty/bmbl4/b4ai.htm)

GUIDELINES FOR WORK WITH TOXINS OF BIOLOGICAL ORIGIN

In recognition of the growing number of microbiological and biomedical laboratories working with toxins of biological origin, the following is provided as a guideline for working with these toxins. The material below is adapted from the Biological Defense Safety Program, Technical Safety Requirements (DA Pamphlet 385-69) (1) and Appendix A of the United States Department of Labor Occupational Safety and Health Association rule "Occupational Exposure to Hazardous Chemicals in Laboratories'' (2). Laboratory managers and facility safety officials are encouraged to utilize the references listed below and to consult with subject matter experts before using any toxin, to ensure that appropriate facilities, containment equipment, policies and procedures, personnel training programs and medical surveillance protocols specific to the toxin and the laboratory are in place.

General The laboratory facilities, equipment, and procedures appropriate for work with toxins of biological origin must reflect the intrinsic level of hazard posed by a particular toxin as well as the potential risks inherent in the operations performed. If both toxins and infectious agents are used, both must be considered when containment equipment is selected and policies and procedures are written. If animals are used, animal safety practices must also be considered. Standard Practices Standard practices listed under BSL 2 and BSL 3 (pages 20 and 27) should be reviewed and incorporated as appropriate into protocols for work with toxins.

Special Practices Special practices listed under BSL 2 and BSL 3 (pages 21 and 28) should be reviewed and incorporated as appropriate into protocols for work with toxins.

1. Each laboratory should develop a chemical hygiene plan specific to the toxin(s) used in that laboratory. The chemical hygiene plan should 1) identify the hazards that will be encountered in normal use of the toxin, and those that could be encountered in case of a spill or other accident, and 2) specify the policies and practices to be used to minimize risks (e.g., containment and personal protective equipment, management of spills, management of accidental exposures, medical surveillance).(3) 2. Training specific to the toxin(s) used should be required and documented for all laboratory personnel working with toxins, before starting work with the toxin and at intervals thereafter. 3. An inventory control system should be in place. 4. Toxins should stored in locked storage rooms, cabinets, or freezers when not in use. 5. Access to areas containing toxins should be restricted to those whose work assignments require access. 6. Preparation of primary containers of toxin stock solutions and manipulations of primary containers of dry forms of toxins should be conducted in a chemical fume hood, a glove box, or a biological safety cabinet or equivalent containment system approved by the safety officer. HEPA and/or charcoal filtration of the exhaust air may be required, depending on the toxin. 7. The user should verify inward airflow of the hood or biological safety cabinet before initiating work.

vi 8. All work should be done within the operationally effective zone of the hood or biological safety cabinet. 9. When toxins are in use, the room should be posted to indicate "Toxins in UseAuthorized Personnel Only." Any special entry requirements should be posted on the entrance(s) to the room. Only personnel whose presence is required should be permitted in the room while toxins are in use. 10. All high risk operations should be conducted with two knowledgeable individuals present. Each must be familiar with the applicable procedures, maintain visual contact with the other, and be ready to assist in the event of an accident. 11. Before containers are removed from the hood, cabinet, or glove box, the exterior of the closed primary container should be decontaminated and placed in a clean secondary container. Toxins should be transported only in leak/spill-proof secondary containers. 12. Contaminated and potentially contaminated protective clothing and equipment should be decontaminated using methods known to be effective against the toxin before removal from the laboratory for disposal, cleaning or repair. If decontamination is not possible/practical, materials (e.g., used gloves) should be disposed of as toxic waste. Materials contaminated with infectious agents as well as toxins should also be autoclaved or otherwise rendered non-infectious before leaving the laboratory. 13. The interior of the hood, glove box, or cabinet should be decontaminated periodically, for example, at the end of a series of related experiments. Until decontaminated, the hood, box, or cabinet should be posted to indicate that toxins are in use, and access to the equipment and apparatus restricted to necessary, authorized personnel.

Safety Equipment The safety equipment guidelines listed under BSL 2 and BSL 3 (see Section III) should be reviewed and incorporated as appropriate into protocols for work with toxins.

1. When using an open-fronted fume hood or biological safety cabinet, protective clothing, including gloves and a disposable long-sleeved body covering (gown, laboratory coat, smock, coverall, or similar garment) should be worn so that hands and arms are completely covered. 2. Eye protection should be worn if an open-fronted containment system is used. 3. Other protective equipment may be required, depending on the characteristics of the toxin and the containment system. For example, use additional respiratory protection if aerosols may be generated and it is not possible to use containment equipment or other engineering controls. 4. When handling dry forms of toxins that are electrostatic: a. Do not wear gloves (such as latex) that help to generate static electricity b. Use glove bag within a hood or biological safety cabinet, a glove box, or a class III biological safety cabinet. 5. When handling toxins that are percutaneous hazards (irritants, necrotic to tissue, or extremely toxic from dermal exposure), select gloves that are known to be impervious to the toxin. 6. Consider both toxin and diluent when selecting gloves and other protective clothing.

If infectious agents and toxins are used together in an experimental system, consider both when selecting protective clothing and equipment. Laboratory Facilities Laboratory facility recommendations listed under BSL 2 and BSL 3 (See Section III) and OSHA standards(4) should be reviewed and incorporated as appropriate into protocols for work with toxins. When vacuum lines are used with systems containing toxins, they should be protected with a HEPA filter to prevent entry of toxins into the lines. Sink drains should be similarly protected when water aspirators are used.

vii References

1. Department of the Army, DOD. 32 CFR Parts 626, 627 Biological Defense Safety Program. 2. United States Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910 - Occupational Safety and Health Standards. 3. United States Department of Labor, Occupational Safety and Health Administration. 29 CFR Part 1910. 4. United States Department Occupational Safety and Health Administration. 29 CFR Part 1910.

Additional References

American Industrial Hygiene Association - Biosafety Reference Manual. Heinsohn P.A.; Jacobs R.R.; Concoby B.A. (eds.). American Industrial Hygiene Association, Fairfax. 1995. National Research Council. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. National Academy Press, Washington D.C. 1995. CRC handbook of Toxicology. Derelanko M.J., Hollinger M.A. (eds.). CRC Press, Boca Raton 1995. Ellenhorn's Medical Toxicology: Diagnosis and Treatment of Human Poisoning. Ellenhorn M.J., Schonwald S., Ordog G., Wasserberger J..Williams and Wilkins, Baltimore 1997. CDC Home PageOHS Internet Home PageBiosafety DocumentsBMBL Table of Contents

viii EXAMPLE METHODS FOR EXPERIMENTAL EXPOSURES

A series of exposure studies will be undertaken to document, test, and validate the toxicity of various samples, and cultures. Samples to be tested include isolates from non-toxic cultures, and water and sediment specimens taken from "hot spots" within the Great Wicomico River, other locations in the river, and elsewhere in Virginia. Standardized exposures will consist of placing ten fish in aquaria (38 L, containing artificial seawater at 15 ppt in de-ionized water, Whisper filters filled with autoclaved calcite and aquarium floss, 20º C) previously inoculated with isolates from culture, water or sediment samples. Fish will be fed regularly, and monitored daily for the appearance of lesions, other signs of morbidity, and mortality. After eight weeks, any sample not resulting in overt toxic pathology will be assessed as nontoxic. Controls will consist of fish in replicate treatments using spiked samples (with toxic forms of Pfiesteria or Pfiesteria-like organisms) (these will be housed in the Exposure areas), and fish in aquaria without inoculants (unexposed controls).

Initially, mummichogs, striped bass hybrids, and tilapia will be used to investigate the toxicity of samples. The species of fish will be standardized after controlled exposures to assess pathology, morbidity, and mortality to cultures that are known to produce toxins. Two sets of experiments will determine which fish species is most susceptible to the development of lesions, and/or mortality. In the first experiment, 10 fish of each species will be placed together in an aquarium containing known quantities of toxin producers (as estimated below). The time course of the outbreak, and the first species to show overt signs of distress will be monitored. In the second experiment, 10 fish of the same species will be placed together in an aquarium containing known quantities of toxin producers. The time course of the outbreak, and the time to first kill will be monitored. Each experiment will be replicated six times in individual aquaria. Data on time to the occurrence of lesions, time to overt signs of morbidity and mortality will be recorded. Histological specimens will be taken to further aid in the standardization procedure, and to provide a detailed look at the pathology associated with acute exposures.

Since menhaden appear to have a higher prevalence of lesions than other fish, juvenile menhaden will be used in exposure studies with cultures known to produce toxins. Menhaden will be housed in circular tanks with a low recirculating velocity of water, or in covered tanks with aeration. Exposures will be undertaken as above.

Cell densities of heterotrophic dinoflagellates will be monitored regularly. Water (dinospore) and bottom (cyst) samples of known volume (2 ml) will be collected, and filtered. Heterotrophic forms will be counted while unstained or stained (calcein, DAPI) with an epifluorescence microscope. Toxicity will be measured as cell densities per mL of water.

Established cultures of toxic stages will be used to develop an assay to locate and identify cysts in spiked sediment samples. Two different approaches will be followed. First, sediments in aquaria will be spiked with known quantities of cysts (from a toxin producer). Fish bioassays will be run as above. Sediment and water samples will be processed at regular intervals to monitor cell growth. Second, cysts will be harvested from toxic cultures and inoculated into preweighed sediment samples. The sediments will then be processed and enumerated using density centrifugation, or flotation.

ix TRAINING CHECKLIST FOR PFIESTERIA TOXICITY STUDIES AT VIMS

Microbiological methods

1. Personal protective equipment (PPE) 2. Sterilization techniques 3. Autoclaves 4. Aseptic techniques 5. Sterile field 6. Sterile culture 7. Biosafety hoods 8. Biological wastes 9. Biosafety level 3 practices

Hazardous materials methods

1. Personal protective equipment (PPE) (latex vs. nitrile vs. polypropylene) 2. Classification and types of hazardous materials 3. Ventilation & fume hoods 4. Respirators 5. Handling hazardous materials 6. Hazardous wastes 7. Biosafety level 3 practices 8. Emergency responses

Additional training

1. Hazard Communication Standard Training (general lab safety) 2. HazMat Awareness at VIMS 3. HazMat Operations at VIMS (optional)

Signed: Dated:

Supervisor: Dated: