standard operating procedures safe autoclave …the purpose of this document is to provide standard...

EH&S. July 2015 Contact Information: 480-965-5389 or email

Department of Environmental Health & Safety Biosafety & Biosecurity

STANDARD OPERATING PROCEDURES Safe Autoclave Operations The purpose of this document is to provide standard operating procedures for the safe use of autoclaves. Autoclaving is a process used to destroy microorganisms and decontaminate biohazardous waste and microbiological equipment used at Biosafety Level 1, 2, 3 and 4.

Autoclave Make/Model:

Person Responsible and Phone #:

Located in Building/Room: HAZARDS Autoclaves use high pressure and high temperature steam for sterilization. The potential safety risks for the operators include:

x Heat burns from hot materials and autoclave chamber walls and door. x Steam burns from residual steam coming out from autoclave and materials on completion of cycle. x Hot fluid scalds from boiling liquids and spillage in autoclave. x Hand and arm injuries when closing the door. x Body injury if there is an explosion.

SAFETY To insure the health and safety of personnel using the autoclave, it is important for each department to maintain autoclaves and to train personnel in their proper use.

x The name of the person responsible for the autoclave shall be posted near the autoclave. This SOP should be posted on the outside to the autoclave.

x It is the supervisor's responsibility to ensure employees are trained before operating any autoclave unit. x Procedural and instructional documents provided by the manufacturer must be followed. x Personal protective clothing and equipment must be worn when loading and unloading the autoclave. x Autoclaves must be inspected at least annually. Inspection services may be managed by your

manufacturer’s preventative maintenance contract. A basic visual inspection should be performed monthly by the person responsible for the autoclave. The inspection, service and repair records should available upon request.

x Spore strips may be used to validate autoclave effectiveness. PERSONAL PROTECTIVE EQUIPMENT Equipment to protect against scalds and burns include:

x Heat-insulating gloves that provide complete coverage of hands and forearm x Lab coat x Eye protection x Closed-toe footwear

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David Nielsen

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OPERATOR INSTRUCTIONS Training All personnel who use autoclaves must have successfully completed a training session from their supervisor on the safe operating procedures. This requirement applies to both new and experienced personnel. Material Preparation Ensure that the material is safe for autoclaving:

x Samples containing solvents or substances that may emit toxic fumes should not be autoclaved. x Do not autoclave bleach!

Glassware must be inspected for cracks prior to autoclaving. Prepare and package material suitably:

x Loose dry materials must be wrapped or bagged in steam-penetrating paper or loosely covered with aluminum foil. Wrapping too tightly will impede steam penetration, decreasing effectiveness of the process.

x Loosen all lids to prevent pressure buildup. All containers must be covered by a loosened lid or steam-penetrating bung.

x Containers of liquid must not exceed two-thirds (2/3) full, with lids loosened. x Glassware must be heat-resistant borosilicate. x Plastics must be heat-resistant, i.e., polycarbonate (PC), PTFE (“Teflon”) and most polypropylene (PP)

items. x Discarded sharps must be in a designated ‘Sharps’ container. x All items must be tagged with autoclave tape.

Place items in secondary containers to secure and contain spills:

x Items should be placed in a stainless steel pan or other autoclavable container for their stability and ease of handling.

x Place containers of liquid, bags of agar plates, or other materials that may boil over or leak, into a secondary pan in the autoclave.

x The pan must be large enough to contain a total spill of the contents. x Bags must not be tightly sealed as steam cannot penetrate.

Biohazardous waste must be processed according to ASU guidelines. Loading Autoclave

x Wear lab coat, eye protection, heat-insulating gloves, and closed-toe shoes. x Place material in autoclave. Do not mix incompatible materials. x Do not overload; leave sufficient room for steam circulation. If necessary, place the container on its side

to maximize steam penetration and avoid entrapment of air. x Close and latch door firmly.



Operating Autoclave

x Close and lock door. x Choose appropriate cycle (e.g., gravity, liquid, or dry cycle) for the material. Consult the autoclave

manual for assistance in choosing a cycle. The manuals for operation of the autoclave should be located near the autoclave.

x Set appropriate time and temperature if you are using a customized cycle. x Start your cycle and fill out the autoclave user log with your contact information. A completed cycle

usually takes between 1-1.5 hours, depending on type of cycle. x Do not attempt to open the door while autoclave is operating. x If problems with your autoclave are perceived, abort cycle and report it to your PI immediately.

Unloading Autoclave

x Wear heat-insulating gloves, eye protection, lab coat, and closed-toe shoes. x Ensure that the cycle has completed and both temperature and pressure have returned to a safe range. x Wearing Personal Protective Equipment (PPE), stand back from the door as a precaution and carefully

open door no more than 1 inch. This will release residual steam and allow pressure within liquids and containers to normalize.

x Allow the autoclaved load to stand for 10 minutes in the chamber. This will allow steam to clear and trapped air to escape from hot liquids, reducing risk to operator.

x Do not agitate containers of super-heated liquids or remove caps before unloading. x Wearing heat-insulated gloves remove items from the autoclave and place them in an area which clearly

indicates the items are ‘hot’ until the items cool to room temperature. x Shut autoclave door.

Autoclave Use Log

x Entries must be placed in the log book each time the autoclave is used. These records are used for maintenance/service schedules and reporting of incidents, accidents and/or faults.

x Entries should include: operator's name, phone number, date, time and duration. x The log book must be kept adjacent to the autoclave. x An Autoclave Use Log example is provided in this document.

Maintenance and Repair

x No person shall operate the autoclave unless the autoclave is in good repair. x Only qualified professionals are permitted to make repairs. x Report possible malfunctions to . x Repairs are performed by your service contract or any other contractor you choose to hire.

CONTINGENCY PLANS Equipment Malfunction

x If the autoclave does not operate exactly as expected, do not attempt to fix the problem. A notice shall be placed on the autoclave indicating that it is not to be used until the problem is diagnosed and corrected.

x Record the problem in the autoclave log book. x Contact or your supervisor to report the problem. x Only qualified professionals are permitted to make repairs.



Incident Response

x All incidents, including a spill or release of biohazardous materials and recombinant or synthetic nucleic acid molecules, must be reported to your supervisor and Biosafety & Biosecurity.

x If any injury occurs seek first aid or, if necessary, seek medical assistance at ASU Health Services or by dialing 911.

x If clothing is soaked in hot water/steam, remove clothing and place the injury in cool water. x Place a notice on the autoclave indicating that it is not to be used until the cause of the incident is

determined, procedures enacted to prevent future incidents, and the autoclave is deemed safe for operation.

Spill Clean-up

x Spills may occur from a boil-over or breakage of containers. x No operation of the autoclave is allowed until the spill is cleaned up. x The operator is responsible for clean-up of spills. Contain the spilled material using paper towels. Use

your laboratory’s spill kit if necessary. Wait until the autoclave and materials have cooled to room temperature before attempting clean-up.

x Review the Safety Data Sheet if appropriate, to determine appropriate PPE, spill cleanup and disposal protocols that are necessary.

x Dispose of the waste following the protocol appropriate for the material (e.g., red biohazard bag). If materials have been intermingled, follow the clean-up and disposal protocol for the most hazardous component of the mixture.

x Cracked glassware must be disposed of properly. x Record the spill and clean-up procedure in the autoclave log book.


ASU Autoclave Training Video The EH&S Biosafety/Biosecurity team is pleased to introduce the ASU Autoclave Training Video. The video provides instructions on how to properly load and unload an autoclave, the requisite personal protective equipment to be worn, the waste disposal procedures to be followed, and other important autoclave safety topics. For your convenience, this training video is now available on Blackboard in five separate modules: Module 1: Overview of Autoclaves Module 2: Preparing for Autoclave Use Module 3: Loading the Autoclave Module 4: Operating the Autoclave Module 5: Unloading the Autoclave This training video is intended to serve as supplemental instruction to hands-on training and is recommended for all individuals using autoclaves at ASU. Please be aware that individuals must receive hands-on training for the autoclaves assigned to their laboratory before using them. Consult with your laboratory manager for this hands-on training. To self-enroll in the Blackboard training, please click on either of the following links. To receive credit for completing this training, please watch the video modules and take the corresponding test in Blackboard. A score of 80% or higher is required to pass the test. EH&S Training Determination Link: https://cfo.asu.edu/ehs-training Self-Enrollment Link: http://www.asu.edu/go/blackboard/selfenroll/?cid=320120 If proof-of-training is not required for your laboratory, the ASU Autoclave Training Video is also available on YouTube: https://www.youtube.com/watch?v=rM_JTgLSKXk


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Autoclave Use Log Print Name (No initials) Contact Number Date Time Cycle Comments



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STANDARD OPERATING PROCEDURES Biological Safety Cabinets The purpose of this guideline is to detail the safe operation of biological safety cabinets (BSC) in ASU laboratories and to ensure adequate containment of biological materials. The most common type of BSC at ASU is a Class II. Class II BSCs provide the microbe-free work environment necessary for cell culture propagation and handling of infectious organisms.

Biological Safety Cabinet Make/Model:

Person Responsible and Phone #:

Located in Building/Room: DEFINITIONS Biological Safety Cabinet: A piece of equipment designed to protect the operator, the laboratory environment and work materials from exposure to infectious aerosols and splashes that may be generated when manipulating substances containing infectious agents, such as viruses, bacteria and primary tissue cultures. High Efficiency Particulate Air (HEPA) Filter: Filter that traps 99.97% of particles of 0.3 μm in diameter and 99.99% of particles of greater or smaller size, thus capturing all infectious agents and ensuring only microbe-free air is exhausted from the cabinet or directed to the work surface. RESPONSIBILITIES Principal Investigator The Principal Investigator (PI) is responsible for ensuring personnel under their supervision are trained on the safe and proper use of the BSC and that correct procedures are followed. Designated Person The PI may designate a member of the lab to oversee the correct operation and maintenance of the BSC. This person shall:

x Periodically inspect the BSC to ensure its operational performance. x Coordinate yearly BSC certification by the certified vendor. The dates of the most recent certification must

be posted on the front of the BSC. x Make any necessary arrangements for repairs and annual certification of the BSC. x Report to PI unsafe practices by BSC users. x Label the BSC when it is not operating correctly.

BSC Users

x BSC users must receive proper training from their PI on the safe and proper use of the BSC prior to use. x BSC users must follow correct procedures. x BSC users shall report any injuries, accidents or spills to their PI. x BSCs users shall report any defects or malfunctions of the BSC to their PI or the designated person.


David Nielsen

GWC 693

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David Nielsen 5-4113

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Procedures Prior to Operation

x Read the Safety Data Sheets (SDS) for materials being used in a BSC. Note any precautions regarding the use of the chemical or microorganisms in the BSC.

x Lift the sash to the recommended height. x Turn on the BSC fan 10 minutes before beginning work to allow adequate air filtration. x Check the air intake and exhaust grilles for obstructions, and check the pressure gauge reading indicating

HEPA filter load. If you do not know the acceptable reading for the cabinet, ask the certifier or consult your manufacturer’s manual.

x If the cabinet is equipped with an alarm, test the alarm and switch it to the “ON” position. x Confirm inward airflow by holding a piece of tissue at the middle of the edge of the viewing panel and

ensuring it is drawn in. x Decontaminate the cabinet surface and surface-decontaminate all materials to be placed inside the BSC

(refer to the Cleaning and Disinfection section). x Bulky items, such as waste containers and suction collection flasks, should be placed to one side of the

interior of the cabinet. x Keep the work area of the BSC free of unnecessary equipment or supplies. Clutter inside the BSC may

impede proper airflow and the level of protection provided. Consider using wire racks or shelving to increase airflow around front and rear grills.

x Wear eye protection, a lab coat, closed-toe shoes and disposable gloves to protect yourself as well as your samples from contamination.

x Respirators may be required for some procedures. The nature of the Personal Protective Equipment (PPE) worn may vary according to the findings obtained from the biological risk assessment.

Operation

x Active work should flow from clean to contaminated areas across the work surface. x Arms should be moved in and out slowly, perpendicular to the front opening to minimize disruption of the

air curtain and laminar flow. x Work as far to the back as possible, but within comfortable reach. x Always use mechanical pipetting aids. Mouth pipetting is not allowed. x Heat sources such as Bunsen burners are strictly prohibited inside the BSCs as they significantly disrupt

the laminar flow of air. x To sterilize bacteriological loops, micro-burners or electric “furnaces” may be used. x Do not work in a BSC while a warning light or alarm is signaling. x Locate liquid waste traps inside cabinet and use an in-line HEPA filter to protect the vacuum line. If traps

must be located on the floor, place them in a secondary container (such as a durable plastic tray or box) to prevent breakage.

Cleaning and Disinfection

x When work is completed, all equipment and supplies from the BSC should be decontaminated and removed from the cabinet.

x The interior surfaces should also be wiped with an appropriate disinfectant that would kill any microorganisms that could be found in the cabinet. Corrosive chemicals such as bleach should be avoided, but if used, should be followed with a wipe down of sterile water or 70% ethanol.

x Allow the cabinet to run for 10 minutes.



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Ultraviolet (UV) Lights

x Ultraviolet (UV) lamps are not recommended in BSCs nor are they necessary. x If installed, UV lamps must be cleaned weekly to remove any dust and dirt that may block the germicidal

effectiveness of the ultraviolet light. x The lamps should be checked weekly with a UV meter to ensure that the appropriate intensity of UV light

is being emitted. x UV lamps must be turned off when the room is occupied to protect eyes and skin from UV exposure,

which can burn the cornea and cause skin cancer. x If the cabinet has a sliding sash, close the sash when operating the UV lamp.

Spills In the event of a spill within a BSC, follow these steps:

1. Alert the other laboratory employees. 2. Leave the cabinet turned on. 3. While wearing gloves, spray or wipe cabinet walls, work surfaces and equipment with disinfectant

equivalent to 1:10 bleach solution. If necessary, flood the work surface, as well as drain-pans and catch basins below the work surface, with disinfectant for a contact time of at least 20 minutes.

4. Report the spill to the laboratory’s PI, who will report the spill to the Responsible Official if a select agent or toxin is involved.

5. Soak up disinfectant and spill with paper towels. Drain catch basin into a container. Lift front exhaust grill and tray and wipe all surfaces. Ensure that no paper towels or solid debris are blown into the area beneath the grill.

6. Autoclave all clean-up materials before disposal in the biohazard waste container. 7. Wash hands and any exposed surfaces thoroughly after the clean-up procedure.

Other Considerations

x All repairs to the BSC must be performed by a qualified technician. Any malfunction of the BSC must be reported and repaired before the BSC is used again.

x The BSC should be positioned in an isolated corner to minimize disruption of the air intake arising from traffic around the biosafety cabinet or drafts from doors, windows and/or air conditioning.

x The BSC must be certified by an NSF/ANSI Standard 49 qualified technician. x The BSC must be decontaminated before filter changes and before being moved. The most common

decontamination method is by fumigation with formaldehyde gas. BSC decontamination may only be performed by a certified contractor.

x Contact Biosafety & Biosecurity if you have any questions.


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I. PURPOSE 1. This procedure outlines methods for safe handling and disposal of all discarded syringes, needles, or sharps used by research staff and students. II. RESPONSIBILITY 1. It is the responsibility of all program staff to contribute to and comply with these procedures. 2. It is the responsibility of the Principal Investigator to provide and maintain a sufficient number of properly labeled sharps containers in procedural areas throughout the lab and as described in this procedure. III. PROCEDURES All needles, syringes, scalpel blades, disposable instrument, etc. will be disposed of at the point of use in rigid, leak proof sharps containers, labeled as biohazardous waste with the biohazardous symbol and phrase. Always wear gloves and other appropriate personal protective equipment when performing procedures using needles and syringes. Disposable syringe-needle units should be used for the injection or aspiration of infectious/hazardous materials. Upon completion of a procedure involving use of a syringe and needle, discard entire needle and syringe system into a clearly labeled puncture – resistant hazardous waste container located in the work area. Needles should never be recapped, bent, broken, removed, or otherwise manipulated by hand.

Biohazardous Sharps Handling and Disposal

All sharps must be handled and disposed in a manner that protects you and others from exposure and possible injury. This procedure applies to the handling and disposal of sharps and must be followed to: 1. Prevent personal injury 2. Prevent contamination of personnel or the environment. 3. Ensure proper containment of laboratory and infectious waste during collection, transfer, and disposal.

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Sharps are items that can easily puncture the skin. Examples include needles, razor blades, and broken glass. All sharps must be handled and disposed in a manner that protects you and others from exposure and possible injury.

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When a Sharps container is three-fourth’s full, don gloves, seal container, and place in the designated area for collection and disposal. Syringe disposal containers will be checked and changed on an as needed basis. If a needle stick should occur, wash area with soap and water, report incident to your supervisor, and seek medical attention as soon as possible. ALL NEEDLE STICKS WILL BE REPORTED AT ONCE TO YOUR SUPERVISOR.

General Precautions Substitute glassware for plastic-ware whenever possible. Routinely inspect glassware and remove from service items that are damaged, starred, cracked, or chipped. Make sure lighting is adequate and the work space is not crowded for the task at hand. Be alert at all times when handling sharps. Don’t look away or become otherwise distracted while handling a sharp object. Select rounded or blunt end devices when practicable. Do not handle sharp objects (i.e., broken glass) with bare hands. Use mechanical devices. Do not leave unprotected sharps (i.e., razor blades, scalpel tips, etc.) on bench tops or loose in drawers. Use protective shields, cases, Styrofoam blocks, tube holders, etc. Use needle syringes only when absolutely necessary. If a needle syringe is absolutely necessary to the procedure. Do not try to recap the needle of a syringe. An accidental puncture might occur. If absolutely necessary, use the one-handed technique. Keep a sharps disposal container immediately accessible. Read the authorized sharps container manufacturer’s instructions and recommended user training information prior to use. Do not allow it to get overfilled. Do not try to retrieve items from sharps disposal containers. Broken glass must be discarded into a plastic-lined container with the label “Broken Glass”, unless it is contaminated with biological materials. Broken glass contaminated with biological materials must be discarded in a red sharps disposal container.

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Precautions for the Disposal of Sharps Ensure that appropriate sharps disposal containers are available in the immediate work area: The sharps disposal container must be labeled with a biohazard symbol. Sharps disposal containers must be rigid, leak and puncture proof, and sealable. Needles and needle-syringe units, whether infectious or not, must be placed in the sharps disposal container. Sharps disposal containers storing needles and syringes contaminated with biological materials cannot be placed in the normal trash. Sharps disposal container must not be overfilled. This hazard should be reported to the lab manager or PI immediately upon notice. If the sharps container is overfilled, obtain a new container and use forceps or tongs to remove protruding devices and place them in the new container. Make sure the sharps container being used is large enough to accommodate the entire device. Notify EH&S for assistance in removing the hazard, if necessary. Do not try to retrieve items from sharps disposal containers. Never force materials into a sharps container. Safety hazards identified with the sharps disposal container, i.e. needles protruding from the container, needles not freely falling into the container, must be immediately reported to the lab manager or PI. Disposal of Sharps Used with Pathological Agents Ensure that appropriate sharps containers are available in the immediate work area:

• Must be labeled as “Sharps.” • Must be labeled with a biohazard symbol. • Containers must be rigid, leak and puncture proof, and sealable. A commercially

available plastic container that cannot be easily opened after sealing must be used for sharps that are contaminated with potentially infectious materials.

Contaminated sharps cannot be placed in the normal trash until effectively decontaminated, usually by steam autoclaving. Use autoclave indicator tape to demonstrate decontamination.

You may mix non-infectious sharps with potentially infectious sharps only if the entire contents of the container is treated as infectious and it is decontaminated before disposal, as described above. Disposal of Sharps that Have Not Been Used with Pathological Agents Appropriate containers must be available in the immediate work area. Containers must be rigid, leak and puncture proof, sealable, and labeled as “Sharps.”

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I. PURPOSE 1. This procedure outlines methods for safe handling and clean-up of biohazardous liquid spills throughout the lab. As the nature of biohazardous waste spills associated with our research activities will primarily constitute live liquid cultures containing bacteria and yeast and will be of sufficiently small volume (typically < 100 mL and always <3 L) the below information has been tailored to this end. II. RESPONSIBILITY 1. It is the responsibility of all program staff to contribute to and comply with these procedures. 2. It is the responsibility of the facility manager to provide and maintain adequate materials needed for the decontamination and clean-up of biohazardous liquid spills. III. PROCEDURES

1) Minor Biological Spills If the spill occurred inside a biological safety cabinet, close the sash and allow the cabinet to operate for at least 15 minutes before proceeding with the spill cleanup. Alert people in area of spill and secure the affected area.

Biohazardous Liquid Spill Clean-up

The purpose of this guideline is to detail procedures to be utilized as a basis for cleaning spills of biological materials including potentially infectious agents, and/or recombinant or synthetic nucleic acid molecules, including 1) Minor Biological Spills and 2) Major Biological Spills.

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Minor Biological Spill: one that the laboratory staff is capable of handling safely without the assistance of EH&S or external emergency personnel. Minor biological spills involve low risk materials (e.g. BSL-1) or spills of a small volume of material inside a biological safety cabinet or other primary containment device. Major Biological Spill: one that requires assistance of EH&S and/or external emergency personnel. Major Biological Spills involve releases of BSL-2 or higher materials outside of a biological safety cabinet, or spills of such materials that involve excessive splashing or aerosol formation.

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If any material has been splashed on you, immediately wash the exposed area with soap and water. If the exposed area is the eye, flush with water only for 15 minutes. NOTE: It is not advisable to apply alcohol, bleach or other disinfectants to the exposed area. Remove and disinfect any contaminated clothing. If you have not been exposed, don the appropriate personal protective equipment for the cleanup operation, which include at a minimum liquid barrier gloves, a combination of safety glasses or full face shield and surgical mask, and lab coat. Cover the spill with paper towels or other absorbent material to prevent further aerosol formation. Pour an approved disinfectant gently over the covered spill, working from the outside inwards.

• For microbial and yeast cultures, use a solution of 10% bleach in water.

Wait at least 15 minutes for the disinfectant to penetrate through the contained spill and achieve the required contact time for disinfection. Do not utilize hands if glass or other sharps are involved in the spill. Use a tool (e.g. shovel or forceps) to remove the absorbent material and debris. Place all materials in a biohazard bag for autoclaving and subsequent disposal. Clean the original spill area with detergent. Notify your supervisor of the incident. 2) Major Biological Spill Alert personnel in the laboratory of the spill, and eliminate entrance of additional personnel via notification or posting of area. If any material has been splashed on you, immediately wash the exposed area with soap and water. If the exposed area is the eye, flush with water only for 15 minutes. NOTE: It is not advisable to apply alcohol, bleach or other disinfectants to the exposed area. Remove and disinfect any contaminated clothing. Notify your supervisor and ASU Environmental Health and Safety (EH&S). If the situation involves an imminently life-threatening injury call 911. Have persons knowledgeable of incident and laboratory available to assist notifying additional EH&S and/or emergency personnel.

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I. PURPOSE 1. This procedure outlines methods for safe handling and disposal of all biohazardous waste materials generated by research staff in the lab. II. RESPONSIBILITY 1. It is the responsibility of all program staff to contribute to and comply with these procedures. 2. It is the responsibility of the facility manager to provide and maintain adequate and sufficient materials for the safe handling, decontamination and disposal of biohazardous wastes throughout the lab and as described in this procedure. III. PROCEDURES All biological waste in the laboratory will be handled and disposed of in accordance with federal, state, and local regulations as well as University policy. Waste will be chemically disinfected (e.g., bleach, quaternary ammonia compounds), thermally inactivated (e.g., steam autoclave), or collected in biohazard containers for EH&S pickup. Any materials to be decontaminated outside the laboratory will be transported in a durable, leak-proof, closed container. All personnel who handle biological waste will successfully complete “Biosafety and Bloodborne Pathogens Training” and “Hazardous Waste Management Training” through EH&S. Enrollment procedures for these trainings are available here: https://cfo.asu.edu/ehs-training. For the research in our laboratory, we will have liquid and solid biological waste. Procedures for waste management in our laboratory are provided below:

Biohazardous Waste Disposal

The purpose of this guideline is to detail procedures for the proper disposal of biohazardous wastes, including: 1) liquid cultures, 2) solid cultures, and 3) contaminated consumables.

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Biohazardous waste includes any materials containing or that have been in contact with microorganisms, materials derived from microorganisms (e.g., recombinant DNA, RNA, proteins), and/or synthetically-derived nucleic acids or nucleotides, as well as any waste material that is potentially infectious to humans, plants, or animals.

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For liquid biological waste, an appropriate, chemically compatible disinfectant, or steam autoclave will be used to decontaminate the waste. An approved disinfectant (e.g., 10% bleach/water solution - 1 part concentrated bleach to 9 parts water) will be added to the liquid waste to obtain the appropriate final concentration and allowed to sit for at least 30 minutes. Alternatively, a steam autoclave can be used to disinfect the liquid biological waste only if no bleach is present in the solution. If approved for drain disposal, the chemically disinfected solution will be drain disposed and a logbook with the following information will be maintained in the laboratory: A) Date of Drain Disposal; B) Type of Waste; C) Volume of Total Waste Disposed; D) Method of Disinfection; E) pH of Final Solution. If the pH is <5 or >10 then drain disposal is not permitted and the waste will be collected in closable, leakproof containers labeled with a biohazard symbol for EH&S to pick-up. For solid biological waste, a steam autoclave will be used. Waste will be collected in a heat-safe autoclave bag (or heat-safe container) and decontaminated using an appropriate cycle in the autoclave. Please consult http://www.asu.edu/uagc/EHS/documents/autoclave-sop.pdf for additional guidance and procedures for proper autoclave use. The autoclaved waste will be collected in a red biohazard bag and subsequently placed into a red biohazard drum. Prior to having the red drum two-thirds full, laboratory personnel will submit an online waste-pickup request at https://cfo.asu.edu/ehs-environmentalaffairs. If waste is not appropriate for chemical disinfection or steam autoclave, it will be collected in double-lined, red biohazard bags and subsequently placed into a red biohazard drum. Prior to having the red drum two-thirds full, laboratory personnel will submit an online waste-pickup request athttps://cfo.asu.edu/ehs-environmentalaffairs. If animal waste is generated in the laboratory, it will be thermally disinfected in a steam autoclave, placed into a red-biohazard bag, and then placed into a yellow drum. If autoclaving is not suggested for the waste, it will be collected in double-lined, red biohazard bags and then placed into a yellow drum. Prior to having the yellow drum two-thirds full, laboratory personnel will submit an online waste-pickup request at https://cfo.asu.edu/ehs-environmentalaffairs. Any spills, injuries, equipment failures, or waste handling problems will be reported to the PI at (480) 965-4113 and EH&S at (480) 965-1823. The EH&S Biosafety and Environmental Affairs groups will be contacted regarding any final waste determinations not addressed by these procedures. Note: Genetically altered living organisms and their products, and materials containing recombinant DNA or synthetic nucleic acids are also considered to be biohazardous waste and will be handled according to the procedures for solid or liquid biohazardous waste.

All requests for waste pick up can be made through ASU’s EHS Assistant portal.

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I. PURPOSE 1. This procedure outlines methods for safe handling and clean-up of chemical spills throughout the lab. II. RESPONSIBILITY 1. It is the responsibility of all program staff to contribute to and comply with these procedures. 2. It is the responsibility of the facility manager to provide and maintain adequate materials needed for the clean-up of chemical spills. III. PROCEDURES 1) Preventing a Spill The possibility of a spill and preparation for handling it should be anticipated in setting up your experiment. Appropriate precautions will alleviate many associated complications. Before using a hazardous chemical:

• Familiarize yourself with the potential hazards of that chemical. Material Safety Data Sheets (MSDS's) are a valuable source of information. Each laboratory has a notebook of MSDS's of the hazardous chemicals used in that laboratory. Whenever you add a new chemical, you should add the MSDS sheet to that notebook.

• Evaluate the type of toxicity of the hazardous chemical (i.e., corrosive, irritant, sensitizer, carcinogen) and the possible routes of exposure (i.e., inhalation, skin absorption, ingestion, injection). Evaluate hazards of flammable and explosive chemicals.

• Select appropriate procedures to minimize exposure. Wear appropriate eye protection and protective apparel.

• Ask yourself, what is the worst that could happen? Ask yourself if you are prepared to handle such a situation. Do not underestimate risks, and consider substituting less hazardous materials, techniques, and equipment.

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The purpose of this guideline is to detail steps to be taken (1) to prevent the spill of a hazardous chemical, and (2) to respond to a chemical spill that has occurred. (3) Information about spill kits is also given.

Emergency Clean-up of Chemical Spills

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• Be prepared for accidents. Know what specific action you will take in the event of a chemical spill. Know the location of the laboratory spill kit, be familiar with the location of the nearest fire alarm and telephone, and know emergency telephone numbers.

• Have a knowledgeable colleague review your experimental design and safety procedures to judge the adequacy of the precautions and emergency steps.

• Purchase only the amount of hazardous material that will be used within a reasonable period, in the smallest container that is practical.

• Plan the transportation of hazardous materials to avoid heavy traffic areas and times. Use hazardous materials inside the chemical fume hood.

• Use secondary containers, metal cans, or plastic-coated bottles for storing and transporting.

• Do not place glass containers of chemicals on the floor. • Take precautions to avoid fallen or leaking gas cylinders.

(2) What to do when a Spill Occurs Whoever causes a minor spill must clean it up. However, some spills may be too large or too dangerous for laboratory personnel to handle, in which case ASU EH&S will work with you to arrange for a professional spill team to come in. Minor Chemical Spill

• Alert people in the area that a spill has occurred. • Use personal protective equipment (provided in the spill kits located in each

laboratory) to protect yourself during the clean up. Avoid breathing vapors from the spill.

• Avoiding risks of injury or contamination, confine the spill to a small area. Prevent the spill from entering any drains.

• Neutralize/absorb the spill with shakers for acids, caustics, and solvents using the spill kits located in each laboratory.

• Collect residue, place in an appropriate container, label with a tag, and dispose of as hazardous waste. Schedule for a hazardous waste pickup using the EHS Assistant.

• Clean the spill area with water. Major Chemical Spill

• Alert people in the lab that a spill has occurred and ask them to evacuate; close doors to the affected area.

• If spilled material is flammable, turn off ignition and heat sources if it can be done safely.

• Attend to injured or contaminated persons and remove them from exposure. If necessary, request help by calling the Campus Police (480-965-3456), or for life threatening emergencies call 911.

3

• If you feel you have been exposed to any hazardous material, report to ASU Medical for an evaluation.

• Contact ASU EH&S (480-965-1823) for cleanup assistance after caring to affected employees.

• If it can be done safely, block off any drains where the spill may enter. • Have a person knowledgeable about the incident and laboratory standby to

assist emergency personnel. If chemicals are known, provide MSDS' s for the emergency response team.

(3) Spill Kits Each laboratory is equipped with spill kits which contains:

• Personal protective equipment: goggles and gloves that are chemical-resistant. • Spill control pillows and wipes • Disposal bags to collect the spilled material and contaminated clean-up

materials. Be aware of the location of spill kits. They should be checked on a regular basis.

Benzene,CAS#71-43-2

ASUDepartmentofEnvironmentalHealth&Safety Page|1

Standard Operating Procedure

Benzene

Print a copy and insert into your Laboratory Safety Manual and Chemical Hygiene Plan.

Refer to instructions for assistance.

School/Department: SEMTE

SOPPreparationDate: 5/11/2016 SOPApprovalDate: Clickheretoenteradate.

PrincipalInvestigator: DavidNielsen

LabManagerName: Clickheretoentertext.

LaboratoryPhone: Clickheretoentertext. OfficePhone: 5-4113

EmergencyContact: FredPena ContactPhone: 5-4122

LaboratorylocationscoveredbythisSOP(building/roomnumber):

GWC693

TypeofSOP: ☐ Process ☒ HazardousChemical ☐ HazardousClassPurposeBenzeneisanOSHAregulatedcarcinogen.Itisamutagenicflammableliquid.Itisanorganicsolventandprecursorintheproductionofdrugs,plastics,syntheticrubber,polymers,explosives,anddyes.PhysicalandChemicalProperties/DefinitionofChemicalGroupCAS: 71-43-2

Class: Flammableliquid,regulatedcarcinogen,mutagenMolecularFormula: C6H6Form(physicalstate): LiquidColor: ColorlessBoilingPoint: 80.0–80.2°C(176.0–176.4°F)PotentialHazards/ToxicityPotentialHealthEffects

Benzene,CAS#71-43-2


Maycausegeneticdefects,maycausecancer.TargetOrgans: Blood,eyes,femalereproductivesystem,bonemarrowInhalation: Maybeharmfulifinhaled,causesrespiratorytractirritation.Skin: Maybeharmfulifabsorbedthroughskin,causesskinirritation.Eyes: Maycauseeyedamage,causeseyeirritation.

Ingestion: Maybeharmfulifswallowed.Aspirationhazardifswallowed–canenterlungsandcausedamagetolungtissue.

PersonalProtectiveEquipment(PPE)

RespiratoryProtectionWhereriskassessmentshowsair-purifyingrespiratorsareappropriate,useafull-facerespiratorwithmulti-purposecombination(US)respiratorcartridgesasabackuptoengineeringcontrols.Respiratorsshouldbeusedonlyunderanyofthefollowingcircumstances:• Asalastlineofdefense(i.e.,afterengineeringandadministrativecontrolshavebeenexhausted).

• WhenPermissibleExposureLimit(PEL)hasexceededorwhenthereisapossibilitythatPELwillbeexceeded.

• Regulationsrequiretheuseofarespirator.• Anemployerrequirestheuseofarespirator.• Thereispotentialforharmfulexposureduetoanatmosphericcontaminant(intheabsenceofPEL)

• AsPPEintheeventofachemicalspillclean-upprocessLabpersonnelintendingtouse/weararespiratormaskmustbetrainedandfit-testedbyEH&S.Thisisaregulatoryrequirement.(http://www.asu.edu/uagc/EHS/documents/asu_respriatory_protection_plan.pdf)HandProtection• SilvershieldorVitonglovesarerecommended.NOTE:ConsultwithyourpreferredglovemanufacturertoensurethattheglovesyouplanonusingarecompatiblewithHydrofluoricacid.

Refertogloveselectionchartfromthelinksbelow:http://www.ansellpro.com/download/Ansell_8thEditionChemicalResistanceGuide.pdfORhttp://www.allsafetyproducts.biz/page/74172OR

Benzene,CAS#71-43-2


http://www.showabestglove.com/site/default.aspxORhttp://www.mapaglove.com/EyeProtection

• WearNIOSHapprovedsafetyglassesorgogglesSkin&BodyProtection

• Flameresistantorretardantlabcoatsshouldbeworn–labcoatsmustbebuttonedtotheirfulllengthandcoatsleevesmustbeofasufficientlengthtopreventskinexposurewhilewearinggloves.

• Full-lengthpants• Close-toedshoes–theareaofskinbetweentheshoeandankleshouldnotbeexposed

HygieneMeasures

Handleinaccordancewithgoodindustrialhygieneandsafetypractice.Avoidcontactwithskin,eyes,andclothing.Washhandsbeforebreaksandimmediatelyafterhandlingtheproduct.

EngineeringControls

Alloperationsinvolvingbenzeneanddilutionsshouldbecarriedoutinaventilatedenclosurefumehoodtokeepairborneconcentrationsbelowrecommendedexposurelimits.Chemicalfumehoodsusedascontainmentareasforparticularlyhazardouschemicalsmusthaveafacevelocityof100cfm,averagedoverthefaceofthehoodandmustbecertifiedannually.Laboratoryroomsmustbeatnegativepressurewithrespecttothecorridorsandexternalenvironment.Thelaboratory/roomdoormustbekeptclosedatalltimes.

FirstAidProcedures

Ifinhaled…Movetofreshair.Ifthepersonisnotbreathing,giveartificialrespirationandcall911fromacampusphoneor(480)965-3456.CallEH&Sat(480)965-1823.Incaseofskincontact…Minorskincontactrequireswashingwithsoapandwater.Soakingofflushingcontaminatedareasoftheskinwithwaterforperiodsupto15minutesisrequiredifalargeareacomesintocontactwiththechemical,orifprolongedcontactoccurs.Contaminatedclothingmayholdthechemicalsincontactwiththeskinwithoutbeingimmediatelynoticed.Manychemicalsareabsorbedthroughtheskin,anddermatitismaylaterappearonskinwhichappearstobeclean.Incaseofeyecontact…Intheeventofeyecontact,theeyeshouldbeimmediatelyflushedwithwater.Ifthechemicalisveryirritating,itislikelythattheaffectedindividualwillrequire

Benzene,CAS#71-43-2


assistancetoholdtheeyeopenduringtheflushing.Usenearestemergencyeyewashimmediately.Call911fromacampusphoneor(480)965-3456fromacellphone.CallEH&Sat(480)965-1823.Ifswallowed…Givemilkorwatertoinducevomitingifconscious.Nevergiveanythingbymouthtoanunconsciousperson.Getmedicalattentionimmediately,call911fromacampusphoneor(480)965-3456fromacellphone.CallEH&Sat(480)965-1823.

SpecialStorage&HandlingRequirements

Storage• Ensurethecontaineristightlyclosedatalltimes.• Storeinadryandwell-ventilatedplace.• Useexplosion-proofequipment.• Keepawayfromsourcesofignition–nosmoking.• Containerswhichareopenedmustbecarefullyresealedandkeptuprighttopreventleakage• Signageisrequiredforthecontainer,designatedworkareaandstorage.• Signwordingmuststate“Danger,CancerHazard.”Handling• Avoidcontactwithskinandeyes.• Avoidinhalationofvaporandmist.• Takemeasurestopreventthebuild-upofelectrostaticcharge.• EnsureyouarewearingthefollowingminimumPPE:NIOSHapprovedsafetyglassesor

goggles,labcoat,fulllengthpants,close-toedshoesSpillandAccidentProcedure

PersonalprecautionsAvoidbreathingvapors,mistorgas.Ensureadequateventilation.Evacuatepersonneltosafeareas.Donotattemptclean-upwithoutminimumPPE.Ifchemicalspillonbodyorclothes–removeclothingandrinsebodythoroughlyinemergencyshowerforatleast15minutes,seekmedicalattention,andnotifysupervisorandEH&Sat(480)965-1823.Ifchemicalsplashintoeyes–immediatelyrinseeyeballandinnersurfaceofeyelidwithwaterfromtheemergencyeyewashstationfor15minutesbyforciblyholdingeyeopen,seekmedicalattention,andnotifysupervisorandEH&Sat(480)965-1823.

EnvironmentalprecautionsPreventfurtherleakageorspillage–ifsafetodoso.Donotallowproducttoenterdrains.

Methodsandmaterialsforcontainmentandclean-upConsidermaterialcompatibilitypriortoclean-up.Verifyspillkitisavailable.

Benzene,CAS#71-43-2


1. Immediatelyassessamountspilled,followpostedASUEmergencyResponseGuide

proceduresforhazardousmaterialsincidents.2. Ifachemicalexposurehasoccurred,afellowlabworkershallcall9-1-1andEH&Sat(480)

965-1823.3. DoncompatibleglovesandotherprotectivePPEifnotalreadybeingworn.4. Secure/restrictaccesstotheareaofthespilltopreventspreadofthechemical.5. Usetheavailablespillkittostopandcontainthespill.Bagthecollectedmaterial.6. Labelandtagashazardouswasteandsubmitapick-uprequesttoEH&SusingEHSAssistant.

Decontamination/WasteDisposalProcedure

Allwastestreamscontainingbenzenewillbecollectedashazardouswaste.Decontaminateworkspacewith70-75%ethanol.Washhandsandarmswithsoapandwaterafterfinished.Contaminatedpipettips,eppendorftubes,andglovesshouldbediscardedashazardouswasteinaccordancewithASUEH&Swastedisposalprocedures.Labelwaste• AttachacompletedASUHazardousWastetagtoallwastecontainersassoonasthefirst

dropofwasteisaddedtothecontainer.Storewaste• Storehazardouswasteinclosedcontainers,insecondarycontainmentandinadesignated

storagelocation.• Double-bagdrywasteusingsealabletransparentbags.• Wastemustbeunderthecontrolofthepersongeneratinganddisposingofit.

Disposeofwaste• Disposeofregularlygeneratedchemicalwastewithin90days.• UseEHSAssistantonlinehazardouswastepick-uprequestsystem.• ContactASUEH&Sat(480)965-1823withquestions.

SafetyDataSheet(SDS)Location InsideGWC693,nexttothedoorinyellowbinders.Protocol/Procedure

Laboratory-specificproceduresAlldispensingofbenzeneshouldtakeplaceinthechemicalhood.Useonlysmallvolumes.Ifsmallspillsoccur,closehoodsashandallowtoevaporate.

Benzene,CAS#71-43-2


DocumentationofTraining

• Priortoconductinganyworkwiththismaterial,PrincipalInvestigatorordesigneemustprovidetohis/herlaboratorypersonnelspecifictothehazardsinvolvedinworkingwiththissubstance,workareadecontamination,andemergencyprocedures.

• ThePrincipalInvestigatormustprovidehis/herlaboratorypersonnelwithacopyofthisSOPandacopyoftheMSDSprovidedbythemanufacturer.

• ThePrincipalInvestigatormustensurethathis/herlaboratorypersonnelhaveattendedappropriate/requiredlaboratorysafetytrainingorrefreshertrainingwithinthelastoneyear.

IhavereadandunderstandthecontentofthisSOP.

EmployeeName ASUAffiliateNo. Signature DateDavidNielsen 1202345876 5/11/2016Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.Clickheretoentertext. Clickheretoentertext. Clickheretoenteradate.

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