Biosafetymonitoring devicesfor biotechnology processes Peter Hambleton, Allan M. Bennett, Geoffrey Leaver and John E. Benbough Compared with the chemical and nuclear industries, traditional industrial biotechnology processes have until now been considered to pose no nlore of a hazard to workers or the envimnment thaq, for instance, a flour mill or an oilseed processing plant’. This y:iew Seems well founded, since the biotechnology and pharmaceutical industries have good safety records based on experience and procedures developed over many years. Ahhough the relatively recent industrial application ofrecombinant DNA technology has now focused attention on the question of the safety of the biotechnology process industry. there is little to suggest that the new biotechnology processes are inherently any more or less safe than the more traditional ones?. Nevertheless, the way the industry responds to the public concern tivcr biosafbty issues will determine future attitudes on the social acceptability ofbiotechnology and its products and, ultimately, the commercial viability of the industry. Regulating bioteehndogy Current interest in biosafety is manifest in the increasing number of national and international regulations designed to control the biotechnology industries (See Table 1)3-l?. Although reguiations were introduced initially on a national basis, in Europe at least, there is now considerable momentum towards standardization. Current regulations covering the industrial use of genetically modified microorganisms (GMMOs) are being added to by regulations designed to control the impact on worker safety and the environment of any hazardous biological substance or organism. In the UK, biotechnology, like other industries, is covered by the Health & Safety at Work Act (1974)3 that requires employers, as far as is reasonably possible, to protect employees corn injury. The Control of Substances Hazardous to Health (COSHH) (1988) regulations~ further require that assessments be carried out on all processes involving the use of such substances in order to determine the level of control required to make that process safe. These regulations specifically include microorganisms as ‘substances potentially hazardous to health’ and include the industrial use of microorganisms.
TiBrEcH
Simikuly, the revised UK genetic manipulation mgulationsj require all centres engaged in genetic manipulation to notify the Heakh & Safety Executive (HSE), and to carry out risk assessments. The Organization of Economic Co-operation and Development (OECD) issued guidelines7 on the use of genetically modified organisms (GMOs) that formed the primary source of information on the containment levels for GMOs. Most importantly, these guidelines make specific recommendations as to the physical containment required for different categories of microorganisms. They recognize, for example, that, generally, industrially used GMOs will have intrinsically low hazard, 2nd duly recommend a level of control based on established good industrial large-scale practice (GILSP). Where higher hazard situations might occur, additional control/containment options are recommended. There are, at present, three European Community (EC) Directives concerned with environmental or health aspects of biotechnology. Of these, two [covering (1) contained usea, and (2) deliberate releas? of GMOs (including GMMOs)] have been implemented, and European governments are currently bringing their national 1egisIation into lint with the Directives’ requirements. Denmark has already completed this process; in the UK there is presently discussion on the proposed regulations to be included in the Environmental Protection Act (EPA) (1991p. The new European regulations will require notification of all operations involving GMOs to a competent authority within member states and environmental risk assessments to be carried out. The other EC directive”’ on the protection of workers from risks related to exposure to biological agents at work has reached the common agreement stage. The directive appears to be a mechanism for incorporating the principles of COSHHI and the OECD guidelines7 into unified legislation covering all microorganisms. The employer’s obligation under this Directive is to protect adequately the health and safety of the workers concerned by reducing the risk of exposure to as low a level as is necessary. In terms of hiosafety these various regulations require industry to take steps to prevent the exposure ofworkers to biohazards and to prevent environmental contamination. A clear requirement is for risk assessment to be carried out on each process involving the use ofz substance hazardous to health or to the environment. If an assessment shows that a process car&s I risk of exposure to a biohazard, then that process must be subject to additional constraints to
193
f eatrrre fable CHeakh and safety regulatiow relevant to the eliminate the risk or reduce it to an acceptable level. A major problem with implementing the burgeoning biosafcty legislation is that, unlike the chemical and nuclear industries, there are few, ifany, rccommcnded exposure limits for microorganisms and their products, nor are there many performance standards rel ;:ed to biosafety. This means that ic can be very difficult to assess whether or not a process is hazardous. There are various ways in which the obligation to reduce the risk of exposure to biohazards may be achieved; for rtxampic: ??by reducing the number of workers at risk of exposure, e by designing processes and equipment to prcvcnt or minimize rclcase, ??by emcrgcncy planning and safe dsposal of waste.
A major conscquencc of the regulations will be a requirement to test for the presence of process organisms and/or products outside the primary physical containment, a feature already in place in the UK within the COSHH replations’. This last rcquiremcnt places on industry an obligation to incorporate environmental biological monitoring into its normal working practices. To do th%, industry must first accept the,nature of the hazards and risks of their procc;scs and implemene appropriate mL~1icoring techniques and strategies. Industry should now be dcfining its current operating performance using the data obtained to de&c iii&try operating standards that can be used to measure compliance with the regulatory demands. Is there a problem? Despite the widespread perceptions of the safety of the tradirional biotechnology and pharmaceutical industries, it must be recognized that many ofthc various unit processes of manufacture have the potential to generate biohazards. This is demonstrated by the various health problems that have been associated with biotechnology manufacturing processes. Although infection is not a feature of industrially acquired iilness, there are many examples of non-infectious illness arising from industrial biotechnology processes (Table 2)‘3.‘4. Any microorganism or biological product is potentially able to induce allergic symptoms, including asthma and dermatitis, in exposed worker@. In addition, symptoms may be induced by exposure to lower concentrations of allergen than were expcricnccd during sensitization 16.Non-allergic illnesses can result from exposure to biotechnology products. For example, Gram-negative bacterial endotoxin may inducr non-allergic respiratory symptoms as wcli as stomach and kidney pains’7.1x. and exposure to antibiotics can result in microbial flora changes in workers resulting in candidiasis”. Experience with laboratory and industrial accidents suggests that inhalation is the most significant mode of entry of microbes or microbial products into the body”-19. While contamination by ingestion or skin contact may occur, this can
bi&chnology industry Regulation/guideline
lmptementation date
Ref.
UK Health and Safety at Work Act
1974
3
Control of Substances Hazardous to Health (CGSHH) regulations
1938
4
Advisory Committee for Genetic Manipulation (ACGMI revised guidelines
1989
5
Environmental Protection Act (EPA)
1991 (Oct.)
6
1982
7
Directives on the Contained Use and De!iberate Release of Genetically Modified Organisms
1991
e,9
Directive on Worker Safety in Biotechnology
1993
10
1990 (revised)
11
National Environmental Policy Act
1970
11
Occupational Safety & Health Act
extant
11
EC OECD guidelines (GILSPP
US6; NIH rDNA guidelines (55 Fed. Reg. 74381
Other Research Involving Recombinant 12 DNA Technology, PAHOa Guidelines 1987 -.“Abbreviations: GILSP,G;od Industrial Large-Scab Practice; PAHO, Pan AmericanHealth Organit&sn.
readily be prevented by good standards of operating practice; ill health due to transmission by these routes usually occurs as a result of gross carelessness. Past espericncc demonstrates that health hazards do arise from biotechnology processes and that problems are as likely to arise fi-om exposure to the product of a biological process. as t?-om exposure to process organisms. Most, if nor all, of the unit operations comprising a biotechnology production process can generatr health hazards through the generation of aerosols or gross environmental contamination as shown by reports of illnesses limited to specific industrial processes (Table 3)_ Iniection or allergic illncssL5 have resulted from release ofmaterials during centrifugation in labo unnil’x considered that in the extraction of intracellular enzymes, the grcatcst demands on biosafcey involve processes handling large quantities of cell debris. With many biological products, the risk of allergic reaction is greatest after the product is more concentrated i.e. during purification and packaging’a. An cffcctivc monitoring strategy should involve environn~cntal assessment of emissions at 3U stages of a manufacturing process and critical assessment of the biosafcty performance of process eqdipmcnt whilst maintaining adequate surveillance ofworker health. Types ofhazardous
The potential for release of liquids exists at many stages of prodcction, although these should be limited in a well-maintained plant. Although spillages and leaks can cause gross local contamination ofequipment surfaces and of buildings, uncontrolled release beyond the production area can be limited by drains, bunds etc. and by the use of efiictive decontamination proccdurcs. Personnel exposed to such emissions arc generally at risk of being contaminated by ipT_stion or by skin contact. These arc both relaiively easily avoided by the provision and use of suitable protective clothing and the application of appropriate instructions for work (Standard Operating i’rocedures). Monitoring 1992 NOL 10)
E$j%fent
disdtarge
Whcrc uncontrolled liquid etllucnt discharge is unacceptable on environmental grounds, then cffluent treatment is nrccssary. Guidelines7 refer to the use of validated means of effluent treatment which usually means the use of heat or chemical trearmcnt. Treat.. mcnt procedures should be validated for all types of effluent likely to bc treated. The e&ctivcncss of effluent treatment should be confirmed prior to discharge tither parametrically or by direct analysis of rhc treated cfflucnt for sterility and/or product destruction. In cithcr case, environmental monitoring downstream of the discharge point might also be undertaken if required. For example, possible interactions bctwecn discharged process organisms or gcnctic elements and normal environmental populations could be monitored using specific tests, e.g. for antibiotic resistancr,
Table 3. BiohazareSpotential of typical biotechnologyprocess ofierations Process step Operation
Type of hazard
Raw materials handling
Weighing, mixing dissolving
Generation of allergenic dusts or aerosols
Bioreactor
Fermentation
Aerosols of reactor contents, spillage, effluent contamination, off gases
Biomass separation
Centrifugation
Aerosols, spillage
Filtration
Leakage
Homogenization
Aerosols
Centrifugaticn concentration, dialvsis, chromatography
Aerosols, spillage
emission
Spillages/leuks
TIBTECH JWE
for such emission is best achieved by a combination of visual inspection and application of microbiological/product assays to surface swabs or washings. Asscss~nc~~t methods that are appropriate to this aspect of monitoring include classical microbiological pro&ures rcgether with more specific immunological, biochemical or genetic identification techniques such as arc discussed below.
Product purification
Product finishing
Blending, filtration, Aerosols, dust, filling, spillage freeze/spray drying
Effluent handling
Sterilization discharge
Discharge of untreated effluent
virulence factors or spcci&~ :w,ue sequences. Althougll such monitoring might, m practice, be pcdormcd by watcr/sewagc treatment organizations, the responsibility for preventing untreated discharge would clearly lie with the process operator. Aerosol tran$mission Inhalation of acrosoIs is probably the most significant route whereby process organisms, extrinsic antigcns or other products gain access to the body”. This route of exposure is not restricted to workers, since aerosols are not easily confined and may cause environmental contamination or illness in surrounding populations”. Airborne transmission can bc d&cult CD detect or prevent since the presence of microorganisms or products in the atmosphere will not be obvious in most instances. it is for these reasons that we consider that detection and asscssmcnt of releases into the air must be considcrcd as the most important aspect of biosafcty monitoring and why this topic forms a majoi clement of this article. Proper&s ofaerosols The health risks associated with biological aerosols depend not only on the concentration of hazardous ma&al but also on the size-distribution ofthe aerosol particle9. Most man-made aeroro!s 2nd industrial dusts contain particles of a wide range of sizes. The size, shape and density of the particle determines the site of deposition in the body. Energy used to stir or move a liquid generates a new surf&c and overc6mcs the viscous forces in the liquid. Any process such as stirring or bubbling results in the formation of a thread offilm ofhquid which subsequently breaks down into small droplets which then evaporate to form an aerosol. The physics of aerosol gencration is beyond the scope of this article and was well reviewed by Green and Lam+. However, it should be noted that whilst a large energy input is rcquircd to generate small droplets, only a moiierate energy input will generate larger ones. For this reason many biotechnology process steps have the potential to generate hazardous aerosols (see Table 3; and below). Aerosols generated from liquids containing microorganisms and/or dissolved solids will contain particles of a range of sizes. Such particles will equilibrate rapidly with water vapour in the atmosphere, and the smaller the droplet the more rapidly it will equilibrate. The size of particles in an aerosol is critical in determining the length of time a particle will remain airborne, the site of deposition in the respiratory tract, the survival and infectivity of microorganisms and the allergic response to dusts and protcinaceous material. For aerosols derived from liquid suspensions, the ultimate particle size of the droplet depends not only on the initial droplet size, but also on the mass concentration (i.e. the concentration of all soli& present, including solute) of the original suspension]‘. When the effect of the aerosol depends upon the mass concentration of the airborne material, as with allergic response to pollens and moulds, the larger aerosr.1 par-
ticks arc of significance since they o6hstitutc the largest part ofthc mass. When, howcvcr, the rffect of the aerosol depends upon a small number ofdroplcts, such as in airborne infection, then the sl;rali particles arc ofsignificance because oftheir greater number and the greater probability. of their being inhaled. For these reasons ICIS imperative that any monitoring and control measures arc based upon an undcrstanding of the particle size of the aerosol or dust giving rise to the hazard. Sampling aerosols Accurate mcasu;cmcnt of aerosols is depcr_‘s:nt upon obtaining a rcprescntative sample from I’. r xir and also minimizing or quantifying losses from the probe to the measuring instrument. This subject itself is complex, and a previous rcvicw” summarizes the factors involved in sampling. particularly from moving air: these include (1) adequacy of mixing; and (3) ensuring that all particle sites enter the sample nozzle. The latter depends, in turn, upon the velocity of air in the sampling probe relative to the vclacir~ of the airstream, and the angle of the probe to the actual sample relative to the axis of part& motion. May25 devised a n>cans of minimizing these problems by siting a b.&lr behind the air sampler (rclativc to the wind direction) to reduce the air v&city to zero. This cnab!r_’ representative sampling to be donr I0 organisms per litrej
Sample processing necessary. No? suitable fo? kw concentrations (~20 organisms per Mrej
lmpingers
Pot-ton
11
Easy to use, sma!!
Fragile, San& evaporatial, violent sam&lg
May three-stage
55
Particle size information, gentle sampling
Fragile, SW_,dardized manuf?:rure difficult
Wers Cyck .e (impinger)
750
Suitdble for wide range cl concentrations
No particle size inform&on
Personal filter samplers
P-4
Suitable for high concentra+;sns ., estimate; worker exposu;e
Low volume, sample recovery and processing difficulties
Settle plates (impdctor)
N/A
Easy to use
Inefficient, qualitative
-,,~*r-d-ii-;--~-~__i.___ “‘_, ;3;iLi:ilCration by large_ ~z-~2ki;i~ jampie?%, or filtration, is necessary.
Monitoting ofa hioprocwC!g mit There arc few descriptions of planned +-l+--~~c~Giit; .‘- -*“Y
+LIL~,
n;or:itorin~
but iscnl~(*tt PI 01.”
dcsctibcd a systematic asscssmcnt carried out using the principles described above. The mcnitorcd plant was operating and inrvit:lhlv tJ*p tnn&nrin~r nrocommercial)y. grd”me was constrained b; the requircrn&t~ of defined ope-ating procedures. For example, space constraints allowed only single samplers to be used in some situations, and decisions had to be made on whether the priority should be CC? r:::nr:! t!~ rime and -~:::-riiz; 3f;‘;.;iJ~~~ub, co measure particle size clan:-bution, the concentrations of material Y~~-1gs’. dlc Ilcgh@d~ UildCf‘ilufi;Mi cl~t~g.., ~ys_t_~lm circumstances. However, it is known that fermenter oprrations can result m slgni(icmt asro& &clrciaikli in the event of incorrect operationJ-’ or accident+. The Ucnnctt study’7 did confirm that centrifugation reprcscnts a likely source of contamination in the bioprocrs2mg +z;,;t. r A s&ii&cant bcneflt of the study was that the information obtained rnablrd procedural changes to be introduced which reduced the risk of worker exposure (A. M. Bennett, unpublished). Bennett et al.” found that qualitatively similar information was obtni& with different samplers, showing tiut there can be some flexib&ty in the choice ofmonitoring device. It is, however, not yet possible to make fiml recommendations for samp!ing devices. As mcntioned above, the user must s&l take into consideration the pcrformancc characteristics of the sampler and the nature of the informat%:l rcq&~~J iz pl,iiiii;,lti an effective rlL;;iLo;irrg srraccgy. The future The biotechnology industry can ill afford to be complacent about its safety standards. Rightly or wrongly, in western countries public perception and acccptat&y of the industry and its products will largely be governed by what the public bclie*:e may go wrong rather than ,Nhat does not go wrong. In contrast, in Japan thrvp 5 -F‘;,~ .P;:., _.
TiBrEcH
Simikuly, the revised UK genetic manipulation mgulationsj require all centres engaged in genetic manipulation to notify the Heakh & Safety Executive (HSE), and to carry out risk assessments. The Organization of Economic Co-operation and Development (OECD) issued guidelines7 on the use of genetically modified organisms (GMOs) that formed the primary source of information on the containment levels for GMOs. Most importantly, these guidelines make specific recommendations as to the physical containment required for different categories of microorganisms. They recognize, for example, that, generally, industrially used GMOs will have intrinsically low hazard, 2nd duly recommend a level of control based on established good industrial large-scale practice (GILSP). Where higher hazard situations might occur, additional control/containment options are recommended. There are, at present, three European Community (EC) Directives concerned with environmental or health aspects of biotechnology. Of these, two [covering (1) contained usea, and (2) deliberate releas? of GMOs (including GMMOs)] have been implemented, and European governments are currently bringing their national 1egisIation into lint with the Directives’ requirements. Denmark has already completed this process; in the UK there is presently discussion on the proposed regulations to be included in the Environmental Protection Act (EPA) (1991p. The new European regulations will require notification of all operations involving GMOs to a competent authority within member states and environmental risk assessments to be carried out. The other EC directive”’ on the protection of workers from risks related to exposure to biological agents at work has reached the common agreement stage. The directive appears to be a mechanism for incorporating the principles of COSHHI and the OECD guidelines7 into unified legislation covering all microorganisms. The employer’s obligation under this Directive is to protect adequately the health and safety of the workers concerned by reducing the risk of exposure to as low a level as is necessary. In terms of hiosafety these various regulations require industry to take steps to prevent the exposure ofworkers to biohazards and to prevent environmental contamination. A clear requirement is for risk assessment to be carried out on each process involving the use ofz substance hazardous to health or to the environment. If an assessment shows that a process car&s I risk of exposure to a biohazard, then that process must be subject to additional constraints to
193
f eatrrre fable CHeakh and safety regulatiow relevant to the eliminate the risk or reduce it to an acceptable level. A major problem with implementing the burgeoning biosafcty legislation is that, unlike the chemical and nuclear industries, there are few, ifany, rccommcnded exposure limits for microorganisms and their products, nor are there many performance standards rel ;:ed to biosafety. This means that ic can be very difficult to assess whether or not a process is hazardous. There are various ways in which the obligation to reduce the risk of exposure to biohazards may be achieved; for rtxampic: ??by reducing the number of workers at risk of exposure, e by designing processes and equipment to prcvcnt or minimize rclcase, ??by emcrgcncy planning and safe dsposal of waste.
A major conscquencc of the regulations will be a requirement to test for the presence of process organisms and/or products outside the primary physical containment, a feature already in place in the UK within the COSHH replations’. This last rcquiremcnt places on industry an obligation to incorporate environmental biological monitoring into its normal working practices. To do th%, industry must first accept the,nature of the hazards and risks of their procc;scs and implemene appropriate mL~1icoring techniques and strategies. Industry should now be dcfining its current operating performance using the data obtained to de&c iii&try operating standards that can be used to measure compliance with the regulatory demands. Is there a problem? Despite the widespread perceptions of the safety of the tradirional biotechnology and pharmaceutical industries, it must be recognized that many ofthc various unit processes of manufacture have the potential to generate biohazards. This is demonstrated by the various health problems that have been associated with biotechnology manufacturing processes. Although infection is not a feature of industrially acquired iilness, there are many examples of non-infectious illness arising from industrial biotechnology processes (Table 2)‘3.‘4. Any microorganism or biological product is potentially able to induce allergic symptoms, including asthma and dermatitis, in exposed worker@. In addition, symptoms may be induced by exposure to lower concentrations of allergen than were expcricnccd during sensitization 16.Non-allergic illnesses can result from exposure to biotechnology products. For example, Gram-negative bacterial endotoxin may inducr non-allergic respiratory symptoms as wcli as stomach and kidney pains’7.1x. and exposure to antibiotics can result in microbial flora changes in workers resulting in candidiasis”. Experience with laboratory and industrial accidents suggests that inhalation is the most significant mode of entry of microbes or microbial products into the body”-19. While contamination by ingestion or skin contact may occur, this can
bi&chnology industry Regulation/guideline
lmptementation date
Ref.
UK Health and Safety at Work Act
1974
3
Control of Substances Hazardous to Health (CGSHH) regulations
1938
4
Advisory Committee for Genetic Manipulation (ACGMI revised guidelines
1989
5
Environmental Protection Act (EPA)
1991 (Oct.)
6
1982
7
Directives on the Contained Use and De!iberate Release of Genetically Modified Organisms
1991
e,9
Directive on Worker Safety in Biotechnology
1993
10
1990 (revised)
11
National Environmental Policy Act
1970
11
Occupational Safety & Health Act
extant
11
EC OECD guidelines (GILSPP
US6; NIH rDNA guidelines (55 Fed. Reg. 74381
Other Research Involving Recombinant 12 DNA Technology, PAHOa Guidelines 1987 -.“Abbreviations: GILSP,G;od Industrial Large-Scab Practice; PAHO, Pan AmericanHealth Organit&sn.
readily be prevented by good standards of operating practice; ill health due to transmission by these routes usually occurs as a result of gross carelessness. Past espericncc demonstrates that health hazards do arise from biotechnology processes and that problems are as likely to arise fi-om exposure to the product of a biological process. as t?-om exposure to process organisms. Most, if nor all, of the unit operations comprising a biotechnology production process can generatr health hazards through the generation of aerosols or gross environmental contamination as shown by reports of illnesses limited to specific industrial processes (Table 3)_ Iniection or allergic illncssL5 have resulted from release ofmaterials during centrifugation in labo unnil’x considered that in the extraction of intracellular enzymes, the grcatcst demands on biosafcey involve processes handling large quantities of cell debris. With many biological products, the risk of allergic reaction is greatest after the product is more concentrated i.e. during purification and packaging’a. An cffcctivc monitoring strategy should involve environn~cntal assessment of emissions at 3U stages of a manufacturing process and critical assessment of the biosafcty performance of process eqdipmcnt whilst maintaining adequate surveillance ofworker health. Types ofhazardous
The potential for release of liquids exists at many stages of prodcction, although these should be limited in a well-maintained plant. Although spillages and leaks can cause gross local contamination ofequipment surfaces and of buildings, uncontrolled release beyond the production area can be limited by drains, bunds etc. and by the use of efiictive decontamination proccdurcs. Personnel exposed to such emissions arc generally at risk of being contaminated by ipT_stion or by skin contact. These arc both relaiively easily avoided by the provision and use of suitable protective clothing and the application of appropriate instructions for work (Standard Operating i’rocedures). Monitoring 1992 NOL 10)
E$j%fent
disdtarge
Whcrc uncontrolled liquid etllucnt discharge is unacceptable on environmental grounds, then cffluent treatment is nrccssary. Guidelines7 refer to the use of validated means of effluent treatment which usually means the use of heat or chemical trearmcnt. Treat.. mcnt procedures should be validated for all types of effluent likely to bc treated. The e&ctivcncss of effluent treatment should be confirmed prior to discharge tither parametrically or by direct analysis of rhc treated cfflucnt for sterility and/or product destruction. In cithcr case, environmental monitoring downstream of the discharge point might also be undertaken if required. For example, possible interactions bctwecn discharged process organisms or gcnctic elements and normal environmental populations could be monitored using specific tests, e.g. for antibiotic resistancr,
Table 3. BiohazareSpotential of typical biotechnologyprocess ofierations Process step Operation
Type of hazard
Raw materials handling
Weighing, mixing dissolving
Generation of allergenic dusts or aerosols
Bioreactor
Fermentation
Aerosols of reactor contents, spillage, effluent contamination, off gases
Biomass separation
Centrifugation
Aerosols, spillage
Filtration
Leakage
Homogenization
Aerosols
Centrifugaticn concentration, dialvsis, chromatography
Aerosols, spillage
emission
Spillages/leuks
TIBTECH JWE
for such emission is best achieved by a combination of visual inspection and application of microbiological/product assays to surface swabs or washings. Asscss~nc~~t methods that are appropriate to this aspect of monitoring include classical microbiological pro&ures rcgether with more specific immunological, biochemical or genetic identification techniques such as arc discussed below.
Product purification
Product finishing
Blending, filtration, Aerosols, dust, filling, spillage freeze/spray drying
Effluent handling
Sterilization discharge
Discharge of untreated effluent
virulence factors or spcci&~ :w,ue sequences. Althougll such monitoring might, m practice, be pcdormcd by watcr/sewagc treatment organizations, the responsibility for preventing untreated discharge would clearly lie with the process operator. Aerosol tran$mission Inhalation of acrosoIs is probably the most significant route whereby process organisms, extrinsic antigcns or other products gain access to the body”. This route of exposure is not restricted to workers, since aerosols are not easily confined and may cause environmental contamination or illness in surrounding populations”. Airborne transmission can bc d&cult CD detect or prevent since the presence of microorganisms or products in the atmosphere will not be obvious in most instances. it is for these reasons that we consider that detection and asscssmcnt of releases into the air must be considcrcd as the most important aspect of biosafcty monitoring and why this topic forms a majoi clement of this article. Proper&s ofaerosols The health risks associated with biological aerosols depend not only on the concentration of hazardous ma&al but also on the size-distribution ofthe aerosol particle9. Most man-made aeroro!s 2nd industrial dusts contain particles of a wide range of sizes. The size, shape and density of the particle determines the site of deposition in the body. Energy used to stir or move a liquid generates a new surf&c and overc6mcs the viscous forces in the liquid. Any process such as stirring or bubbling results in the formation of a thread offilm ofhquid which subsequently breaks down into small droplets which then evaporate to form an aerosol. The physics of aerosol gencration is beyond the scope of this article and was well reviewed by Green and Lam+. However, it should be noted that whilst a large energy input is rcquircd to generate small droplets, only a moiierate energy input will generate larger ones. For this reason many biotechnology process steps have the potential to generate hazardous aerosols (see Table 3; and below). Aerosols generated from liquids containing microorganisms and/or dissolved solids will contain particles of a range of sizes. Such particles will equilibrate rapidly with water vapour in the atmosphere, and the smaller the droplet the more rapidly it will equilibrate. The size of particles in an aerosol is critical in determining the length of time a particle will remain airborne, the site of deposition in the respiratory tract, the survival and infectivity of microorganisms and the allergic response to dusts and protcinaceous material. For aerosols derived from liquid suspensions, the ultimate particle size of the droplet depends not only on the initial droplet size, but also on the mass concentration (i.e. the concentration of all soli& present, including solute) of the original suspension]‘. When the effect of the aerosol depends upon the mass concentration of the airborne material, as with allergic response to pollens and moulds, the larger aerosr.1 par-
ticks arc of significance since they o6hstitutc the largest part ofthc mass. When, howcvcr, the rffect of the aerosol depends upon a small number ofdroplcts, such as in airborne infection, then the sl;rali particles arc ofsignificance because oftheir greater number and the greater probability. of their being inhaled. For these reasons ICIS imperative that any monitoring and control measures arc based upon an undcrstanding of the particle size of the aerosol or dust giving rise to the hazard. Sampling aerosols Accurate mcasu;cmcnt of aerosols is depcr_‘s:nt upon obtaining a rcprescntative sample from I’. r xir and also minimizing or quantifying losses from the probe to the measuring instrument. This subject itself is complex, and a previous rcvicw” summarizes the factors involved in sampling. particularly from moving air: these include (1) adequacy of mixing; and (3) ensuring that all particle sites enter the sample nozzle. The latter depends, in turn, upon the velocity of air in the sampling probe relative to the vclacir~ of the airstream, and the angle of the probe to the actual sample relative to the axis of part& motion. May25 devised a n>cans of minimizing these problems by siting a b.&lr behind the air sampler (rclativc to the wind direction) to reduce the air v&city to zero. This cnab!r_’ representative sampling to be donr I0 organisms per litrej
Sample processing necessary. No? suitable fo? kw concentrations (~20 organisms per Mrej
lmpingers
Pot-ton
11
Easy to use, sma!!
Fragile, San& evaporatial, violent sam&lg
May three-stage
55
Particle size information, gentle sampling
Fragile, SW_,dardized manuf?:rure difficult
Wers Cyck .e (impinger)
750
Suitdble for wide range cl concentrations
No particle size inform&on
Personal filter samplers
P-4
Suitable for high concentra+;sns ., estimate; worker exposu;e
Low volume, sample recovery and processing difficulties
Settle plates (impdctor)
N/A
Easy to use
Inefficient, qualitative
-,,~*r-d-ii-;--~-~__i.___ “‘_, ;3;iLi:ilCration by large_ ~z-~2ki;i~ jampie?%, or filtration, is necessary.
Monitoting ofa hioprocwC!g mit There arc few descriptions of planned +-l+--~~c~Giit; .‘- -*“Y
+LIL~,
n;or:itorin~
but iscnl~(*tt PI 01.”
dcsctibcd a systematic asscssmcnt carried out using the principles described above. The mcnitorcd plant was operating and inrvit:lhlv tJ*p tnn&nrin~r nrocommercial)y. grd”me was constrained b; the requircrn&t~ of defined ope-ating procedures. For example, space constraints allowed only single samplers to be used in some situations, and decisions had to be made on whether the priority should be CC? r:::nr:! t!~ rime and -~:::-riiz; 3f;‘;.;iJ~~~ub, co measure particle size clan:-bution, the concentrations of material Y~~-1gs’. dlc Ilcgh@d~ UildCf‘ilufi;Mi cl~t~g.., ~ys_t_~lm circumstances. However, it is known that fermenter oprrations can result m slgni(icmt asro& &clrciaikli in the event of incorrect operationJ-’ or accident+. The Ucnnctt study’7 did confirm that centrifugation reprcscnts a likely source of contamination in the bioprocrs2mg +z;,;t. r A s&ii&cant bcneflt of the study was that the information obtained rnablrd procedural changes to be introduced which reduced the risk of worker exposure (A. M. Bennett, unpublished). Bennett et al.” found that qualitatively similar information was obtni& with different samplers, showing tiut there can be some flexib&ty in the choice ofmonitoring device. It is, however, not yet possible to make fiml recommendations for samp!ing devices. As mcntioned above, the user must s&l take into consideration the pcrformancc characteristics of the sampler and the nature of the informat%:l rcq&~~J iz pl,iiiii;,lti an effective rlL;;iLo;irrg srraccgy. The future The biotechnology industry can ill afford to be complacent about its safety standards. Rightly or wrongly, in western countries public perception and acccptat&y of the industry and its products will largely be governed by what the public bclie*:e may go wrong rather than ,Nhat does not go wrong. In contrast, in Japan thrvp 5 -F‘;,~ .P;:., _.
