CEB/Biotech/Petexm.31811(Rev) 

Release and Exposure Assessment in Support of Proposed TSCA 5(h)(4) Exemption for 

Bacillus amyloliquefaciens 

Joe Avcin 

Chemical Engineering Branch 

February 18, 2000 (Revision 1) 

April 21, 2010 (Revision 2) 

March 18, 2011 (Revision 3) 

June 3, 2011 (Revision 4) 

Addendum to: 

“Analysis of Environmental Releases and 

Occupational Exposure in Support of 

Proposed TSCA 5(h)(4) Exemption”, Reilly, 1991 

I. INTRODUCTION 

 This report is an addendum to the engineering report prepared by CEB in 1991 in support 

of the proposed TSCA 5 (h)(4) exemption (CEB, 1991). In that report, CEB presented an 

assessment of potential environmental releases and occupational exposures for a typical closedsystem fermentation process. Since 1991, CEB has reviewed additional biotechnology PMN 

submissions and gathered additional information on closed system processes. This led to the 

development of a generic scenario for closed system fermentation processes which provides a 

standardized assessment approach (CEB, 1997). 

 The purpose of this addendum is to update the 1991 report by using the generic scenario 

to revise the estimates of potential exposure and release. This addendum can be used by OPPT 

for risk assessments to evaluate petitions to add microorganisms to the list that are eligible for 

the 5(h)(4) exemption. This addendum presents estimates for both an uncontrolled process and 

also for a process which meets the 5(h)(4) criteria for waste inactivation which became rule 

when EPA promulgated its Final Rule for Regulation of Microbial Products under TSCA, 

published in June of 1997 (62 FR 17910 - 17958). 

 This addendum focuses specifically on the estimates of release and exposure. The 

estimates are based on a large-scale closed system fermentation process which operates 350 

days/yr. Based on the generic scenario and case history, the number of batches/yr is 100, the 

CFU concentration in the fermentation broth is 1 x 1011 CFU/ml, and the volume of the 

fermentation broth is 70,000 L. The process consists of the main steps of laboratory 

propagation, fermentation and then recovery where filtration operations separate out the biomass 

from the concentrated desired product. The operations, and sources of exposure and release are 

described in more detail in the above referenced documents. 

II. RELEASES 
CEB/Biotech/Petexm.31811(Rev) 

A. Air

1. Fermentor Offgas 

 a. “Contained” system that treats exhaust gases = 2.0 x 107

 CFU/site-day 

 b. Uncontrolled system = 2.0 x 109 CFU/site-day 

Basis: 

a. From generic scenario: 

 Arfo = CFUb AF (1-nR) (Vb) 

Where: 

 Arfo = Air release from fermentor off-gas (CFU/bt) 

 CFUb = CFU concentration in final fermentation broth (CFU/ml) 

 AF = Aerosolization factor ((aerosol CFUs)/(CFU/ml in fermentation broth)) 

 nR = removal efficiency of control technology or inactivation technique 

 Vb = volume of fermentation broth 

from generic scenario: 

 CFUb = 1 x 1011 CFU/ml 

 AF = 1 x 10-9

nR = assume 99% efficient control technology, with no subsequent inactivation 

(cyclone, scrubber, etc.) 

 Vb = 70,000 L = 70,000,000 ml 

 Arfo = (1 x 1011 )(1 x 10-9 )(1-0.99)(70,000,000) 

 = 7.0 x 107 CFU/bt 

 CFU/site-day = 7.0 x 107

 CFU/bt x (100 bt/yr)/350 days/yr 

 = 2.0 x 107

 

 

b. If no control technology, then 
CEB/Biotech/Petexm.31811(Rev) 

 CFU/site-day = 2.0 x 107

 /(1-0.99) = 2.0 x 109 

2. Rotary drum filter 

 a. “Contained” system that treats exhaust gases = 7.0 x 107

 CFU/site-day 

 b. Uncontrolled system = 7.0 x 109 CFU/site-day 

Basis: 

 The generic scenario suggests the same approach for estimating releases from process 

unit operations equipment such as a rotary drum filter. 

B. Water and Solids

1. Wastewater from Cleaning Operations 

 a. “Contained” system meeting exemption criteria of 6 log reduction 

 = 1.4 x 1011 CFU/site-day 

 

 b. Uncontrolled = 1.4 x 1017 CFU/site-day 

Basis: 

a. From generic scenario 

 WRcw = CFUb (0.02) (Vb)(1-ni ) 

Where: 

 WRcw = Water release from cleaning wastewater (CFU/site-day) 

 CFUb = CFU concentration in final fermentation broth (CFU/ml - 1 x 1011 ) 

 0.02 = 2% loss is standard CEB default for total process cleanup. Note: generic 

scenario suggests 1% for fermentor cleanup which is CEB standard assumption 

for single vessel cleanup. 2% is used here to account for situations where other 

equipment may be cleaned, generating an aqueous waste. 

ni = inactivation or sterilization efficiency = 99.9999% for processes meeting 

exemption criteria 

 Vb = volume of fermentation broth = 70,000,000 ml 
CEB/Biotech/Petexm.31811(Rev) 

 WRcw = (1 x 1011 )(0.02)(1-0.999999)(70,000,000) 

 = 1.4 x 1011 CFU/bt 

 CFU/site-day = 1.4 x 1011 CFU/bt x (100 bt/yr)/100 days/yr 

 = 1.4 x 1011 

 

b. Uncontrolled. 

 WRcw = (1 x 1011 )(0.02)(1-0)(70,000,000) 

 = 1.4 x 1017 CFU/bt 

 CFU/site-day = 1.4 x 1017 CFU/bt x (100 bt/yr)/100 days/yr 

 = 1.4 x 1017 

2. Wastewater from Fermentor 

a. “Contained” system that treats exhaust gases = 6.9 x 109 CFU/site-day 

 b. Uncontrolled system = 0 CFU/site-day 

Basis: 

a. From generic scenario 

 WROG = ([CFU]B)(AF)(nR)(VB) 

Where: 

WROG = Water release from fermentor or rotary drum off-gas treatment 

wastewater(CFU/site-day) 

 CFUb = CFU concentration in final fermentation broth (CFU/ml - 1 x 1011 ) 

AF = Aerosolization factor ((aerosol CFUs)/(CFU/ml in fermentation broth)) 

 nR = removal efficiency of control technology or inactivation technique 

Vb = volume of fermentation broth 

from generic scenario: 
CEB/Biotech/Petexm.31811(Rev) 

 CFUb = 1 x 1011 CFU/ml 

 AF = 1 x 10-9

nR = assume 99% efficient control technology, with no subsequent inactivation 

(cyclone, scrubber, etc.) 

 Vb = 70,000 L = 70,000,000 ml 

WROG = (1 x 1011 )(1 x 10-9 )(0.99)(70,000,000) 

 = 6.9 x 109

 CFU/bt 

 CFU/site-day = 6.9 x 109 CFU/bt x (100 bt/yr)/100 days/yr 

 = 6.9 x 109

 

b. If no control technology, then nR = 0 

CFU/site-day = (1 x 1011 )(1 x 10-9 )(0)(70,000,000) = 0 

3. Wastewater from Rotary Drum Off-Gas Treatment 

a. “Contained” system that treats exhaust gases = 6.9 x 109

 CFU/site-day 

 b. Uncontrolled system = 0 CFU/site-day 

Basis: 

 The generic scenario suggests the same approach for estimating releases from process 

unit operations equipment such as a rotary drum filter. 

4. Solid Wastes 

 a. “Contained” system meeting exemption criteria of 6 log reduction 

 = 7.0 x 1012 CFU/site-day 

 

 b. Uncontrolled = 7.0 x 1018 CFU/site-day 

Basis: 
CEB/Biotech/Petexm.31811(Rev) 

a. From generic scenario 

 LR = CFUb (Vb)(1-ni )(nR) 

Where: 

 LR = Land release from (CFU/site-day) 

 CFUb = CFU concentration in final fermentation broth (CFU/ml - 1 x 1011 ) 

 ni = inactivation or sterilization efficiency = 99.9999% for processes meeting 

 exemption criteria 

 Vb = volume of fermentation broth = 70,000,000 ml 

 nR = removal efficiency. Standard assumption used by CEB in biotech assessments is 

that the combination of rotary drum filtration and plate and frame biological 

filters removes “100%” of biomass from the product stream and is therefore 

available for release to land. 

 

 LR = (1 x 1011 )(70,000,000)(1-0.999999)(1) 

 = 7.0 x 1012 CFU/bt 

 CFU/site-day = 7.0 x 1012 CFU/bt x (100 bt/yr)/100 days/yr 

 = 7.0 x 1012 

 

b. Uncontrolled. 

 WRcw = (1 x 1011 )(70,000,000)(1-0)(1) 

 = 7.0 x 1018 CFU/bt 

 CFU/site-day = 7.0 x 1018 CFU/bt x (100 bt/yr)/100 days/yr 

 = 7.0 x 1018 

C. Summary

 A summary of the release estimates is presented below in Table 1. 

Table 1: Summary of Release Estimates for a Typical Fermentation Process 
CEB/Biotech/Petexm.31811(Rev) 

Media Controlled/Treated 

(Full Exemption) 

(CFU/site-day) 

Uncontrolled/ 

Untreated 

(CFU/site-day) 

Days/yr 

AIR 

 Fermentor exhaust 

 Rotary drum filter 

< 2.0 x 109 

< 7.0 x 109

2.0 x 109

7.0 x 109

350 

100 

WATER - to w.w.t. 

 Cleanup 

 Fermentor exhaust 

treatment 

 Rotary drum 

exhaust treatment 

1.4 x 1011 

6.9 x 109 

6.9 x 109

1.4 x 1017 

0 

0 

100 

100 

100 

LAND - to landfill 

 Filter cakes 7.0 x 1012 7.0 x 1018 100 

III. OCCUPATIONAL EXPOSURE 

Total No. of workers 36 

Basis: Average workers per facility (from CEB’s generic scenario: 4/shift involved with 

fermentation; 4/shift for recovery; 3/shift for lab; 1 supervisory; 3 shifts/day) 

Days/yr 250 (CEB standard maximum per year per worker) 

# workers with inhalation exposure: 36 total 

Estimates are presented below for workers in 4 areas: labs, fermentation, recovery, and 

supervision (note that the estimates in this addendum assume a worst case exposure 
CEB/Biotech/Petexm.31811(Rev) 

potential due to 12 hour shifts). 

Dermal:

Dermal exposures are assessed for laboratory propagation, fermentation, and recovery 

processes; supervisory workers are not expected to have dermal contact with the GEM. 

1) Laboratory Propagation 

<9.0 x 1010 CFU/day over 250 days/yr (9 workers) 

Basis: 

· CFU concentration during laboratory propagation (CFU/ml – unknown, final 

broth concentration = 1 x 1011 ); 

· < 0.9 ml/day (CEB dermal exposure to liquids for bench scale activities); 

(1 x 1011 CFU/ml)(< 0.9 ml/day) 

= <9.0 x 1010 CFU/day 

2) Fermentation Area 

<9.0 x 1010 CFU/day over 250 days/yr (12 workers) 

Basis: 

· Final CFU concentration during fermentation (CFU/ml = 1 x 1011 ); 

· < 0.9 ml/day (CEB dermal exposure to liquids from sampling, 1-hand incidental 

contact). 

(1 x 1011 CFU/ml)(< 0.9 ml/day) 

= <9.0 x 1010 CFU/day 

3) Recovery Area 

1.2 x 1011 – 3.7 x 1011 CFU/day over 250 days/yr (12 workers) 

Basis: 

· Final CFU concentration during fermentation (CFU/ml = 1 x 1011 ); 

· 0.6 – 1.8 ml/day (CEB dermal exposure to liquids from sampling, 2-hand dermal 

contact). 

(1 x 1011 CFU/ml)(0.6 to 1.8 ml/day) 
CEB/Biotech/Petexm.31811(Rev) 

= 6..0 x 1010 – 1.8 x 1011 CFU/day 

4) Summary 

 A summary of potential dermal dose rates is presented below in Table 2. 

Table 2: Summary of Dermal Exposures for a Typical Fermentation Process 

Area of Plant # of workers/site Dermal Potential Dose Rate 

(CFU/day)1

1. Laboratory 9 <9.0 x 1010

2. Fermentation 12 <9.0 x 1010

3. Recovery 12 6.0 x 1010 – 1.8 x 1011

Notes: 

1 - exposure estimates were rounded to 2 significant digits 

Inhalation: 

Estimates are based on air concentrations of microorganisms from NIOSH area sampling 

conducted at 3 closed system fermentation facilities in 1986. 

Note: As discussed in (CEB, 1991), typically, the Chemical Engineering Branch would 

not use area monitoring data to estimate occupational exposure levels since the correlation 

between area concentrations and worker exposure is highly uncertain. However, personal 

sampling data is not available at the present time. Therefore, area sampling data is an appropriate 

means for assessing potential exposures. 

 The area monitoring data is expressed in units of CFU/m3

. In order to convert the 

airborne concentrations to an estimate of daily exposure, assumptions need to be made of the 

hours per day a worker could receive that exposure. For this assessment, the following 

assumptions regarding worker activity patterns are used. 

 Laboratory 8 hrs 

 Fermentor sampling 2 hrs 

 Time near unit used for recovery operation 2 hrs 

 Control room 8 hrs 

 Exposure estimates are as follows. 
CEB/Biotech/Petexm.31811(Rev) 

1) Laboratories 

 530 CFU/day (average) over 250 days/yr(9 workers) 

Basis: 

· 53 CFU/m3

 (average of CFU concentrations in the laboratory at 3 facilities 

sampled by NIOSH - generic scenario) 

· 1.25 m3

/hr standard breathing rate; 

· 8 hrs of exposure at this level; 

(53 CFU/m3)(1.25 m3/hr)(8 hr/day) 

= 530 CFU/day (average) 

2) Fermentation area 

 660 CFU/day (average) over 250 days/yr(12 workers) 

Basis: 

· 263 CFU/m3 (average of CFU concentrations around fermentor sample port at 3 

plants sampled by NIOSH - generic scenario); 

· 1.25 m3/hr standard breathing rate; 

· 2 hrs of exposure at this level; 

(263 CFU/m3)(1.25 m3/hr)(2 hr/day) 

= 660 CFU/day (average) 

3) Recovery area 

 9800 CFU/day (average) over 250 days/yr(12 workers) 

Basis: 

· 3,906 CFU/m3

 (geometric mean of CFU concentrations around filter press when 

closed at facility sampled by NIOSH - generic scenario); 

· 1.25 m3

/hr standard breathing rate; 

· 2 hrs of exposure at this level; 

(3906 CFU/m3

)(1.25 m3/hr)(2 hr/day) 

= 9800 CFU/day 

4) Supervision 
CEB/Biotech/Petexm.31811(Rev) 

 81 CFU/day (average) over 250 days/yr(3 workers) 

Basis: 

· 8.1 CFU/m3

 (geometric mean of CFU concentrations in control room at facility 

sampled by NIOSH - generic scenario); 

· 1.25 m3/hr standard breathing rate; 

· 8 hrs of exposure at this level; 

(8.1 CFU/m3

)(1.25 m3/hr)(8 hr/day) 

= 81 CFU/day (average) 

5) Summary 

 A summary of potential inhalation dose rates of bioaerosols is presented below in Table 

3. 

Table 3: Summary of Inhalation Exposures for a Typical Fermentation Process 

Area of Plant # of workers/site Inhalation to bioaerosols 

Potential Dose Rate 

(CFU/day)1

1. Laboratory 9 530 

2. Fermentation 12 660 

3. Recovery 12 9800 

4. Control Room 3 81 

Notes: 

1 - exposure estimates were rounded to 2 significant digits 
CEB/Biotech/Petexm.31811(Rev) 

IV. REFERENCES 

1. CEB, 1991. Analysis of Environmental Releases and Occupational Exposure in Support of 

Proposed TSCA 5(h)(4) Exemption. Prepared by Breeda Reilly, Chemical Engineering Branch 

of the Economics, Exposure and Technology Division, Office of Pollution Prevention and Toxic, 

U.S. EPA, Washington, DC. 

2. CEB, 1997. Generic Scenario for Biotechnology Premanufacture Notices - Final Report. 

Prepared by Eastern Research Group (ERG) for the Chemical Engineering Branch of the 

Economics, Exposure and Technology Division, Office of Pollution Prevention and Toxic, U.S. 

EPA, Washington, DC. under EPA contract 68-D4-0092.