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 anassessment of potential environmental releases and occupational exposures for a typical closedsystem fermentation process. Since 1991, CE B 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 10 11 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. RELEASESCEB/Biotech/Petexm.31811(Rev) A. Air

1. Fermentor Offgas

a. “Contained” system that treats exhaust gases = 2.0 x 10 7 CFU/site-day b. Uncontrolled system = 2.0 x 10 9 CFU/site-day

Basis:

a. From generic scenario:

Ar fo = CFU b AF (1-n R ) (V b )

Where:

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

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

n R = removal efficiency of control technology or inactivation technique

V b = volume of fermentation broth

from generic scenario:

CFU b = 1 x 10 11 CFU/ml

AF = 1 x 10 -9

n R = assume 99% efficient control technology, with no subsequent inactivation (cyclone, scrubber, etc.)

V b = 70,000 L = 70,000,000 ml

Ar fo = (1 x 10 11 )(1 x 10 -9 )(1-0.99)(70,000,000)

= 7.0 x 10 7 CFU/bt

CFU/site-day = 7.0 x 10 7 CFU/bt x (100 bt/yr)/350 days/yr

= 2.0 x 10 7

b. If no control technology, thenCEB/Biotech/Petexm.31811(Rev) CFU/site-day = 2.0 x 10 7 /(1-0.99) = 2.0 x 10 9

2. Rotary drum filter

a. “Contained” system that treats exhaust gases = 7.0 x 10 7 CFU/site-day b. Uncontrolled system = 7.0 x 10 9 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 10 11 CFU/site-day

b. Uncontrolled = 1.4 x 10 17 CFU/site-day

Basis:

a. From generic scenario

WR
cw = CFU b (0.02) (V b )(1-n i )

Where:

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

CFU b = CFU concentration in final fermentation broth (CFU/ml - 1 x 10 11 ) 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.

n i = inactivation or sterilization efficiency = 99.9999% for processes meeting exemption criteria

V b = volume of fermentation broth = 70,000,000 mlCEB/Biotech/Petexm.31811(Rev) WR
cw = (1 x 10 11 )(0.02)(1-0.999999)(70,000,000)

= 1.4 x 10 11 CFU/bt

CFU/site-day = 1.4 x 10 11 CFU/bt x (100 bt/yr)/100 days/yr

= 1.4 x 10 11

b. Uncontrolled.

WR
cw = (1 x 10 11 )(0.02)(1-0)(70,000,000)

= 1.4 x 10 17 CFU/bt

CFU/site-day = 1.4 x 10 17 CFU/bt x (100 bt/yr)/100 days/yr

= 1.4 x 10 17

2. Wastewater from Fermentor

a. “Contained” system that treats exhaust gases = 6.9 x 10 9 CFU/site-day b. Uncontrolled system = 0 CFU/site-day

Basis:

a. From generic scenario

WR OG = ([CFU] B )(AF)(n R )(V B )

Where:

WR OG = Water release from fermentor or rotary drum off-gas treatment wastewater(CFU/site-day)

CFU b = CFU concentration in final fermentation broth (CFU/ml - 1 x 10 11 ) AF = Aerosolization factor ((aerosol CFUs)/(CFU/ml in fermentation broth))

n R = removal efficiency of control technology or inactivation technique

V b = volume of fermentation broth

from generic scenario:CEB/Biotech/Petexm.31811(Rev) CFU b = 1 x 10 11 CFU/ml

AF = 1 x 10 -9

n R = assume 99% efficient control technology, with no subsequent inactivation (cyclone, scrubber, etc.)

V b = 70,000 L = 70,000,000 ml

WR OG = (1 x 10 11 )(1 x 10 -9 )(0.99)(70,000,000)

= 6.9 x 10 9 CFU/bt

CFU/site-day = 6.9 x 10 9 CFU/bt x (100 bt/yr)/100 days/yr

= 6.9 x 10 9

b. If no control technology, then n R = 0

CFU/site-day = (1 x 10 11 )(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 10 9 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 10 12 CFU/site-day

b. Uncontrolled = 7.0 x 10 18 CFU/site-day

Basis:CEB/Biotech/Petexm.31811(Rev) a. From generic scenario

LR = CFU b (V b )(1-n i )(n R )

Where:

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

CFU b = CFU concentration in final fermentation broth (CFU/ml - 1 x 10 11 )

n i = inactivation or sterilization efficiency = 99.9999% for processes meeting exemption criteria

V b = volume of fermentation broth = 70,000,000 ml

n R = 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 10 11 )(70,000,000)(1-0.999999)(1)

= 7.0 x 10 12 CFU/bt

CFU/site-day = 7.0 x 10 12 CFU/bt x (100 bt/yr)/100 days/yr

= 7.0 x 10 12

b. Uncontrolled.

WR
cw = (1 x 10 11 )(70,000,000)(1-0)(1)

= 7.0 x 10 18 CFU/bt

CFU/site-day = 7.0 x 10 18 CFU/bt x (100 bt/yr)/100 days/yr

= 7.0 x 10 18

C. Summary

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

Table 1: Summary of Release Estimates for a Typical Fermentation ProcessCEB/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 10 9

< 7.0 x 10 9 2.0 x 10 9

7.0 x 10 9 350

100

WATER - to w.w.t.

Cleanup

Fermentor exhaust

treatment

Rotary drum

exhaust treatment 1.4 x 10 11

6.9 x 10 9

6.9 x 10 9 1.4 x 10 17

0

0 100

100

100

LAND - to landfill

Filter cakes 7.0 x 10 12 7.0 x 10 18 100

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 exposureCEB/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 10 10 CFU/day over 250 days/yr (9 workers)

Basis:

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

broth concentration = 1 x 10 11 ); · < 0.9 ml/day (CEB dermal exposure to liquids for bench scale activities);

(1 x 10 11 CFU/ml)(< 0.9 ml/day)

= <9.0 x 10 10 CFU/day

2) Fermentation Area

<9.0 x 10 10 CFU/day over 250 days/yr (12 workers)

Basis:

· Final CFU concentration during fermentation (CFU/ml = 1 x 10 11 ); · < 0.9 ml/day (CEB dermal exposure to liquids from sampling, 1-hand incidental contact).

(1 x 10 11 CFU/ml)(< 0.9 ml/day)

= <9.0 x 10 10 CFU/day

3) Recovery Area

1.2 x 10 11 – 3.7 x 10 11 CFU/day over 250 days/yr (12 workers)

Basis:

· Final CFU concentration during fermentation (CFU/ml = 1 x 10 11 ); · 0.6 – 1.8 ml/day (CEB dermal exposure to liquids from sampling, 2-hand dermal contact).

(1 x 10 11 CFU/ml)(0.6 to 1.8 ml/day)CEB/Biotech/Petexm.31811(Rev) = 6..0 x 10 10 – 1.8 x 10 11 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 10 10

2. Fermentation 12 <9.0 x 10 10

3. Recovery 12 6.0 x 10 10 – 1.8 x 10 11

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), typicall y, 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/m 3 . 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/m 3 (average of CFU concentrations in the laboratory at 3 facilities sampled by NIOSH - generic scenario)

· 1.25 m 3 /hr standard breathing rate; · 8 hrs of exposure at this level;

(53 CFU/m 3 )(1.25 m 3 /hr)(8 hr/day) = 530 CFU/day (average)

2) Fermentation area

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

Basis:

· 263 CFU/m 3 (average of CFU concentrations around fermentor sample port at 3 plants sampled by NIOSH - generic scenario);

· 1.25 m 3 /hr standard breathing rate; · 2 hrs of exposure at this level;

(263 CFU/m 3 )(1.25 m 3 /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/m 3 (geometric mean of CFU concentrations around filter press when closed at facility sampled by NIOSH - generic scenario);

· 1.25 m 3 /hr standard breathing rate; · 2 hrs of exposure at this level;

(3906 CFU/m 3 )(1.25 m 3 /hr)(2 hr/day) = 9800 CFU/day

4) SupervisionCEB/Biotech/Petexm.31811(Rev) 81 CFU/day (average) over 250 days/yr(3 workers)

Basis:

· 8.1 CFU/m 3 (geometric mean of CFU concentrations in control room at facility sampled by NIOSH - generic scenario);

· 1.25 m 3 /hr standard breathing rate; · 8 hrs of exposure at this level;

(8.1 CFU/m 3 )(1.25 m 3 /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 digitsCEB/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 Divisi on, Office of Pollution Prevention and Toxic, U.S. EPA, Washington, DC.

2. CEB, 1997. Generic Scenario for Biotechnol ogy Premanufacture Notices - Final Report. Prepared by Eastern Research Group (ERG) for the Chemical Engineering Branch of the Economics, Exposure and Technology Division, Offi ce of Pollution Prevention and Toxic, U.S. EPA, Washington, DC. under EPA contract 68-D4-0092.