Evaluation of Exposure 

to the Chemosterilant 

Bisazir Among Biological 

Technicians – Michigan

Carlos Aristeguieta, MD, MPH

James Couch, CIH, MS, REHS/RS

Health Hazard Evaluation Report

HETA 2010-0012-3125

March 2011

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

Workplace

Safety and Health

 National Institute for Occupational 

Safety and Health

This Health Hazard Evaluation (HHE) report and any recommendations made herein are for the specific 

facility evaluated and may not be universally applicable. Any recommendations made are not to be 

considered as final statements of NIOSH policy or of any agency or individual involved. Additional HHE 

reports are available at http://www.cdc.gov/niosh/hhe/
The employer shall post a copy of this report 

for a period of 30 calendar days at or near 

the workplace(s) of affected employees. The 

employer shall take steps to insure that the 

posted determinations are not altered, defaced, 

or covered by other material during such 

period. [37 FR 23640, November 7, 1972, as 

amended at 45 FR 2653, January 14, 1980].
Health Hazard Evaluation Report 2010-0012-3125 Page i

Acknowledgments

Report Abbreviations.......................................................................ii

Highlights of the NIOSH Health Hazard Evaluation............ iii

Summary ............................................................................iv

Introduction..........................................................................1

Assessment.........................................................................6

Results.................................................................................8

Discussion .........................................................................11

Conclusions.......................................................................13

Recommendations.............................................................13

References ........................................................................15

Contents

Acknowledgments and Availability of Report.....................17
Page ii Health Hazard Evaluation Report 2010-0012-3125

μg microgram

μg/m3 micrograms per cubic meter

μg/L micrograms per liter

ACGIH® American Conference of Governmental Industrial Hygienists

cm centimeter

cm2 square centimeter

CFR Code of Federal Regulations

DNA Deoxyribonucleic acid

GA General area

HHE Health hazard evaluation

IgG Immunoglobulin G

LOD Limit of detection

LOQ Limit of quantitation

MDC Minimum detectable concentration

mL Milliliter

MSDS Material safety data sheet

NAICS North American Industry Classification System

ND Not detected

NIOSH National Institute for Occupational Safety and Health

OEL Occupational exposure limit

OOHS Office of Occupational Health and Safety

OSHA Occupational Safety and Health Administration

PAPR Powered air purifying respirator

PBZ Personal breathing zone

PEL Permissible exposure limit

PPE Personal protective equipment

REL Recommended exposure limit

STEL Short term exposure limit

TLV® Threshold limit value

TMF 3-trifluoromethyl-4-nitrophenol

TWA Time-weighted average

WEEL Workplace environmental exposure level

Abbreviations
Health Hazard Evaluation Report 2010-0012-3125 Page iii

What NIOSH Did

● We evaluated the biological station in May 2010.

● We watched employees work and spoke with them about 

their work practices and health concerns.

● We tested air, water, surfaces, and work gloves for bisazir.

● We tested urine samples from three employees for bisazir.

What NIOSH Found

● We found no bisazir in the air, water, surface, or work glove 

samples.

● We found no bisazir in the urine samples.

● Employees wore the correct personal protective equipment 

(PPE) and followed procedures.

What Managers Can Do

● Continue to talk to employees about the hazards of bisazir 

and teach them safe handling practices.

● Determine if PPE can be downgraded in future sterilization 

seasons.

What Employees Can Do

● Attend training about handling bisazir safely.

● Follow recommended work practices and PPE procedures.

The National Institute 

for Occupational Safety 

and Health (NIOSH) 

received a request for a 

health hazard evaluation 

at a biological station 

in Michigan. A federal 

government agency 

concerned about 

possible health effects to 

employees handling the 

chemosterilant bisazir 

submitted the request. 

Bisazir is used to control 

sea lampreys, an aquatic 

parasite of fish in the 

Great Lakes.

Highlights of the

NIOSH Health

Hazard Evaluation
Page iv Health Hazard Evaluation Report 2010-0012-3125

In October 2009, NIOSH received an HHE request from a federal 

government agency concerning employees’ potential exposure 

to the chemosterilant bisazir at a biological station in Michigan. 

Bisazir is used to sterilize male sea lampreys, an invasive aquatic 

parasite of the Great Lakes.

We evaluated the biological station on May 19–20, 2010. We 

walked through the facility and observed work processes, practices, 

and conditions. We spoke with employees about health and 

workplace concerns related to bisazir and collected samples for 

bisazir from air, water, surfaces, gloves, and urine.

We detected no bisazir in general area air samples (MDC ≤ 0.8 μg/

m3), PBZ air samples (MDC ≤ 2.0 μg/m3), or cotton glove samples 

(LOD = 10 μg/sample). No bisazir was detected in the employees’ 

urine samples. No bisazir was detected in bulk water samples taken 

from tanks that housed bisazir-treated sea lampreys (LOD = 0.4 

μg/L). We found that employees were aware of the potential risks 

from exposure to bisazir and that they followed administrative 

procedures and PPE recommendations.

Because we sampled over just 2 workdays and during only one 

sea lamprey spawning season, additional air and surface sampling 

during the upcoming 2011 season would be useful to confirm 

these results. If no bisazir is detected in these additional samples 

and the good work practices and engineering controls we observed 

continue, management could consider downgrading the level of 

PPE required for working in the injection and tank rooms. The 

agency had previously used semen analysis to identify possible 

adverse reproductive effects, but discontinued this program in 

2009 as semen analysis is not an appropriate measure of the effects 

of bisazir exposure.

NIOSH evaluated 

potential exposure to the 

chemosterilant bisazir 

among employees of a 

biological station. We 

detected no bisazir in 

environmental or urine 

samples. Engineering and 

administrative measures 

and PPE effectively 

protected employees.

Summary

Keywords: NAICS 924120 (Administration of Conservation 

Programs), sea lamprey, bisazir, alkylating agent, chemosterilant
Health Hazard Evaluation Report 2010-0012-3125 Page 1

Figure 1. Photograph of two male sea lampreys that escaped from the 

transfer basket, lying by the side of the tank truck. The arrow indicates 

the missing dorsal fin, which was cut off to mark the sea lamprey as 

sterilized.

Introduction

In October 2009, NIOSH received an HHE request from a federal 

government agency concerning potential employee exposure to 

the chemosterilant bisazir that is used at a biological station in 

Michigan. Bisazir is used to sterilize male sea lampreys, an invasive 

aquatic parasite of the Great Lakes.

Sea Lampreys 

The sea lamprey (Petromyzon marinus, Figure 1) is an eel-like aquatic 

animal with a suction-cup-like jawless mouth. Adult sea lampreys 

measure 12 to 48 inches in length. They parasitize fish by adhering 

to them with their mouth, filing a hole with their tongue, and 

suctioning blood and body fluids. Sea lampreys began invading the 

Great Lakes in the mid-1800s, entering from the Atlantic Ocean 

through the Erie Canal, reaching Lake Superior in the 1940s and 

causing great damage to the fish population [Christie and Goddard 

2003]. In 1955, Canada and the United States created the Great 

Lakes Fisheries Commission to protect the fisheries of the Great 

Lakes. The U.S. Department of the Interior Fish and Wildlife 

Service is the U.S. agency represented on the Commission.
Page 2 Health Hazard Evaluation Report 2010-0012-3125

Introduction 

(continued)

Figure 2. Photograph of the injection room, with fume hood on the right, 

an employee operating the automated injection machine in the center, 

and bisazir storage refrigerator between them. Sea lampreys are held in 

the tank on the left before sterilization.

Control of sea lampreys has been achieved primarily through 

application of the selective larvicide TMF in tributaries of the 

Great Lakes, migratory barriers, and trapping. A secondary 

technique of releasing sterile male sea lampreys to compete with 

fertile ones was implemented in 1991. This sterile male release 

technique relies on trapping large numbers of sea lampreys in 

tributary rivers as they begin their spawning run in the spring 

[Twohey et al. 2003].

Sterilization Process 

The sea lamprey sterilization facility operates during the sea 

lamprey spawning season, which extends from the end of April 

to early July. This custom-built sterilization facility houses the 

injection room (Figure 2), a large tank room with holding tanks 

and a water pump system, a decontamination room, bathrooms 

with lockers and showers, an office, and a storage room with 

workshop. The sterilization room has a general area ventilation 

system and a fume hood equipped with activated carbon and high 

efficiency filters. Both systems exhaust 100% of the ventilated air 

outside the building. During the spawning season the sterilization 

facility operates daily. At the end of the season the facility is 

decontaminated and shut down for the remainder of the year.
Health Hazard Evaluation Report 2010-0012-3125 Page 3

Introduction (continued) The number and trend of sea lampreys trapped over the spawning 

run gives managers an indication of how many sterilization sessions 

are needed. Up to 350 sea lampreys are sterilized in one session, 

with up to four sessions per day, for a total of 1,400 sea lampreys 

per day. On average 26,500 sea lampreys are sterilized per season, 

with a maximum of 43,184 in 2000 [Bergstedt and Twohey 2007].

Eight employees (all but one male) were working with bisazir at the 

time of our visit. These employees received an annual occupational 

health exam post season, including pulmonary function test, and 

because some job tasks exposed them to a wilderness environment, 

Lyme disease serology. In the past, male employees who worked 

with bisazir had an annual semen analysis to identify possible 

adverse reproductive effects. A review of the program in 2009 by 

the agency revealed that this was an inappropriate test to detect 

adverse effects from bisazir, and it was discontinued.

Employees entering the injection room and tank room areas 

were required to wear a 3M Breathe-EasyTM loose-fitting PAPR 

with cartridges protective against organic vapors, sulfur dioxide, 

chlorine, and hydrogen chloride. The PAPR was selected because 

of comfort and ease of use during long periods, and because it does 

not require fit testing. The site had a written respiratory protection 

program that included respirator medical clearance for new hires, 

continuing medical clearance evaluations, proper respirator 

maintenance and storage procedures, and cartridge selection in 

accordance with OSHA respiratory protection standards [CFR 

1910.134].

To minimize skin contact with bisazir, the employees wore DuPont 

Tychem® SL (Saranex) coveralls and butyl rubber gloves and boots. 

The gloves and boots were sealed to the Saranex coveralls with 

tape. To sterilize sea lampreys, two employees suited up to enter the 

containment area. Following a schedule, they alternated during the 

day with two other employees. Employees worked 10 consecutive 

days in the sterilization facility, followed by 4 days off.

The sea lampreys were transported in trucks to the biological 

station where males and females were manually separated. Female 

sea lampreys were shipped off to become dissection specimens for 

biology classes. Male sea lampreys were placed into holding tanks 

and transported into the sterilization room. A technician placed 

an individual sea lamprey on a scale to determine the weightdependent (100 mL per kilogram) dose of a 1% solution of bisazir 

(P, P-bis(1-aziridinyl)-N-methylphosphinothiotic amide) to inject. 
Page 4 Health Hazard Evaluation Report 2010-0012-3125

Introduction 

(continued) The sea lamprey was then inserted by hand into the automated 

injection machine where the injection occurred in a closed, 

ventilated box (Figure 3). The injection machine then released the 

sea lamprey into a holding tank outside the sterilization room. Sea 

lampreys were then held in tanks for 48 hours to allow them to 

metabolize the bisazir.

Figure 3. Photograph of the automated injection machine. Sea lampreys 

were weighed on the scale, then inserted tail first through the inlet. The 

machine pulled the sea lamprey in, injected it with the correct dose of 

bisazir, and released it into a holding tank. The machine was usually 

closed during operation but is open in the photograph for mechanical 

troubleshooting.

The bisazir solution was prepared in a Hamilton SafeAir® fume 

hood in the sterilization room. Pure bisazir powder was received 

from the manufacturer in premeasured packets and stored cold. 

To make a 1% solution, the bisazir was removed from refrigerated 

storage, placed inside the fume hood, and then opened and 

weighed. The bisazir powder was emptied into a flask containing 

the premeasured saline and a magnetic stirrer. The flask was then 

placed on the magnetic stirrer platform and mixed for at least 2 

hours to ensure complete solution of the bisazir powder. While 

preparing bisazir solution within the fume hood, employees 

wore an additional clean pair of unlined nitrile gloves that were 

stored and kept inside the fume hood to prevent bisazir powder 
Health Hazard Evaluation Report 2010-0012-3125 Page 5

Introduction (continued) contamination. The bisazir solution was then manually transported 

to the automated injection machine and loaded into a closed 

delivery system. Decontamination was accomplished by using 

muriatic acid (pH = 2) to neutralize the bisazir.

After 48 hours in holding tanks, employees marked the sterilized 

male sea lampreys by removing part of their dorsal fin before 

release into the St. Mary’s river. Once released, sterile male sea 

lampreys resume their spawning run, swimming back to the rivers 

to find and mate with females. Although sterile male sea lampreys 

produce milt and fertilize the eggs of the females, the resulting 

embryos are not viable.

Water from Lake Huron supplied the sea lamprey holding tanks. 

The water was circulated back to the lake after passing through 

a carbon filtration system to prevent contaminating the lake 

with bisazir. The outflow water was tested weekly for bisazir at a 

laboratory across the street.

In 2002, prompted by the use of bisazir and the larvicides TMF 

and Bayluscide, the agency evaluated the biological station’s 

operation and drafted employee medical standards and a medical 

examination protocol. The agency re-evaluated these procedures 

in 2009, after an employee had been medically restricted from 

working with bisazir because of abnormal semen analysis results. 

The 2009 evaluation led to this HHE request that asked (1) what 

is the potential for exposure to bisazir at the facility?, (2) are there 

adverse health effects related to exposure to bisazir?, (3) are the 

proper tests being used to monitor exposure?, and (4) is there a 

better way to monitor potential exposure to bisazir?
Page 6 Health Hazard Evaluation Report 2010-0012-3125

Prior to our site visit we talked with the requestors by phone to 

review previous evaluations and to request an MSDS for bisazir 

and a copy of the medical standards. We also requested copies 

of medical records from one employee with abnormal semen 

analysis. We reviewed the 2002 and 2009 evaluations of the 

facility’s procedures by the agency, the medical standards and 

medical examination protocol, and the OSHA Form 300 Log of 

Work-related Injuries and Illnesses for the facility for the years 

2007–2009.

During our site visit on May 19–20, 2010, we held opening and 

closing meetings with local managers and employee representatives. 

Three of the four employees who were scheduled to work in the 

injection room signed consent forms and agreed to provide urine 

to be tested for bisazir. We spoke with the employees present at the 

time about their work practices when handling bisazir and about 

any health concerns.

We collected PBZ and GA air, wipe, and cotton glove liner samples 

to test for bisazir. The PBZ air samples were taken on employees 

entering into either the injection room or the tank room. 

Employees typically worked 3 to 4 hours in the injection or the 

tank room. GA air samples were placed in the decontamination 

area, sterilization room, and tank room. PBZ and GA air samples 

were taken using AirChek 2000 air sampling pumps calibrated to 

1 liter per minute. PBZ samples were taken for the time spent in 

either the injection room or the tank room (186 to 301 minutes). 

GA samples were taken for the working day (429 to 528 minutes). 

Air samples were collected on OSHA Versatile Sampler-2 sorbent 

tubes and stored on ice immediately after sampling was concluded. 

The OSHA Versatile Sampler-2 tubes were analyzed for bisazir by 

gas chromatography equipped with flame photometric detection 

with an LOD of 0.4 μg of bisazir per sample and an LOQ of 1.4 

μg of bisazir per sample. The MDC of bisazir in PBZ samples was 

2 μg/m3 based on a 267-liter air sample. The MDC of bisazir in 

GA samples was 0.8 μg/m3 based on a 474-liter air sample. The 

sampling method was NIOSH Method 5600 for organophosphorus 

pesticides modified for analysis of bisazir [NIOSH 2011]. The front 

and back sections of the tubes were transferred to separate 4-mL 

vials, and 2 mL of a 10% acetone and 90% toluene solution was 

added. The quartz fiber filter was desorbed together with the front 

sorbent section. The vials were placed on a mechanical shaker for 

60 minutes and subsequently transferred to amber autosampler 

vials for analysis by gas chromatography equipped with flame 

photometric detection.

Assessment
Health Hazard Evaluation Report 2010-0012-3125 Page 7

Assessment (continued) Surface wipe samples were taken using Alpha Texwipe® wipes 

moistened with 100% isopropanol. A 10 cm × 10 cm square 

template was used to determine a 100 cm2 sampling area. The 

surface wipe samples were collected in the injection room, tank 

room, decontamination room, men’s and women’s restrooms, 

office area, and on an outdoor storage tank that held sterilized 

sea lampreys awaiting release. The sample media was analyzed for 

bisazir by gas chromatography equipped with flame photometric 

detection with an LOD of 2 μg of bisazir per sample and an LOQ 

of 8.0 μg of bisazir per sample. The sampling method is NIOSH 

Method 5600 for organophosphorus pesticides modified for 

analysis of bisazir [NIOSH 2011]. The wipe samples were placed in 

individual desorption vials. The wipes were chemically desorbed 

by adding 15 mL of 10% acetone/90% toluene solution. The vials 

were placed in a mechanical shaker for 60 minutes. After shaking, 

the samples were transferred to amber autosampler vials for 

analysis by gas chromatography equipped with flame photometric 

detection.

To evaluate potential dermal exposure we asked employees to wear 

100% cotton gloves (Lab Safety Supply, Janesville, Wisconsin)

under their Tyvek® gloves. The cotton gloves were collected at 

the end of the shift by NIOSH investigators wearing sterile gloves 

and analyzed for bisazir to evaluate potential dermal exposure 

from permeation or leakage through the gloves or contamination 

when the employee donned or doffed them. Each pair of cotton 

gloves was analyzed for bisazir by gas chromatography equipped 

with flame photometric detection. The LOD was 10 μg of bisazir 

per sample, and the LOQ was 35 μg of bisazir per sample. The 

sampling method was NIOSH Method 5600 for organophosphorus 

pesticides modified for analysis of bisazir [NIOSH 2011]. The 

individual gloves were placed into 100-mL wide mouth jars. 

The jars were chemically desorbed by adding 15 mL of 10% 

acetone/90% toluene solution. The jars were placed in a 

mechanical shaker for 60 minutes. After shaking, an aliquot of the 

sample was transferred to amber autosampler vials for analysis by 

gas chromatography equipped with flame photometric detection.

We collected bulk water samples from the retention tanks by 

manually lowering a glass jar into the tank while wearing protective 

gloves. The jar was rinsed three times before collecting the sample. 

Each bulk sample was analyzed for bisazir by gas chromatography 

equipped with flame photometric detection. The LOD was 

0.4μg/L of bisazir per sample, and the LOQ was 1.4μg/L of bisazir 
Page 8 Health Hazard Evaluation Report 2010-0012-3125

Assessment (continued) per sample. The sampling method was Environmental Protection 

Agency Method 8141A for organophosphorus compounds 

modified for analysis of bisazir [Environmental Protection Agency 

1992]. The bisazir was extracted from the water using a separatory 

funnel technique. The water sample was extracted three separate 

times with 60 mL of methylene chloride. The three extractions 

were combined. The extract was evaporated down to approximately 

1 mL under nitrogen using a Turbovap concentrator. The 

methylene chloride was solvent exchanged with n-hexane at this 

point. The extract was concentrated down to a final volume of 1 

mL using the Turbovap concentrator. The extracts were analyzed by 

gas chromatography equipped with flame photometric detection.

Urine samples were collected by employees and submitted for 

analysis. Sample aliquots of 20 mL were extracted and then 2.0 mL 

of methylene chloride was added to each sample. The sample was 

shaken for 10 minutes and then allowed to stand for at least one 

hour. The methylene chloride extract was then dried with sodium 

sulfate prior to the addition of internal standards. Analysis was 

completed by gas chromatography/mass spectrometry. The LOD 

was 25 μg/L of bisazir per sample, and the LOQ was 83 μg/L of 

bisazir per sample.

No OELs exist for bisazir. The scientific literature contains no 

reports of human health effects from exposure to bisazir.

We detected no bisazir in any of the samples we took. No bisazir 

was detected (LOD of 25 μg of bisazir per liter) in the urine of the 

three employees. As shown in Table 1, airborne bisazir was not 

detected; concentrations were below the MDC of 0.8 μg/m3 (for 

a 474-liter GA air sample) and 1.5 μg/m3 (for a 267-liter PBZ air 

sample). No bisazir was detected (LOD = 2 μg of bisazir per 100 

cm2) on any of the surface wipe samples (Table 2). As shown in 

Table 3, no bisazir was detected (LOD =10 μg of bisazir per sample) 

on any of the cotton gloves worn under the protective gloves by 

employees who performed various activities, including mixing 

bisazir in the sterilization chamber, weighing and injecting bisazir, 

and transporting sea lampreys in the tank room.

We observed employees using proper work practices when working 

with bisazir. All observed employees were wearing the proper PPE, 

utilizing engineering controls, and thoroughly decontaminating 

Results
Health Hazard Evaluation Report 2010-0012-3125 Page 9

Results (continued)

Our conversations with employees indicate that they were aware of 

the potential risks from exposure to bisazir (i.e., it is an alkylating 

agent that can damage tissues and have mutagenic and teratogenic 

effects). Employees did not report any recognized breakdowns in 

the engineering controls (automated injector or general ventilation) 

or PPE that could have led to accidental exposure to bisazir.

their equipment with the muriatic acid solution. We observed one 

preparation of bisazir solution during which employees adhered 

strictly to the standard operating procedures posted near the fume 

hood.

Table 1. Bisazir concentrations in air samples collected on May 19–20, 2010

Sample Location Sample Description Time 

(minutes) Conc.

PERSONAL BREATHING ZONE

Injection room Weighing and loading sea lampreys into injection machine 196 ND*

Injection room Weighing and loading sea lampreys into injection 

machine. Also mixed bisazir into solution.

186 ND*

Injection room Weighing and loading sea lampreys into injection machine 354 ND*

Tank room Transferring sea lampreys, emptying holding tank to 

outside tank

297 ND*

Tank room Transferring sea lampreys, emptying holding tank to 

outside tank

301 ND*

Tank room Transferring sea lampreys 270 ND*

GENERAL AREA

Injection room Table across from fume hood near injection machine 471 ND†

Injection room Table across from fume hood near injection machine 474 ND†

Tank room On top of holding tank P1 473 ND†

Tank room On top of holding tank P1 475 ND†

Tank room Far corner near transfer tank from outside 469 ND†

Tank room Far corner near transfer tank from outside 469 ND†

Decontamination room On window sill near hallway to injection room 528 ND†

Decontamination room On window sill near hallway to injection room 429 ND†

*The MDC was 2.0 μg/m3for a 267-liter air sample (average PBZ sample volume).

†The MDC was 0.8 μg/m3 for a 474-liter air sample (average GA sample volume).
Page 10 Health Hazard Evaluation Report 2010-0012-3125

Results (continued)

Table 2. Bisazir surface sample results from May 19–20, 2010

Description Result

(μg/100 cm2)

Door handle exiting tank room into decontamination area† ND*

Side of tank P2 in tank room ND*

Table of injection room ND*

Substrate of fume hood before mixing bisazir ND*

Substrate of fume hood after mixing bisazir ND*

Door handle from restroom to decontamination area† ND*

Men’s restroom floor before entering decontamination area ND*

Decontamination area floor before entering men’s restroom ND*

Women’s restroom floor before entering decontamination area ND*

Hallway floor outside injector room ND*

Side of tank 1 outside holding tank ND*

Office floor next to computer ND*

Women’s restroom floor ND*

*ND = not detected (below the LOD of 2.0 μg per sample)

†The 100 cm2 template could not be used on door handles. Sample was taken from the surface of the door 

handle.

Table 3. Bisazir sample results for cotton glove samples* from May 19–20, 2010

Sample Location Sample Description Result

(μg/sample)

Injection room Weighing and injecting sea lampreys ND†

Injection room Mixing bisazir solution ND†

Injection room Weighing and injecting sea lampreys ND†

Tank room Transferring sea lampreys, emptying holding tank to 

outside tank

ND†

Tank room Transferring sea lampreys, emptying holding tank to 

outside tank

ND†

Tank room Transferring sea lampreys ND†

Tank room Transferring sea lampreys ND†

*Each sample consisted of a pair of cotton gloves worn by employees under their outer gloves.

†ND = not detected (below the LOD of 10 μg of bisazir per sample)
Health Hazard Evaluation Report 2010-0012-3125 Page 11

Results (continued) Review of semen analysis reports from one employee showed 

variability in sperm count from normal to decreased and atypical 

sperm morphology. Most samples were from the winter months, 

beyond the time frame where spermatogenesis would have been 

affected from a springtime exposure to a sterilant. The other 

employees had no reports of semen analysis abnormalities or of 

infertility or adverse reproductive outcomes.

The OSHA Logs for the years 2007–2009 contained reports of 22 

injuries and one illness, a tick bite believed to have caused Lyme 

disease in 2009. These injuries and illness were not sustained at the 

sterilization facility, but rather during field work. No unusual injury 

types or patterns were reported.

Bisazir is an alkylating agent in the nitrogen mustard class developed 

in the 1960s for agricultural pest control applications. It was found to 

be an effective chemosterilant in sea lampreys in tests conducted in 

the 1970s [Hanson and Manion 1980].

Studies on sea lampreys show that bisazir-induced infertility 

results from DNA damage in the sperm cells, while the numbers, 

morphology, and motility of the sperm are unaffected. The damage 

consists of DNA adducts, and DNA-DNA and DNA-protein linkages 

[Ciereszko et al. 2005]. An experiment on mice showed that bisazir 

induced abnormal metaphases in spermatogonia and sperm head 

abnormalities [Devi and Reddy 1985]. These two findings suggest that 

semen analysis is not appropriate for detecting the type of damage 

induced by bisazir in men, assuming the effects are the same as for 

mice. Although bisazir has not been used in humans, similar nitrogen 

mustard compounds (e.g. cyclophosphamide and thiotepa) are used 

as chemotherapeutics for treating cancers. In mice, cyclophosphamide 

causes germ cell phase-specific DNA damage [Codrington et al. 2004]. 

Toxic clinical effects of cyclophosphamide and thiotepa include 

defective spermatogenesis and oogenesis. Adverse effects to a fetus 

may be caused by either male or female treated parent, and they may 

cause infertility in males and females [DrugPoints® System 2009a; 

DrugPoints® System 2009b].

Furthermore, the medical surveillance program for the biological 

station does not address reproductive issues in female employees, who 

are potentially equally at risk of adverse reproductive health effects 

from exposure to bisazir.

Employees are understandably concerned about potential adverse 

reproductive effects and should be informed about what they can 

Discussion
Page 12 Health Hazard Evaluation Report 2010-0012-3125

Discussion (continued) do to minimize their risk of such effects. The Washington State 

Department of Labor and Industries’ Safety and Health Assessment 

and Research for Prevention Program has prepared a comprehensive 

technical report titled “Workplace Hazards to Reproduction and 

Development: A Resource for Workers, Employers, Health Care 

Providers, and Health & Safety Personnel” [http://www.lni.wa.gov/

Safety/Research/files/repro_dev.pdf]. The section on “How Workers 

Can Protect Themselves” (p. 55) can be adapted for the needs of the 

biological station.

Bisazir exposure pathways can potentially include inhalational (from 

powder and liquid forms) and dermal routes. The vapor pressure of 

bisazir is not available from the MSDS. Review of scientific literature 

estimates the vapor pressure of bisazir at 0.005 millimeters of mercury 

at 25oC, which indicates that bisazir is highly volatile [Carlson and 

Bailey1981]. However, the vapor pressure was estimated by comparing 

gas chromatography retention times to n-paraffins, which is not 

an ideal method for determining vapor pressure but is acceptable 

[Letcher and Naicker 2004]. Additional information that may also 

be used to better determine the volatility of bisazir such as Henry’s 

constant (determined by both volatility and water solubility) was not 

available. Bisazir in powdered form can be an inhalational hazard. In 

a saline solution, bisazir should be considered volatile until proven 

otherwise [Streicher 2010]. Bisazir is water soluble as evidenced by the 

chemical structure and the chemical analysis of effluent water from 

the station. Bisazir in liquid form or water contaminated with bisazir 

should be considered a dermal hazard.

Our sampling results did not detect bisazir in the air or on work 

surfaces, and no bisazir was detected in the urine samples from three 

employees. However, because we collected air and surface samples 

over just 2 workdays during only one sea lamprey spawning season, 

additional sampling may be needed to confirm our findings. On the 

basis of observations of protective standard operating procedures, 

good work practices, adequate engineering controls, and negative 

bisazir sample results, the level of PPE may be downgraded in 

subsequent sterilization seasons.

Regarding the four questions in the HHE request, we found that (1) 

the potential for bisazir exposure was low, (2) no adverse health effects 

were attributable to bisazir exposure, (3) semen analysis was not the 

proper test to detect reproductive effects of bisazir, and (4) despite 

limitations in detection limits of chemical analysis, environmental 

and urine sampling for bisazir are better than semen analysis to 

monitor potential exposure to bisazir.

Current Lyme disease assays (i.e., enzyme immune assay-specific IgG 

antibodies to Borrelia burgdorferi) have poor specificity, are not useful 
Health Hazard Evaluation Report 2010-0012-3125 Page 13

Discussion (continued) in screening persons without objective manifestations of disease, and 

are costly [Wormser et al. 2006]. Testing for Lyme disease should be 

guided by the presence of symptoms and signs compatible with Lyme 

disease. Although the biological station is in an area where infected 

ticks are rare [ALDF 2011], employees should be educated about the 

signs and symptoms of Lyme disease because their field tasks expose 

them to ticks.

Employees were protected from bisazir exposure through engineering 

controls (ventilated automated injection device and fume hood), 

administrative practices, and PPE. Nevertheless, given the potential 

toxicity of bisazir, management should continue looking for effective 

chemosterilants that are less toxic to humans. Our sampling results 

did not detect bisazir in the air or on work surfaces, and no bisazir 

was detected in the urine samples from three employees. However, 

because we sampled over just 2 workdays and during only one sea 

lamprey spawning season, additional air and surface sampling during 

the upcoming season would be useful to confirm these results. If no 

bisazir is detected in these additional air and surface samples and 

the good work practices and engineering controls that we observed 

during this evaluation continue, the management of the biological 

station may want consider downgrading the level of PPE required for 

the employees working in the injection and tank rooms.

On the basis of our findings, we recommend the actions listed below 

to create a more healthful workplace. We encourage the facility to use 

an employee-employer health and safety committee or working group 

to discuss the recommendations in this report and develop an action 

plan. Those involved in the work can best set priorities and assess the 

feasibility of our recommendations for the specific situation at the 

facility. Our recommendations are based on the hierarchy of controls 

approach. This approach groups actions by their likely effectiveness 

in reducing or removing hazards. In most cases, the preferred 

approach is to eliminate hazardous materials or processes and install 

engineering controls to reduce exposure or shield employees. Until 

such controls are in place, or if they are not effective or feasible, 

administrative measures and/or personal protective equipment may 

be needed.

Elimination and Substitution

Elimination or substitution of a toxic/hazardous process material 

is a highly effective means for reducing hazards. Incorporating this 

Conclusions

Recommendations
Page 14 Health Hazard Evaluation Report 2010-0012-3125

Recommendations

(continued) strategy into the design or development phase of a project, commonly 

referred to as “prevention through design,” is most effective because it 

reduces the need for additional controls in the future.

● Investigate the use of other effective chemosterilants that are 

less toxic to humans.

Engineering Controls

Engineering controls reduce exposures to employees by removing the 

hazard from the process or placing a barrier between the hazard and 

the employee. Engineering controls are very effective at protecting 

employees without placing primary responsibility of implementation 

on the employee.

● Investigate ways to further isolate the bisazir supply to the 

automated injector such as extending the bisazir delivery system 

directly into the fume hood. This would allow for replacement 

of bisazir vials to occur within the fume hood and eliminate the 

potential for spills while transporting bisazir to the automated 

injection device.

Administrative Controls 

Administrative controls are employer-dictated work practices and 

policies to reduce or prevent exposures to workplace hazards. 

The effectiveness of administrative changes in work practices for 

controlling workplace hazards is dependent on employer commitment 

and employee acceptance. Regular monitoring and reinforcement 

are necessary to ensure that control policies and procedures are not 

circumvented in the name of convenience or production.

● Employees handling bisazir should be counseled regarding 

potential toxicity and reproductive hazards of bisazir, including 

overall risks of common adverse reproductive outcomes in the 

general population.

● Discontinue annual Lyme disease serology.

● Educate employees about signs and symptoms of Lyme disease.

Personal Protective Equipment 

PPE is the least effective means for controlling employee exposures. 

Proper use of PPE requires a comprehensive program, and calls for a 
Health Hazard Evaluation Report 2010-0012-3125 Page 15

Recommendations

(continued) high level of employee involvement and commitment to be effective. 

The use of PPE requires the choice of the appropriate equipment to 

reduce the hazard and the development of supporting programs such 

as training, change-out schedules, and medical assessment if needed. 

PPE should not be relied upon as the sole method for limiting 

employee exposures. Rather, PPE should be used until engineering 

and administrative controls can be demonstrated to be effective in 

limiting exposures to acceptable levels.

● Use the current PPE until exposures are re-evaluated: 3M 

Breathe-Easy Airstream loose-fitting PAPR with organic vapor/

sulfur dioxide/hydrogen chloride/chlorine cartridges, DuPont 

Tychem SL coveralls, and butyl rubber gloves and boots.

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Health Hazard Evaluation Report 2010-0012-3125 Page 17

The Hazard Evaluations and Technical Assistance Branch (HETAB) 

of the National Institute for Occupational Safety and Health 

(NIOSH) conducts field investigations of possible health hazards 

in the workplace. These investigations are conducted under the 

authority of Section 20(a)(6) of the Occupational Safety and Health 

Act of 1970, 29 U.S.C. 669(a)(6) which authorizes the Secretary of 

Health and Human Services, following a written request from any 

employer or authorized representative of employees, to determine 

whether any substance normally found in the place of employment 

has potentially toxic effects in such concentrations as used or found. 

HETAB also provides, upon request, technical and consultative 

assistance to federal, state, and local agencies; labor; industry; and 

other groups or individuals to control occupational health hazards 

and to prevent related trauma and disease.

The findings and conclusions in this report are those of the authors 

and do not necessarily represent the views of NIOSH. Mention of any 

company or product does not constitute endorsement by NIOSH. In 

addition, citations to websites external to NIOSH do not constitute 

NIOSH endorsement of the sponsoring organizations or their 

programs or products. Furthermore, NIOSH is not responsible for 

the content of these websites. All Web addresses referenced in this 

document were accessible as of the publication date.

This report was prepared by Carlos Aristeguieta and James Couch 

of HETAB, Division of Surveillance, Hazard Evaluations and Field 

Studies. Analytical support was provided by Charles E. Neumeister, 

Jennifer L. Roberts, and Robert P. Streicher, Division of Applied 

Research and Technology, and Bureau Veritas North America. Health 

communication assistance was provided by Stefanie Evans. Editorial 

assistance was provided by Ellen Galloway. Desktop publishing was 

performed by Robin Smith.

Copies of this report have been sent to employee and employer 

representatives at the federal government agency in Michigan, the 

state health department, and the Occupational Safety and Health 

Administration Regional Office. This report is not copyrighted and 

may be freely reproduced. The report may be viewed and printed at 

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National Technical Information Service at 5825 Port Royal Road, 

Springfield, Virginia 22161.

Acknowledgments and

Availability of Report
Below is a recommended citation for this report:

NIOSH 2011. Health hazard evaluation report: evaluation of exposure to the 

chemosterilant bisazir among biological technicians – Michigan. By Aristeguieta C, 

Couch J. Cincinnati, OH: U.S. Department of Health and Human Services, Centers 

for Disease Control and Prevention, National Institute for Occupational Safety and 

Health, NIOSH HETA No. 2010-0012-3125.

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