EPA/600/R-07/055
                                           September 2007
Proceedings of the Biosolids Exposure
         Measurement Workshop
               National Exposure Research Laboratory
                Office of Research and Development
               U. S. Environmental Protection Agency
                 26 W. Martin Luther King Drive
                   Cincinnati, OH 45268

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Proceedings  of the  Biosolids Exposure
           Measurement Workshop
                            Edited by
             Michael W. Ware, National Exposure Research Laboratory
              Ann C. Grimm, National Exposure Research Laboratory
                    Workshop Steering Committee

              G. Shay Fout, National Exposure Research Laboratory
              Ann C. Grimm, National Exposure Research Laboratory
              Bruce Mintz, National Exposure Research Laboratory
          James E. Smith, National Risk Management Research Laboratory
          Laurel J. Staley, National Risk Management Research Laboratory
             Michael W. Ware, National Exposure Research Laboratory

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                              Abstract
Sludges are generated during the processing of wastewater.  These sludges are
further treated,  primarily to  reduce the concentration of pathogens, to produce
biosolids which are then  beneficially  used on land.  In the  United States, the
Environmental Protection  Agency regulates biosolids through the 1993 40 CFR
Part 503 regulations. A 2002 National Academy of Sciences-National Research
Council report, "Biosolids  Applied to Land: Advancing Standards and Practices,"
concluded that additional scientific work was needed to reduce uncertainty about
human health effects from exposure to biosolids.  The final Agency response to
this report was published in the Federal Register in 2003. The response included
an action plan with 14 projects related to the treatment and disposal of biosolids.
One of these  projects was to conduct  a Biosolids Exposure Measurement
Workshop.  This  workshop was held March  16-17, 2006, at the Andrew W.
Breidenbach Environmental Research  Center,  Cincinnati, OH.

This document  is a summary of the workshop. It  describes presentations given
by  16  experts  on issues relevant to  measuring  human  exposure to  biosolids
contaminants and outlines the topics covered during a panel discussion session.
It  concludes  with a  list of research  needs that,  if  met,  will  enable  the
environmental  community to better  evaluate human  exposure to  biosolids
contaminants.  In the long-run, the goal of this workshop is to help enable the
Agency to better assess the  risk associated with the land application of biosolids.

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                              Notices
The views expressed in this Summary Report are those of the individual authors
and may not necessarily reflect the  views and policies of the United States
Environmental  Protection Agency (USEPA).   Sections  prepared by Agency
scientists have  been reviewed  in  accordance  with  USEPA's  peer and
administrative review policies and approved for presentation and publication.
Mention of trade names or commercial products does not constitute endorsement
or recommendation by USEPA for use.
                                   in

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                                             Contents
Abstract	ii
Notices 	iii
Acronyms and Abbreviations	vi
Introduction	1
  Workshop Background and Objectives
     Bruce Mintz	1
  Land Application and Sludge Treatment
     James E. Smith	2
  Protocols for the Timely Investigation of Potential Health Incidents Associated with Biosolids Land Application
     Alfred P. Dufour	2
  Community Perspective of Biosolids Land Application
     Maureen Reilly	2
Current Exposure Measurement Methods and Risk Assessment	4
  Multi-media Sampling at a Biosolids Land Application Test Site
     Eric A. Foote	4
  Pathogen Risk Assessment for Biosolids: Recent Developments
     Jeffrey A. Seller	5
Sludge Fate and Transport	6
  Analyzing Biosolids for Microorganisms to Achieve Regulatory Compliance
     MarkC. Meckes	6
  Fate and Transport Models
     Charles P. Gerba	6
Dermal Exposure	7
  Non-invasive Assessment of Dermal Exposure
     Karla D. Thrall	7
Odors and Irritants	8
  Odor, Irritation and Health Symptoms from Biosolids
     Pamela H. Dalton	8
  Odor Measurements  and Impacts from an Experimental Biosolids Land Application Site
     Robert H. Forbes, Jr	8
Particulates	9
  Airborne Particulates: Technologies and Measurement Issues
     Patricia D. Millner	9
Pathogens and Endotoxins	10
  Biomarkers of Viral Exposure
     G. Shay Fout	10
  Community and Occupational Risk from Bioaerosols During Land Application of Biosolids
     Ian  L. Pepper	10
  Improving the Efficiency of an Impinger for Collecting Bacteria During a Biosolids Application Field Study
     Edwin F. Barth	11
  Endotoxins
     Nancy C. Burton	11
  Occupational Exposure Assessments at WWTPs
     Nancy C. Burton	11
                                                   IV

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                                     Contents (continued)
Panel Discussion Session	13
Bibliography	15
Appendices
  A    Biosolids Exposure Measurement Workshop Agenda	16
  B    Participant List	18
  C    Update on Incident Response Project	24
  D    Technical Papers	25
       D-1    Non-Invasive Assessment of Dermal Exposure	26
       D-2    Odor, Irritation, and Health Symptoms from Biosolids Land Applications	30
       D-3    Detection and Measurement of Odor, Ammonia, and Hydrogen Sulfide Emissions as part of USEPA
              Biosolids Land Application Research Project	34
       D-4    Community and Occupational Risk from Bioaerosols during Land Application of Biosolids	37
       D-5    Lessons Learned Regarding the Use of an Impinger for Collecting Airborne Bacteria during a
              Biosolids Application Field Study	40

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                              Acronyms and Abbreviations
AGI          All glass impinger
CDC         Centers for Disease Control and Prevention
CFR         Code of Federal Regulation
D/T          Dilution/threshold
ELISA        Enzyme-Linked Immunosorbent Assay
FT           Foot
FTIR         Fourier Transform Infrared Spectroscopy
HPC         Heterotrophic plate counts
M            Meter
MIN          Minute
MRA         Microbial risk assessment
MAS         National Academy of Sciences
NCEA        National Center for Environmental Assessment (USEPA)
NOT         National Decontamination Team (USEPA)
NERL        National Exposure Research Laboratory (USEPA)
NHEERL     National Health and Environmental Effects Research Laboratory (USEPA)
NIOSH       National Institute for Occupational Safety and Health
NRC         National Research Council
NRMRL      National Risk Management Research Laboratory (USEPA)
OW          Office of Water (USEPA)
OW-OWM     Office of Water Office of Wastewater Management (USEPA)
OW-OST     Office of Water Office of Science and Technology (USEPA)
PBPK        Physiologically based pharmacokinetic
PCR         Polymerase chain reaction
PI            Principal Investigator
PM2.5        Particulate matter 2.5 urn or smaller
PPM         Parts per million
PPMV        Parts per million by volume
QA           Quality assurance
QAPP        Quality assurance project plan
QC           Quality control
RFP         Request for proposal
SEC         Second
USDA        United States Department of Agriculture
USEPA      United States Environmental Protection Agency
UV           Ultraviolet
VOC         Volatile organic compound
VIC          Volatile inorganic compound
WERF        Water Environment Research Foundation
WWTP       Wastewater treatment plant
                                                VI

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                                            Introduction
The pathogen and vector attraction reduction of treated
sludge is regulated by the U.S. Environmental Protection
Agency (USEPA) through the Subpart D requirements of
the 40  CFR Part  503  Rule  (USEPA,  1993).    This
regulation was promulgated in 1993  under the authority
of the Clean Water Act.  A  National Academy  of
Sciences (NAS)-National Research Council (NRC) 2002
report entitled,  "Biosolids Applied to Land: Advancing
Standards and  Practices," noted that there  is a  limited
amount of information  on the effect of  biosolids  land
application on individuals living or working near such
sites.   Although the  report  states that there  is no
documented scientific evidence that Part 503 regulations
have  failed to protect public  health, it concluded that
additional scientific work is needed to reduce uncertainty
about the potential  for  adverse  human  health  effects
from  exposure  to   biosolids.   The research needed
includes  gathering   epidemiological  data, investigating
allegations of health incidents  and  conducting additional
exposure  and risk  studies in  community  populations.
The report suggested  conducting preplanned exposure
assessment studies  to characterize the effect of biosolid
exposure on the community, to identify chemicals and
microorganisms  in  collected  field  samples, and  to
measure   endotoxin  exposure   (bacterial   cell  wall
constituents).

These research priorities were further  delineated by
participants in the USEPA-Water Environment Research
Foundation (WERF)  Biosolids Research Summit in 2003
(WERF,  2004).   Several high-priority research  needs
were  identified,  including:  (a) the  development  and
deployment  of  a rapid  incident  response  process  to
evaluate   possible   health effects  associated  with  a
biosolids  land application;  (b) the  characterization  of
bioaerosols  associated  with  the  land  application  of
biosolids;  and   (c)  the  identification  of  the   odor
compounds emitted  by sludge  in the various stages from
generation to end   use, and  the  specification of  their
sensory potencies and mechanisms of generation and
release.
The Agency described its action plan for responding to
the NRC report in the Federal Register notice entitled
"Final  Agency  Response to the  National  Research
Council Report on Biosolids Applied to Land and  the
Results of  EPA's Review of Existing  Sewage Sludge
Regulations" (USEPA, 2003). The plan included a list of
14 projects, with the goal  being the strengthening of the
technical basis for  sewage  sludge  use and disposal
regulations.  One of these projects was to conduct a
workshop  that would examine  issues  relating to  the
measurement of exposures associated with biosolids.

To address  this  need, the USEPA hosted a Biosolids
Exposure  Measurement  Workshop  on  March  16-17,
2006  at the Andrew W. Breidenbach  Environmental
Research  Center in Cincinnati,  OH.   Seventy-seven
people, including researchers from  government  and
academia,  regional  sludge coordinators,  state sludge
coordinators, and  other stakeholders, participated in the
event.  This report provides a summary of the workshop,
which included presentations by 16 experts, followed by
a panel discussion session.  The primary focus of the
workshop was to identify current measurement tools and
to prioritize research  on human health effects related to
land application of biosolids.  The workshop agenda and
list of participants are provided in Appendices A and B,
respectively. Appendix C is an update on the status of
the WERF incident  response project.   Five technical
proceedings papers are found in Appendix D.

Workshop Background and Objectives
Bruce Mintz

Mr. Mintz, an Assistant Laboratory Director for USEPA's
National   Exposure  Research  Laboratory   (NERL),
opened the workshop by discussing its background and
objectives.  Public concern about this issue is of major
importance,  particularly  when it  is  unclear whether
reported incidences of adverse human health effects are
attributable to  the land application of  biosolids.   The
NRC  Report (2002) found  that "there is a  lack  of
exposure and health information on populations exposed

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to biosolids," and recommended that "EPA promote and
support  response   investigations,  targeted  exposure
surveillance   studies,   and   a  few   well-designed
epidemiological investigations of exposed populations."

Measuring  exposure  to  biosolids  is  very  complex
because  of the number of possible contaminants and
exposure pathways.  The  Section 503  Rules identified
14  exposure  pathways  related to  land application  of
biosolids.   This workshop focused  primarily  on the
inhalation and  dermal absorption  pathways  because
most  of the published complaints describe symptoms
that would  be associated  with  these pathways.   The
objectives of the group panel discussion  were to identify
protocols for exposure measurement and modeling and
to recommend research priorities.  The group  tried  to
reach a consensus on priority contaminants, sampling
procedures and  frequency, methods of analysis, and
fate and transport measurement and modeling.

Land Application and Sludge Treatment
James E.  Smith

Dr.  Smith,  with  USEPA's  National  Risk  Management
Research Laboratory (NRMRL), discussed  biosolids and
issues associated  with land application.   Wastewater
treatment plants  (WWTP) are  designed to  remove
sewage contaminants, resulting in a clean effluent which
is discharged into the environment. These  contaminants
are concentrated in sludge  which can be treated to form
biosolids  that are  then  beneficially   used  on  land.
Approximately  60% of WWTPs treat  and land  apply
biosolids.  This accounts for 40% of the  total amount of
sludge produced in the U.S.  Collectively,  this provides
more  than four million dry  tons  of material per year for
land application.  [Note: additional data,  not available  at
the  time  of  the  workshop,  can   be  found  at:
www.nebiosolids.org]  Biosolids  applied to  land  must
meet  federal  and  state disinfection, vector attraction
reduction, and chemical  concentration  requirements
(primarily metals) for protecting  the public health.  The
minimal metal concentrations are largely  achieved by
pretreating  industrial wastes.    The  Section  503 Rule
approach  for  public  health protection uses  multiple
barriers,  including  disinfection and stabilization (vector
attraction  control)  to  reduce  pathogens below the
detection limit (Class A).   If indicator  organisms are
present  in  sufficient  densities,  the  biosolids  are
considered  Class B and land  use restrictions are then
imposed.  These restrictions are designed to allow for
natural decay of the pathogens.

More  information would aid in determining  the best way
to  use biosolids.  Some  issues  to address  include
studying  the  aerosols and  dusts generated  as the
material  dries, identifying  the  fate of  pathogens, and
verifying  the  effectiveness  of  Class  B  disinfection
processes in inactivating pathogens.  In addition, some
biosolid treatment  and application processes tend  to
emit odors  and aerosols and so it would  be helpful  to
determine if the public concerns  about  land  applied
biosolids are due more to aesthetic issues or to human
health effects.

Protocols for the Timely  Investigation  of
Potential Health  Incidents  Associated with
Biosolids Land Application
Alfred P. Dufour

Dr.   Dufour,   with   USEPA's   NERL,   discussed  a
December,  2004  WERF  workshop on rapid incident
response to health complaints  from land application  of
biosolids.   Participants in  the meeting developed  a
request for proposal  (RFP),  based on  input  about
previous  cases  of  reported  health  effects  from the
perspective of those affected.

The  objectives of the RFP  will be addressed in three
phases.  Phase 1 will involve the development of a
protocol  for rapid  incident  response.   This will  be
achieved by:   reviewing  previous  incidents,  collecting
information  on  all adverse  environmental  outcomes,
developing  a  data  collection/investigative instrument,
consulting with states about conducting investigations,
providing investigation guidance, and  communicating
with health practitioners. In Phase 2, the protocol will be
refined after pilot testing  at three localities.  After the
protocol is optimized, recommendations will be made  to
WERF on how to roll out the  protocol  nationwide.   In
Phase 3, a database of biosolids-related investigations
will be developed. In  addition, guidance will be provided
on the  communication of the  protocol  to  the  public
through community dialogs and  other mechanisms.

An update  on  the Rapid  Incident Response  Project  is
provided in Appendix C.

Community Perspective  of Biosolids Land
Application
Maureen Reilly

Ms.  Maureen  Reilly,  with  Sludgewatch,  outlined her
concerns about biosolids policies and practices from her
perspective as a member  of  a  community in  which
biosolids have been land applied.  In her view:

   •   The health, water supplies, quality of life,  and
       property values of rural communities have been
       compromised by land application of biosolids.
   •   There  is not  an  effective  mechanism  for the
       public to express  their  concerns about adverse

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health effects that they believe to be caused by
biosolids applications.
The  WERF  rapid  incident  response  project,
which will formulate a  mechanism to respond to
complaints, has been delayed too long.
The oversight of land application is complicated
by the  multiple  regulations and governmental
authorities    (federal,    state,   county,   and
municipal).
The 40 CFR Part 503  regulations need rigorous
review.
Since the 40 CFR Part 503  regulations were
promulgated they have been made more lenient
and are enforced less aggressively.
There  has  not  been  adequate  research on
pathogens  or  on  the  synergistic  effects  of
contaminants that may be in sewage sludge.
There is the perception that the USEPA  is both
the regulator and  the chief promoter  of land
application.
Ms. Reilly made  several suggestions that would help
address  her  concerns.   First,  she suggested  that  a
permitting process would  allow  for better compliance
monitoring and should be  implemented.  Currently, the
40 CFR Part  503  regulations are self-implementing and
no  written  permit is required.   Second,  Ms.  Reilly
suggested  the  risks from  land application can be
reduced  by  separating  medical  and industrial wastes
from the  general waste  stream,  and  by monitoring
application   sites   for   contaminants.    Finally,  she
suggested  that complaints  should  be  systematically
recorded, responded  to,  and if required, investigated.  In
conclusion, in her view, the small  amount  of  money
saved by land application is offset by increased  illness,
hospital costs, and contamination of soil and water.

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          Current Exposure Measurement Methods and Risk Assessment
Multi-media  Sampling at  a Biosolids Land
Application Test Site
Eric A. Foote

Mr. Foote, with Battelle, reported  on an approach utilized
for onsite  sampling  at a biosolids land application site.
The research was a  collaboration of the USEPA, the US
Department  of Agriculture  (USDA), the  North  Carolina
Department of Agriculture, Battelle, and other supporting
groups, agencies, and organizations.  This field study,
which is one of the projects described in the December
2003 USEPA action plan, was conducted in 2004 on a
two-acre  plot  of grass pasture  in  Salisbury, North
Carolina.  This site was selected because it  had not
received previous biosolids applications.  Air  and soil
sampling were conducted prior to and during the surface
application of anaerobically digested  and  dewatered
biosolids,  and  some  sampling  continued  for  several
months.

The goals of this research study were to develop a multi-
media  sampling approach  for airborne and soil-bound
contaminants  resulting  from  the  land  application  of
biosolids, and to optimize this sampling approach for use
in future studies. The study measured air emissions and
their   short-range  transport,   and   soil  microbial
concentrations at and around the test site, with  a focus
on   selected    constituents    including   particulates
(endotoxins),   microorganisms   (bacteria,   fungi,  and
viruses),   and   volatile    compounds   (including
malodorants).   Prior to  application, the  biosolids  were
analyzed  for these compounds.   Biomass loadings,
microbial  population  dynamics,  endocrine  disrupter
concentrations,  and agronomic characteristics also were
determined.

The objectives of the bioaerosol and particulate matter
sampling  plan  developed  by  the  USEPA were  to
characterize the  type   and  concentrations of select
biological  agents and  particulates  at several exposure
points  within 50 meters of the applied biosolids.  The
analyses used  included ones to detect and measure:
heterotrophic bacteria, fecal conforms, Escherichia coli,
Salmonella, Staphylococcus aureus, Enterococcus sp.,
Clostridium  perfringens,   total   bacterial   bioburden,
fungus,  enteric virus, coliphage  (MS2),  and particulate
size  fractions.   The  sampling  process utilized  both
mobile and stationary bioaerosol sampling  units which
included SKC BioSamplers® (impingers) and Andersen 6
stage samplers (impactors).

The objectives of the volatile organic compounds (VOC),
volatile inorganic compounds (VIC), and odor monitoring
measurements were  to determine  the presence  and
concentration of select compounds, both  upwind  and
downwind  of   the   application   area.     A  gas-
chromatography  method was  used to measure sulfur
and nitrogen compounds.  A Fourier Transform Infrared
Spectroscopy (FTIR)  unit was used to take real-time
measurements of VOCs in the  field.   Flux chambers
were used in the field to measure VOCs. Flux samples
were taken directly from the ground prior to application,
from the biosolids pile, and from the ground one and two
days  after application.  A  portion of the flux samples
were  analyzed by an  odor panel; odor measurements
were  also made in the field with the Nasal Ranger™
Olfactometer.

The objectives of the soil monitoring were to measure:
the amount and  distribution of biosolids applied to the
field;   the   concentration   of   various   microbial
contaminants over time; the concentration of endocrine
disruptive chemicals as a function of depth and time; the
microbial community  as a  function  of depth and  time;
and  ecotoxicity  before  and after biosolids  application.
Measurements were  taken to detect the presence of
fecal  conforms,  some  pathogenic bacteria,  enteric
viruses, coliphage (MS2), and viable helminth ova.

This study required a considerable level of coordination
and  communication among the  research collaborators
because  of  the   large   study  site.      Quality
assurance/quality control (QA/QC) was complicated and
involved different agencies and offices.  Data from this
project  are  a  landmark  for  multi-media  information
gathered from one site and may serve as a  baseline for
future studies. A report on this study is in preparation.

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Pathogen  Risk Assessment for Biosolids:
Recent Developments
Jeffrey A. Seller

Mr. Seller, with Seller Environmental, discussed a two-
phase WERF-funded project to develop a methodology
for  assessing  the  risk of pathogen  exposures  from
biosolids.   Microbiological  contaminants  of  concern
include  viruses,  bacteria, protozoan  parasites,  and
helminths from a number  of potential human exposure
pathways.   The  following  exposure  pathways were
investigated in the study:  direct ingestion  of biosolids-
amended  soil,  ingestion  of  groundwater containing
microbes  leached  through  soil  from  land  applied
biosolids; and  exposure  to wind transported microbes
from land applied  biosolids.  Potential important issues
in  determining appropriate microbial  risk assessment
(MRA)   methods   include    both   exposure-specific
parameters,  such  as  magnitude   and  frequency  of
exposure,  and  pathogen-specific  factors,  such  as
infectious dose,  disease, and  infectivity.   The  study
results  indicate that it is both feasible and  reasonable to
apply a dynamic  population-based  MRA  method  to
estimate human  health  risk  from pathogens  in  land
applied biosolids.   The  framework was  completed  in
2002 and  published in a WERF report (2003) and  in
Eisenberg  et  al.,  2004.   A demonstration  of the
methodology has recently been completed.

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                                  Sludge Fate and Transport
Analyzing Biosolids for Microorganisms  to
Achieve Regulatory Compliance
Mark C. Meckes

Mr.  Meckes, with  USEPA's  NRMRL, discussed  an
evaluation of analytical methods for microbial monitoring
in  biosolids materials.   Current federal  regulations
require monitoring  for indicator  microorganisms under
some conditions for Class  B biosolids and  under  all
conditions for Class A biosolids.  Standard protocols  for
quantifying these  organisms were  specified  in  the
regulations; however, these protocols were not designed
for biosolids.  Two  alternatives  for meeting Class A
requirements include monitoring for  enteroviruses and
viable helminth ova.  A guidance document, "Control of
Pathogens and Vector Attraction  in Sewage Sludge"
(USEPA, 1992), includes a plaque assay for detection of
total culturable viruses and a protocol for enumeration of
viable helminth ova in biosolids.

Recently,  methods used for analysis of fecal conforms
and Salmonella were reviewed and a standard protocol
was  developed  for biosolids  applications.   These
protocols were  evaluated by  testing various types of
biosolids at twelve laboratories throughout the U.S. Two
multiple fermentation tube methods were evaluated  for
fecal conforms  in  biosolids,  the  lauryl tryptose broth
(LTB) - EC broth method and the A-1 method. A single
method  for Salmonella  was  evaluated, the modified
semi-solid  Rapaport-Vasiliadis  procedure.     Results
showed considerable variation between  laboratories
depending  upon the biosolids evaluated.   The  fecal
conforms and Salmonella methods have been  included
as USEPA methods under 40 CFR 136.  A similar study
evaluating methods for the detection and enumeration of
viruses  has  been   initiated  with  the  University  of
Cincinnati.

Fate and Transport  Models
Charles P. Gerba

Dr. Gerba, with the University of Arizona, discussed the
major factors to be  considered in modeling fate and
transport of  biosolids  pathogens.    The  purpose  of
developing  these  models  is to  provide  a  better
characterization of the  exposure  component of a risk
assessment.   Some  of the  challenges  in  modeling
microbial transport are that: there  are  many  different
species of microbes, some microbes may multiply in the
environment, and organisms are not evenly distributed in
the   environment   because   they   are  particulate.
Furthermore,  their  survival and   transport rates  are
affected by weather and climate. Factors that need to be
included when  modeling pathogen survival in soil  or
water include temperature,  soil moisture,  and rate of
moisture loss. A major factor influencing virus survival in
the subsurface is  temperature, which  can be  easily
measured  and modeled.  A more  challenging  factor is
that viral migration rates  vary and  are  influenced by soil
type and saturation.

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                                       Dermal Exposure
Non-invasive
Exposure
Karla D. Thrall
Assessment   of    Dermal
Dr.  Thrall, with Pacific Northwest National  Laboratory,
discussed  the   use   of   physiologically   based
pharmacokinetic  (PBPK)  modeling  to  non-invasively
measure  dermal adsorption.   Volatile  chemicals  in
biosolids  may be absorbed through  the skin from the
soil.  This compound can then pass into the bloodstream
and  other tissues.   The absorbed  compound either
remains in the tissue or blood,  or returns in the venous
blood and is exhaled.  With each breath, some of the
compound is volatilized and exhaled, and by measuring
the exhaled air the amount of chemical absorbed can be
predicted.  Breath analysis is conducted  using a mass
spectrometer that takes real-time measurements.  The
sample collection device  is very simple, and has been
used on rats, mice, primates, and human volunteers.
Real-time  breath  analysis and  PBPK  modeling  are
ideally suited to track the kinetics of dermal exposure.
The methodology is sensitive; therefore, human  studies
can  be  conducted  at  low exposure concentrations.
Deployment  of  the  technology with  PBPK  modeling
could improve industrial hygiene  practices by enabling
onsite measurement of human exposure.

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                                       Odors and  Irritants
Odor, Irritation and Health Symptoms from
Biosolids
Pamela H. Dalton

Dr.  Dalton, with  the Monell Chemical  Senses Center,
discussed airborne chemicals and odor and their effects
on  humans.    Volatile  chemicals  associated  with
bioaerosols and  particulates  in biosolids may  produce
ocular/airway irritation as  well  as  emit objectionable
odors.     These  chemicals   can  therefore  cause
annoyance,  stress,  and  perception  of health  risks.
Irritation  response ranges  from mucosal  burning  to
upper respiratory irritation.  However, the odor detection
threshold  is  at  much  lower concentration than  the
irritation threshold.

Dynamic   determinants  affecting  sensitivity  to  odor
include frequency, intensity, duration, and offensiveness.
Women in  particular may become more sensitive with
intermittent, infrequent exposures.  The way people are
exposed to odor affects the response to odors. Duration
of exposure  can dramatically  increase the  sensory
impact and temporal factors play a huge role. One brief
exposure  does  not  predict  the results from longer
exposure.

An  important unresolved  issue  is   whether  odors
associated with land application of biosolids  elicit health
symptoms through direct action on target organs or from
annoyance.  Key data gaps in evaluating the effects of
biosolids  odors include:  documenting  actual exposure
and effects in communities, identifying key odors and
irritants  in  biosolids,  and evaluating  the response
variation  due to temporal factors, individual sensitivity,
and attitudes and expectations.
Odor Measurements  and Impacts from  an
Experimental  Biosolids  Land  Application
Site
Robert H. Forbes,  Jr.

Mr. Forbes, with CH2M  Hill, discussed a study involving
onsite and laboratory  analysis of odor samples taken at
a North Carolina biosolids  land application site  before,
during, and after application.  A certified offsite panel
was used  for  odor  analysis  of  three flux-chamber
samples taken on the  day before (-1), the day of (0), and
the day after (1) land  application.  Hand-held  Nasal
Ranger™ olfactometers were used  for field assessment
of odors.  Chemical sensory tubes were used  for field
measurements  of ammonia  and  a Jerome  gold-film
analyzer was  used for field measurements of hydrogen
sulfide.   The offsite  panel and Nasal  Ranger™ test
results indicated odor increased on day 0 and  1 when
compared to  day  -1.   Odors were not  detected more
than 200 feet from the  application  site.  Odors began
subsiding on  day  2, and were  not  detected  by day 4.
Ammonia and hydrogen sulfide disperse rapidly in air
and  were detected  only  directly   above  the  applied
biosolids  immediately after application.  Most lingering
odors appear to be due to organic sulfur compounds
such  as  methyl  mercaptan,  dimethyl sulfide,  and
dimethyl disulfide, or nitrogen-based compounds such
as trimethyl amine, indole,  and  skatole.  Odors  can be
reactivated by changes in temperature or rain events.

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                                             Particulates
Airborne  Particulates:
Measurement Issues
Patricia D. Millner
Technologies   and
Dr. Millner, with USDA, discussed airborne  participates
and exposure assessment issues. Airborne particles of
concern are those that are breathable and could cause
adverse health  effects.   Whether health effects  occur
after exposure  to particulate matter is dependent on
several  factors,  including:   particle  size,  exposure
intensity and duration, the chemical/biological nature of
the particles, the length of time that a  particle remains in
the air, and the presence of pre-existing conditions (e.g.,
asthma).   A  number of different   air samplers  are
available such as  impingers, cyclones, particle counters,
impactors,  filters,  and  dustfall deposit  gauges.  Each
sampler samples  at  a different rate, ranging from 1.2
L/min to 400 L/min, and has different sample volumes.
Several factors need to be considered when sampling
land applied biosolids for aerosols.  The application site
needs  to be sampled before  biosolids application with
mock conditions and during the application.  An upwind
reference site also needs to be sampled.  Biosolids are
applied by  a mobile source which  typically  applies the
material in a  directional manner;  therefore,  sampling
location and mobility are critical factors  in determining
exposure.   In addition, meteorological data such as wind
speed  and  direction, the analytical  method, background
levels  of   particulates,  and   the  effects  of sample
collection conditions upon the  analyte (desiccation or
shearing forces) need to be considered in the analytical
and sampling plans.

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                                  Pathogens and Endotoxins
Biomarkers of Viral Exposure
G. Shay Fout

Dr.  Fout, with  NERL,  discussed  a USEPA Office  of
Research   and  Development  (ORD)  study  entitled
"Salivary Antibody  Responses  as  an  Indicator  of
Waterborne Infections:  Pilot Community Study  Before
and After Installation of UV Treatment."  The study is
novel both because of its design and because the use of
new   approaches to  determine  pathogen  exposure.
Although this study  is aimed at detecting  exposure  to
pathogens  in water,  it could serve  as a model for future
studies aimed at determining the health risks associated
with the application of biosolids.

This  intervention study  has been designed to correlate
pathogen occurrence in water with exposure and health
effects.  Specifically, individuals will be surveyed before
and  after  a new drinking  water  treatment  plant  with
ultraviolet disinfection goes online.  Volunteer  families
will  provide health data by completing questionnaires
and  by  providing monthly saliva  samples.   These
samples will be tested for antibodies to Cryptosporidium,
norovirus, and  rotavirus. In addition, water samples will
be  taken  to   determine   the  occurrence  of these
waterborne pathogens.

In addition to the unique overall design, this study takes
advantage  of a  new approach to detect exposure by
testing  for the  presence  of  specific   biomarkers
(antibodies) found in saliva samples. Traditional indices
for  estimating  pathogen  exposure  in  a  community
include:  individual  symptom surveys,  school  illness
records, and pharmaceutical sales. Unfortunately, these
indices  are not  effective for estimating the risk  from
biosolids exposure  because  of the lack of a  proper
control  population.    In  addition, these  indices  are
generally not pathogen specific. In contrast, biomarkers
for measuring exposure, such as direct pathogen assays
and  antibody  responses  in serum  or oral  fluids,  can
more  directly  target individuals that are  likely to be
exposed as well as control  individuals who are not
affected.  In addition,  antibody  assays are pathogen
specific and can  be used to approximate the time of
exposure.

The  specific saliva-based assay used  in  this study,
which takes advantage of a novel fluorescent bead array
LiquiChip™  technology  to determine  antibody levels,
has several  advantages.  Unlike a serum-based assay
for  biomarkers, a saliva-based antibody  assay is  not
invasive. This  feature allows  individuals at  a range of
ages to participate,  enhances participation of the entire
community, and provides significantly reduced specimen
collection  costs.  In  addition, the LiquiChip™ assay is
more  sensitive and reproducible  than the  standard
ELISA with lower costs and analysis time.

Community  and  Occupational  Risk  from
Bioaerosols  during  Land  Application  of
Biosolids
Ian L. Pepper

Dr. Pepper, with the  University of Arizona, discussed  two
studies evaluating occupational and community risk from
bioaerosols  during   land  applications  of  biosolids.
Bioaerosol  samples  were  collected   with  SKC
BioSampler®  impingers from land  application  sites
located  across the  U.S., using different  application
practices,  such as liquid spray and "cake" application.
Samples were collected from  downwind and background
sites.   The  concentrations of several  microbes were
measured, including: heterotrophic plate count bacteria
(HPC),    total   conforms,   coliphage,    Clostridium
perfringens,      Escherichia      coli,      endotoxin
(lipopolysaccharide),    enterovirus,   norovirus,   and
hepatitis A  virus (HAV).    Overall,  in 500 samples
analyzed,  the levels of bacteria and phage  were at or
below detection limits by culture methods, and only three
samples  were  positive  for  norovirus  by  reverse
transcription PCR.  Calculated  risk of  infection in  the
community was determined to be at or below a 1:10,000
risk  of  annual  infection.    Endotoxin concentrations
observed  during  land application were similar to those
                                                   10

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observed  from  tractor  operations  without  biosolids,
implying that the endotoxins are from the soil.

Occupational risk studies included three experiments to
characterize   the   concentration   of   aerosolized
microorganisms from  biosolid  applications,  the  plume
created during land application,  and the occupational
risk of infection from land application of biosolids.  More
than  300 air samples  were  collected downwind  of
biosolids application sites  using  liquid  impingers, and
over   100   samples  were   collected   downwind   of
microbially seeded,  land applied  water samples.  The
seeded water served as a model system for tracking the
plume. The  aerosolization  rates from land application of
biosolids were calculated  to  be  less  than  33  plaque
forming units (PFU)  of coliphage and 10 colony forming
units  (CPU)  of conforms per meter traveled, while  the
water aerosolization  rates were much higher.  Exposure
duration  was brief and  limited to  the  time when  the
biosolids were in  the air.  Samples also were taken from
air and biosolids at 10  land application sites from  across
the U.S. and analyzed for conforms,  coliphages, and
HPC.   The application   method  strongly  influenced
aerosolization rates,  while relative humidity, temperature
and wind speed  showed limited effects. Occupational
risks  of  infection from  land  application are  greatest
during the loading of  biosolids.  Both studies indicate
that occupational or  community exposure to viruses and
bacterial  indicators  is  low  during  and   after  land
application of biosolids, at least for the meteorological
conditions evaluated.

Improving the Efficiency of an Impinger for
Collecting  Bacteria   during   a  Biosolids
Application Field Study
Edwin F. Barth

Dr. Barth, with USEPA's NRMRL, discussed the  use of
impingers for bioaerosol sample collection.  Measuring
individual airborne exposure accurately is  challenging
because  of the  difficulty of  matching air sampling
devices to different respiratory mechanisms, deposition
sizes, and personal  breathing heights.   In addition,  the
survival of microorganisms  is dependent upon their size,
the relative  humidity,  time, temperature, and  UV and
ozone exposure.   Traditional  bioaerosol  collection
methods include  filtration,  impaction, and impingement.
Advantages  of impingement include: longer sampling
times (up to 8 hours), the ability  to split  aliquots for
multiple analyses, suitability for molecular methods, and
sample  direction   flexibility.      Disadvantages   of
impingement include the delicate,  relatively expensive
equipment,   and   the  stress-induced  effects  on  the
microorganisms during collection.  Research conducted
in  the field and at the  NRMRL Bioaerosol Wind Tunnel
evaluated  the   performance  of  SKC  BioSampler®
impingers.  The results suggest that modifications to the
standard practices involving  impinger use will improve
recoveries. These modifications include covering of the
impinger apparatus to block UV radiation, washing of the
inlet neck  to prevent drying of the  accrued material
during sampling, and rinsing the inlet neck and jets after
collection to liberate organisms captured  in the neck.

Endotoxins
Nancy C. Burton

Ms. Burton, with  the Center for Disease Control  and
Prevention's National Institute  for Occupational Safety
and   Health    (CDC-NIOSH),   discussed    endotoxin
sampling   techniques    and    analytical    methods.
Endotoxins are  a  lipopolysaccharide complex formed  in
gram negative bacteria that are  chemically and thermally
stable.  Endotoxins can  cause powerful inflammatory
reactions in humans with symptoms including fever, flu-
like  symptoms,  cough,   headache,  asthma,  and/or
respiratory distress.  Other associated health effects
include organic dust syndrome and respiratory disease
such  as  asthma.   Occupational  groups  with  highly
documented exposures  include farmers,  cotton workers,
wastewater treatment  workers,  trash  haulers,   and
poultry and swine handlers.  One method for sampling
endotoxins uses   0.4  urn  pore  size   endotoxin-free
polycarbonate  filters in cassettes,  and filter/cassette
vacuum collectors. The analytical methods used include
the  Limulus  amebocyte  kinetic  assay,  the  kinetic
chromogenic  method,  and  the  gas  chromatography-
mass spectrometry method.

Occupational   Exposure   Assessments   at
WWTPs
Nancy C. Burton

Ms. Burton discussed exposure routes, contaminants,
and   sampling   techniques   used  for  occupational
exposure  assessments  of workers  at WWTPs.  These
assessments are a conservative estimate of community
exposure to biosolids since the contaminant loads are
thought to  be the  highest at the WWTP.  In addition,
these  assessments  are  site  specific because  the
exposure  risks  are dependent  upon   the  incoming
wastes, treatment  process, and job  tasks.   When
identifying potential hazards,  it is necessary to relate the
environmental   evaluation  to   any  reported  health
symptoms. Assessments at WWTPs should include a
consideration of chemical, biological, and onsite physical
hazards. The chemical and biological contaminants are
primarily concentrated in the sludge.

Exposure   routes   for   chemical  and    biological
contaminants are  inhalation,  ingestion,  and  dermal
adsorption.  Chemicals in the waste  stream  include
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gaseous  by-products  (carbon  monoxide,   methane,    of    approaches,   including   cultural   techniques,
ammonia, and  hydrogen  sulfide),  dewatering agents,    microscopic  analysis (spore traps and surface), and
and trace metals. Human  pathogens that may be found    polymerase chain reaction  (PCR) techniques.  Some
in  sludge  include  bacteria,  viruses,   protozoa,  and    considerations  for field  monitoring of biological agents
helminths. There are no specific occupational exposure    include sample storage, transport, aerosol range, and
criteria to infectious agents  in wastewater or sewage.    sampling and analytical methods.
Biological contaminants are identified through a variety
                                                    12

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                                   Panel  Discussion Session
Bruce  Mintz charged the  workshop  participants  to
recommend  an exposure  measurement protocol, and to
identify  research  needs  for measuring exposure.   In
addition,  several  other   topics  related  to  incident
response  and  biosolids  practices  were  discussed.
Because of the complexity of biosolids and the multiple
exposure pathways, an exposure measurement protocol
framework could  not  be  recommended at the present
time.  However, it was agreed that the protocol should
include  multiple  exposure routes and contaminants,
much like the approach used in the North Carolina field
study.    The following  is  a  summary of the  panel
discussion.

The   development   of   an   exposure  measurement
framework is hindered by  a lack of knowledge about the
nature of complaints and  health effects associated with
biosolids  land application.  The lack of an  incident
response    protocol   prevents  health   complaints
associated  with   land   applications   from   being
systematically recorded by local and state authorities. It
also   prevents  the  classification of  complaints   by
symptom  (such as respiratory effects, gastroenteritis,
skin  irritation,  odor/nuisance,  and headaches)  or  by
exposure  pathway (air, soil, or water).  Epidemiological
studies  of the effects of  biosolids land application  are
needed,  with  better  analyses  of baseline  and post-
application exposure and  health information.   In most
situations there are only a few complaints; how well this
correlates with the total number of people who may  be
affected  is unknown.   It  is  also  unclear if there is  a
relationship  between  complaints  and such factors  as
educational  level  or  health  insurance coverage.  To
better evaluate community exposure,  complaints and
health effects should be mapped around the application
site with  the prevailing weather conditions indicated.
Real time measurements  are needed. This information
would  make  it   possible to  define  the   relationship
between the number and  severity  of complaints and the
distance from application site.

The  next theme  discussed  was biosolids treatment
practices. The panel felt many complaints are likely due
to the odor of the applied biosolids. The 503 regulations
were  designed   to   reduce   pathogens  and  vector
attraction but not control odor. The relationship between
vector control,  odor generation, treatment practices, and
digestion level  needs refinement.  In addition, the effects
of various  storage and  curing practices  need  to  be
evaluated when sludges are composted. Many biosolids
products do not generate complaints but some products
and  operations are  problematic.  As such, treatment
practices should be optimized when necessary.   Some
alternatives  to  improving  treatment  would   include
applying biosolids in remote areas or injecting them into
the soil.  The  panel  felt that exposure and complaints
could be greatly reduced by changing the recommended
best  practices  for  sludge treatment  and  biosolids
application processes so that odors were reduced.

The  third theme  discussed was  the  use of biosolids
indicators.  A biosolids indicator could  be used to trace
movement  of  contaminants  after  application, to study
decay rates, and to plot source curves.  Caffeine, fibers,
steroidal aerosols, specific  microbes, fugitive  dust  or
other substances that occur in biosolids are possible
targets.   A true  indicator would need to distinguish
biosolids from  manure or septage. Since indicators  do
not provide  specific  information about the  cause of a
complaint and  do not necessarily  correlate  with the
transport properties of a specific contaminant, they may
be only useful as a screening or research tool.

The   final   themes   discussed   were   exposure
measurement,   analytical   methods,   and   research
priorities.   To answer basic exposure questions, the
broad  and  indicator-based  exposure   measurement
approach  taken  in the North Carolina field  study is
appropriate.  Indicators would not be appropriate  for
incident response  because the sampling and  analytic
approach needs to be specific to the complaint and site.
This  means  that  the   compounds  or  organisms
associated  with  complaints  need  to  be identified.  In
general, analytical  methods may need  to be developed,
and more real-time monitoring is needed before,  during,
and after the application of biosolids both onsite and in
the surrounding community.

During the  panel  discussion, several research  needs
became  apparent, especially in the area of pathogen
                                                    13

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and  odor exposure.   The evaluation of exposure  to
microorganisms  associated  with  biosolids application,
particularly  aerosolized  pathogens,   needs  further
research.  The health  effects of fungi may need to be
evaluated because soil has a higher fungal burden after
biosolids  application.   Adding  to  the  complexity  of
monitoring is the interpretation of results due to the fact
that  microorganisms  are  present  without   biosolids
application; therefore, ambient background levels need
to be determined.  The sample planning process should
carefully consider the number of samples needed so that
the amount  of exposure can be  accurately estimated.
Sampling  methods, such as  impactors  and impingers,
should be evaluated to define both their recovery rate
and  effects  upon  the  stress-sensitive microorganisms
that are trapped.

Measurement  of exposure  to  odors  also  has many
research  gaps,  in  part because irritation and  health
effects  from  chemical  aerosols  are  dependent  upon
dose, duration of exposure, and  concentration.  Other
undefined volatile  chemicals, not just hydrogen sulfide
and  ammonia,  cause odor  in  biosolids.     These
compounds  are  likely to  be  found  at extremely low
concentrations, vary among  sites,  and  have different
migration  rates and detection thresholds than hydrogen
sulfide or ammonia.  The time of day also affects odor
sample results with the odors being strongest  overnight.
In addition,  the health effects caused by  tasteless,
odorless  materials  found   in  biosolids  (PM2.5   or
chemicals) need to be determined.
Priority Research Needs

    1.  Characterize the sites, biosolid products,  and
       biosolids  application  processes that generate
       complaints,   and  change  either  the  sludge
       treatment   or   biosolids    land    application
       processes.
    2.  Identify the  source  of complaints  and  health
       effects;  specifically,  the  microorganisms  or
       chemicals that cause  complaints.   If possible
       correlate field measurements to health effects.
    3.  Improve   data  collection   and   analysis  of
       complaint incidents.
    4.  Measure microbial exposure  levels before  and
       after  land  application in controlled  long-term
       research projects.
    5.  Characterize VOC and odor emissions from land
       application sites, and other agricultural sites.
    6.  Evaluate  and  improve  bioaerosols  exposure
       methods,  including  the  direct comparison of
       microbial sample collection systems.
    7.  Measure real-time endotoxin concentrations at
       application sites and other agricultural sites.
    8.  Measure odor  at land application  sites before,
       during, and after application.  Compare the odor
       levels to upwind and downwind sites.
                                                    14

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                                            Bibliography
Eisenberg,  J.,  Seller, J., Scott,  J.,  Eisenberg, D., and
Colford, J. (2003). A dynamic model to assess microbial
health  risks associated with the  beneficial  uses of
biosolids. WERF: Alexandria, VA.

Eisenberg,  J.,  Seller, J., Scott,  J.,  Eisenberg, D., and
Colford, J. (2004). A dynamic model to assess microbial
health  risks associated with the  beneficial  uses of
biosolids. Risk Analysis 24: 221-236.

NRC.  (2002).  Biosolids  applied  to  land:  advancing
standards  and  practices.  National Academy  Press:
Washington DC.

USEPA.  (1989).  National  primary  drinking  water
regulations; filtration and disinfection; turbidity;  Giardia
lamblia; and heterotrophic  bacteria. Federal Register.
54: 27486-27541.
USEPA  (1992)  Control  of  pathogens  and
attraction in sewage sludge. EPA/625/R-92/013.
vector
USEPA. (1993).  Standards for the use or disposal  of
sewage sludge. 40  Code  of Federal Regulations  Part
503 (40CFR503).

USEPA. (2003). Final Agency response to the National
Research  Council Report  on biosolids applied  to  land
and  the results  of  EPA's review of existing sewage
sludge regulations. Federal Register. 68: 75531-75552.

WERF.  (2004).  Biosolids Research Summit  Report.
WERF: Alexandria, VA.
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                                         Appendix A
                Biosolids Exposure Measurement Workshop Agenda
                         Andrew W. Breidenbach Environmental Research Center
                                           Cincinnati, OH
Thursday, March 16, 2006
Introduction Session
8:30-9:45     Workshop Background and Objectives
                    Bruce Mintz, USEPA
             Land Application and Sludge Treatment
                    James E. Smith, USEPA
             Protocols for the Timely Investigation of Potential Health Incidents Associated with Biosolids Land
             Application
                    Alfred P. Dufour, USEPA
             Community Perspective of Land Application
                    Maureen Reilly, Sludgewatch

9:45-10:00    Break

Current Exposure Measurement Methods and Risk Assessment
10:00-11:00   Multi-Media Sampling at a Biosolids Land Application Test Site
                    Eric A. Foote, Battelle
11:00-11:30   Pathogen Risk Assessment for Biosolids: Recent Developments
                    Jeffrey A. Seller, Seller Environmental

11:30-12:45   Lunch

Sludge and Fate and Transport
12:45-1:15    Analyzing Biosolids for Fecal Conforms and Salmonellae
                    Mark C.  Meckes, USEPA

1:15-1:45     Fate and Transport Models
                    Charles P. Gerba, University of Arizona

Dermal Exposure
1:45-2:15     Non-invasive Assessment of Dermal Exposure
                    Karla D. Thrall, Pacific Northwest National Laboratory

Odors and Irritants
2:15-2:45     Odor, Irritation and Health Symptoms from Biosolids
                    Pamela H. Dalton, Monell Chemical Senses Center

2:45-3:00     Break
                                                16

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3:00-3:30      Odor Measurements and Impacts from an Experimental Biosolids Land Application Site
                     Robert H. Forbes, Jr., CH2M Hill

Particulates
3:30-4:00      Airborne Particulates: Technologies and Measurement Issues
                     Patricia D. Millner, USDA

Pathogens and Endotoxins
4:00-4:30      Biomarkers of Viral Exposure
                     G. Shay Fout, USEPA

Friday, March 17,2006

8:40-9:10      Community and Occupational Risk from Bioaerosols during Land Application of Biosolids
                     Ian L. Pepper, University of Arizona

9:10-9:40      Improving the Efficiency of an Impinger for Collecting Bacteria during a Biosolids Application Field
              Study
                     Edwin F. Barth, USEPA

9:40-10:10     Endotoxins
                     Nancy C. Burton, CDC-NIOSH

10:10-10:30   Break

10:30-11:00   Occupational Exposure Assessments at WWTPs
                     Nancy C. Burton, CDC-NIOSH

11:00-2:00     Panel Discussion Session
              (Lunch during session)
                                                  17

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                                         Appendix B
                                       Participant List
Carolyn Acheson
Chemical Engineer
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7190
Email: acheson.carolyn@epa.gov

Bethany Acquisto
Environmental Engineer
USEPA-NRMRL
26 West Martin Luther King Drive, MS-G75
Cincinnati, OH 45268
Phone:513-569-7690
Email: acquisto.bethany@epa.gov

Adam Balz
Chemist
Pegasus Technical Services, Inc.
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7107
Email: balz.adam@epa.gov

Ed Earth
Environmental Engineer
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7669
Email: barth.ed@epa.gov

Robert Bastian
Senior Environmental Scientist
USEPA-OW-OWM
1200 Pennsylvania Avenue, NW, 4204M
Washington, DC 20460
Phone: 202-564-0653
Email: bastian.robert@epa.gov
Benjamin Blaney
Assistant Laboratory Director
USEPA-NRMRL
26 West Martin Luther King Drive MS-235
Cincinnati, OH 45268
Phone:513-569-7852
Email: blaney.ben@epa.gov

Bryan Boulanger
Environmental Engineer
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7018
Email: boulanger.bryan@epa.gov

Richard Brenner
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7657
Email: brenner.richard@epa.gov

Nichole Brinkman
Microbiolgist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7315
Email: brinkman.nichole@epa.gov

Nancy Burton
Industrial Hygienist
CDC/NIOSH
4676 Columbia Parkway, MS R-11
Cincinnati, OH 45226
Phone:513-841-4323
Email: nburton@cdc.gov
                                                18

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Mark Chappell
ORISE Postdoctoral Fellow
USEPA
809 Heavenly Lane
Cincinnati, OH 45238
Phone:513-922-2442
Email: chappell.mark@epa.gov

Patrick Clark
Physical Scientist
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7561
Email: clark.patrick@epa.gov

Kawana  Cohen
Biosolids Permitting Coordinator
USEPA-OW-OWM
1200 Pennsylvania Avenue, 4203M
Washington, DC  20460
Phone: 202-564-2435
Email: cohen.kawana@epa.gov

Tracy Dahling
Physical Science Technician
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7429
Email: dahling.tracy@epa.gov

Pamela Dalton
Member
Monell Chemical  Senses Center
3500 Market Street
Philadelphia, PA  19104
Phone:215-898-5595
Email: pdalton@pobox.upenn.edu

Armah de la Cruz
Scientist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7224
Email: delacruz.armah@epa.gov

Maura Donohue
Research Chemist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7634
Email: donohue.maura@epa.gov
Alfred Dufour
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7330
Email: dufour.alfred@epa.gov

Eric Foote
Principal Research Scientist
Battelle
505 King Avenue
Columbus, OH 43201
Phone:614-424-7939
Email: foote@battelle.org

Robert Forbes
Senior Technologist
CH2M HILL, Inc.
4824 Parkway Plaza Boulevard, Suite 200
Charlotte, NC 28217
Phone: 704-329-0073 ext. 213
Email: bob.forbes@ch2m.com

Shay Fout
Microbiologist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7387
Email: fout.shay@epa.gov

Charles Gerba
The University of Arizona
2601 East Airport Drive
Tucson, AZ  85706
Phone:520-621-6906
Email: gerba@ag.arizona.edu

Ann Grimm
Branch Chief
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7397
Email: grimm.ann@epa.gov

Alan Hais
Senior Program Director
WERF
635 Slaters Lane
Alexandria, VA 22314
Phone: 703-684-2470 x7146
Email: ahais@werf.org
                                                19

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Margaret Hardebeck
Research Associate, M.L.T.
Pegasus Technical Services, Inc.
26 West Martin Luther King Drive
Cincinnati, Ohio  45268
Phone:513-569-7611
Email: hardebeck.margaret@epa.gov

Evelyn Hartzell
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7728
Email: hartzell.evelyn@epa.gov

Fred Hauchman
Division Director
USEPA-NERL
26 West Martin Luther King Drive
MD-593
Cincinnati, OH 45268
Phone:513-569-7007
Email: hauchman.fred@epa.gov

Richard Haugland
Microbiologist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati, OH 45268
Email: haugland.richard@epa.gov

Gina Hayes
Contractor
USEPA
26 West Martin Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7953
Email: hayes.gina@epa.gov

Christopher Heaney
Doctoral Student
Department of Epidemology CB# 7435
School of Public Health
University of North Carolina at Chapel Hill
Chapel Hill, NC
Phone:919-423-6441
Email: cheaney@email.unc.edu

Michelle Henderson
QA Manager
Shaw Environmental, Inc.
5050 Section Avenue
Cincinnati, Ohio  45212
Phone:513-782-4763
Email: michelle.henderson@shawgrp.com
Gerard Henderson
EHS Monitor
Pegasus Technical Services, Inc.
Center Hill Facility
5995 Center Hill Avenue
Cincinnati, OH  45224
Phone:513-569-7868
Email: henderson.gerard@epa.gov

Ronald Herrmann
Microbiologist
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH  45468
Phone:513-569-7741
Email: herrmann.ronald@epa.gov

Edith Holder
Research Associate
Pegasus Technical Services, Inc.
46 East Hollister Street
Cincinnati, OH  45219
Phone:513-569-7178
Email: holder.edith@epa.gov

Pyoungchung Kim
Doctoral Student
University of Tennessee
1611 Laurel Avenue, #1115
Knoxville, TN 37916
Phone: 865-924-5892
Email: pkim1@utk.edu

Karen Koran
Onsite Manager
Pegasus Technical Services, Inc.
26 West Martin Luther King Drive
ML 421
Cincinnati, OH  45268
Phone:513-569-7304
Email: koran.karen@epa.gov

Drew Luellen
Research Scientist
Virginia Institute of Marine Science
1208 Create Road, Box 1346
Gloucester Point, VA  23062
Phone: 804-684-7749
Email: luellen@vims.edu

Ruth Marfil-Vega
Research Associate
UC/Pegasus Technical Services, Inc
26 West Martin Luther King Drive
Cincinnati, Ohio 45220
Phone:513-807-5332
Email: marfil-vega.ruth@epa.gov
                                                 20

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Jonathan May
Graduate Student
Medical University of Ohio/NOCPH
Phone: 785-545-5821
Email: jmay@medohio.edu

Paul McCauley
Chemist
USEPA-NRMRL
26 West Martin Luther King  Drive
Cincinnati, OH 45468
Phone:513-861-3348
Email: Paul82346@yahoo.com

Deborah McKean
Toxicologist
USEPA-NDT
26 West Martin Luther King  Drive
Cincinnati, OH 45268
Phone:513-487-2435
Email: mckean.deborah@epa.gov

Mark Meckes
Microbiologist
USEPA-NRMRL
26 West Martin Luther King  Drive
Cincinnati, Ohio  45268
Phone:513-569-7348
Email: meckes.mark@epa.gov

Patricia Millner
Research Microbiologist
USDA-ARS-BARC
10300 Baltimore Avenue, Building 001, Room 140
Beltsville, MD  20705-2350
Phone:301-504-7199
Email: millnerp@ba.ars.usda.gov

Bruce Mintz
Assistant Lab Director
USEPA-NERL
109 TW Alexander Drive
RTP, NC 27709
Phone:919-541-0272
Email: mintz.bruce@epa.gov

Felipe Alatriste Mondragon
Environmental Engineering  and Natural
 Resource Management
Camino a la Presa San Jose
2055 Lomas 4a
Seccion, C.P.  78216
San Luis Potosi, S.L.P., Mexico
Phone: +52 444-834-20-00
Email: falatriste@ipicyt.edu.mx
Michael Moore
Manager
Orange County Sanitation District
10844 Ellis Avenue
Fountain Valley, CA 92708
Phone:714-962-2411
Email: mmoore@ocsd.com

Regan Murray
USEPA-NHSRC
26 West Martin Luther King  Drive
Cincinnati, OH 45268
Phone:513-569-7031
Email: murray.regan@epa.gov

Tonya Nichols
USEPA-NHSRC
26 West Martin Luther King  Drive
Cincinnati, OH 45268
Phone:513-569-7805
Email: nichols.tonya@epa.gov

Xuejun Pan
Research Associate
University of Cincinnati
26 West Martin Luther King  Drive
Cincinnati, OH 45268
Phone:513-569-7354
Email: pan.xuejun@epa.gov

Sandhya Parshionikar
Microbiologist
USEPA-OW
26 West Martin Luther King  Drive
Cincinnati, OH 45268
Phone:513-569-7123
Email: parshionikar.sandhya@epa.gov

Bruce Peirano
Toxicologist
USEPA-NRMRL
26 West Martin Luther King  Drive
MD690
Cincinnati, Ohio 45268
Phone:513-569-7540
Email: peirano.bruce@epa.gov

Ian Pepper
Director
The University of Arizona
2601 East Airport Drive
Tucson, AZ 85706
Phone: 520-626-3328
Email: ipepper@ag.arizona.edu
                                                21

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Amit Pramanik
WERF
635 Slaters Lane, Suite 300
Alexandria, VA  22314-1177
Phone: 703-684-2470
Email: apramanik@werf.org

Shuang Qi
Senior Environmental Engineer
Pegasus Technical Services  Inc.
26 West Martin  Luther King Drive
ML 421
Cincinnati, OH  45268
Phone:513-5697819
Email: qi.shuang@epa.gov

Maureen Reilly  (presentation via phone)
Executive Director
Sludgewatch
103 Avenue Road, #601
Toronto, Ontario, Canada
Phone:416-801-4099
Email: maureen.reilly@sympatico.ca

Mark Rodgers
Microbiologist
USEPA-NRMRL
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7225
Email: rodgers.mark@epa.gov

Alan Rubin
Principal
Envirostrategies, LLC.
3304 Mill Cross Court
Oakton, VA
Phone: 703-385-8203
Email: rubinhial@cox.net

James Ryan
Soil Scientist
USEPA-NRMRL
5995 Center Hill Avenue
Cincinnati, OH  45268
Phone:513-569-7653
Email: ryan.jim@epa.gov

Yonggui Shan
Senior Research Associate
University of Cincinnati
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7606
Email: shan.yonggui@epa.gov
Marietta Sharp
Environmental Scientist
Washington Department of Ecology
3190 160th Avenue, S.E.
Bellevue, WA 98008
Phone: 425-649-7258
Email: mars461@ecy.wa.gov

Mano Sivaganesan
Statistician
USEPA-NRMRL
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7118
Email: sivaganesan.mano@epa.gov

James Smith
USEPA-NRMRL
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7355
Email: smith.james@epa.gov

Curtis Snook
Medical Officer
USEPA, NOT
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-487-2434
Email: snook.curtis@epa.gov

Jeff Seller
Principal Scientist
Seller Environmental
3022 King Street
Berkeley, CA 94703
Phone:510-847-0474
Email: jsoller@sollerenvironmental.com

Laurel Staley
Branch Chief
USEPA-NRMRL
26 West Martin  Luther King Drive
Cincinnati, OH 45268
Phone:513-569-7863
Email: staley.laurel@epa.gov

Gerard  Stelma
Senior Science Advisor
USEPA-NERL
26 West Martin  Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7384
Email: stelma.gerard@epa.gov
                                                 22

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Rick Stevens
Biologist
USEPA-OW-OST
1200 Pennsylvania Avenue, 4304T
Washington, DC 20460
Phone:202-566-1135
Email: stevens.rick@epa.gov

Karla Thrall
Staff Scientist
Pacific Northwest National Laboratory
902 Battelle Boulevard, Mail Stop P7-59
Richland, WA  99352
Phone:509-376-6115
Email: karla.thrall@pnl.gov

Eunice Varughese
Microbiologist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati,  OH  45268
Phone:513-569-7628
Email: varughese.eunice@epa.gov

Michael Ware
Microbiologist
USEPA-NERL
26 West Martin Luther King Drive
Cincinnati,  OH  45268
Phone:513-569-7731
Email: ware.michael@epa.gov

Alan Weinrich
USEPA-NHSRC
26 West Martin Luther King Drive
Cincinnati,  OH  45268
Phone:513-569-7860
Email: weinrich.alan@epa.gov
Karen White
Bio Science Technician
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7248
Email: white.karenm@epa.gov

Steve Wing
Associate Professor
UNC Department of Epidemiology
 CB# 7435, School of Public Health
Chapel Hill, NC
Phone:919-966-7416
Email: steve_wing@unc.edu

Stephen Wright
Environmental Scientist
USEPA-NRMRL
26 West Martin Luther King Drive
Cincinnati, OH  45268
Phone:513-569-7610
Email: wright.stephen@epa.gov

Eric (Xueqing) Zhu
Senior Environmental Engineer
Pegasus Technical Services, Inc.
26 West Martin Luther King Drive
ML 421
Cincinnati, OH  45268
Phone:513-569-7571
Email: zhu.xueqing@epa.gov
                                                 23

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                                            Appendix C
                           Update on Incident Response Project
Project Signed, Work Begun on Timely Incident
Response

A  new  WERF  research  effort,   Methodology  for
Implementing a Timely Incident Response Mechanism
(03-HHE-5PP), was started under the lead of Principal
Investigator Steve Wing with the University of North
Carolina. The first phase of the project will develop a
protocol to  be used  in conjunction with  established
public    health   investigation    procedures    and
implemented  through the existing  network of public
health organizations. The  protocol will be designed to
help determine if, among all potential causes, reports
of illness in a community could be associated with
biosolids land  application  or  possibly   other  soil
amendment practices.

Proper implementation of the protocol by  local, state,
and federal agencies could  provide information about
the occurrence of reported symptoms in proximity to
biosolids land applications  sites and the  times  and
places   where   reports   are   more   common.
Implementation of the protocol  could also help provide
the basis for conducting more  definitive  studies of
causal links  between  human  exposures  from  all
potential  sources and  health effects.  The  current
project does not include  implementation.

This project was the highest  ranked priority at the
2003  Biosolids  Research  Summit,  during  which  a
group of  nearly  75 individuals  representing agencies,
conservation groups, wastewater facilities, academia,
and citizens identified their most  pressing  research
needs  regarding land application  of biosolids. This
research  responds to a July  2002  report from the
National  Research Council (NRC)  of  the  National
Academy of Sciences regarding  potential health risks
related biosolids.

Oct. 5, 2006

http://www.werf.org/press/News Events.cfm
                                                   24

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                                           Appendix D
                                       Technical Reports
Appendix D contains five technical reports that expand
upon the information presented at the workshop.  The
titles and authors of the technical papers are:

    •   Non-Invasive    Assessment    of    Dermal
       Exposure by Karla Thrall
    •   Odor, Irritation,  and Health  Symptoms from
       Biosolids Land Applications by Pamela Dalton
    •   Detection   and   Measurement   of  Odor,
       Ammonia, and Hydrogen Sulfide Emissions as
       Part of USEPA  Biosolids  Land Application
       Research Project by Robert Forbes
    •   Community and  Occupational  Risk  from
       Bioaerosols  during  Land  Application  of
       Biosolids by Ian Pepper
    •   Lessons Learned  Regarding  the  Use of an
       Impinger for Collecting  Airborne  Bacteria
       during a Biosolids Application Field Study by
       Edwin Barth

The first four papers were submitted by scientists not
employed by the USEPA. As such, these papers were
not reviewed by the Agency prior to inclusion in these
proceedings.   The last paper  by Edwin Barth, an
Agency scientist, was reviewed before  publication  in
accordance with USEPA policy.
                                                  25

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                                           Appendix D-1
                     Non-Invasive Assessment of Dermal Exposure
Karla D. Thrall
Pacific Northwest National Laboratory

Abstract

Realistic estimates of dermal bioavailability  following
exposures to volatile  solvents in  the  workplace,  or
through contaminated soil and water,  are critical  to
understanding human  health  risk.    Compared  to
dermal exposures with neat or aqueous  compound,
little  is understood about the dermal bioavailability  of
solvents in soil, dust, sludge, or sediment matrices.  A
method has  been developed  to  determine dermal
uptake of solvents under  non-steady state conditions
using real-time breath analysis.  The exhaled  breath
data is subsequently  analyzed  using  physiologically
based pharmacokinetic (PBPK) models to estimate the
dermal permeability  coefficient (Kp).  This approach
has  been utilized in  both  experimental animal and
controlled human  exposures, with  studies conducted
to compare the impact of exposure matrix, occlusion
versus  non-occlusion  exposure  techniques,  and
species-differences.   To  date, studies have  clearly
illustrated that the methodology is sufficiently sensitive
to enable the conduct  of animal and human dermal
studies  at low  exposure concentrations  over  small
body surface  areas, for short periods  of time. Further,
the portability of the system allows the methodology to
be used to conduct occupational and  environmental
exposure assessments to volatile compounds.

Introduction

Traditional exhaled breath analysis techniques have
involved collection of breath  samples  in Tedlar bags  or
stainless  steel canisters,  followed   by  laboratory
analysis  -   generally   by  gas   chromatography.
Disadvantages  of  these  techniques  include the
possibility that the  collection device may  alter the
integrity of the sample, the time-consuming and costly
analysis of the sample, and the delay between sample
collection  and   analysis.     To   overcome  these
disadvantages,  a  breath-inlet device  for  a mass
spectrometer was  developed   to  allow  for  the
continuous real-time analysis of undiluted exhaled air
from experimental  animals  and  humans (Thrall  and
Kenny, 1996; Thrall et al. 2001).  The applications  of
this  system  in   studies ranging  from  occupational
exposure  assessment  to   applied  research  are
described  here.

Exhaled Breath Analysis System

The  breath   monitoring  system  utilized  in  all  the
described  studies  consisted  of  an  inlet  device
connecting a human volunteer directly  with a mass
spectrometer. The subjects' exhaled breath is passed
through a heated large-diameter transfer line into a
heated glass-mixing chamber (1.3-L  volume).    A
breath  sample enters the mixing  chamber via a tube
that bends off to one side, and exits the chamber via a
tube   bending   in  the  opposite  direction,  thus
maximizing   turbulence   and  mixing.     A  mass
spectrometer continually withdraws air samples from
the center of the mixing chamber at a calibrated rate.
Excess exhaled  air is vented from the mixing chamber
via a large diameter bore-hole exit tube with negligible
flow restriction.

For rodent studies, animals  are individually placed  in
small off-gassing chambers.  The animals are awake
and unrestrained while in the chamber. Hospital grade
(grade  D) breathing air  is  supplied to the animal
through the lid of the chamber at a calibrated rate  of
approximately 200 ml/min.  The  mass  spectrometer
continually withdraws  air samples from the mixing
chamber through a port in the chamber lid at the same
rate of approximately 200 ml/min.  The concentration
of compound in the chamber is  used  to  represent
exhalation from the animal.
                                                   26

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                            Inhalation
                            Exposure
                      Intravenous-
                      Infusion







, /
/}
Alveolar Air
Lung Blood

Richly Perfused
Tissue

Slowly Perfused
Tissue

Skin *

Fat

Kidney

Liver
t
Gl Tract
— i" Exhaled Air









Dermal
Exposure
                                                                                Exposure


Figure 1: General  structure of a PBPK model used to describe pharmacokinetics,  tracking uptake from an oral, intravenous
injection, inhalation, or dermal exposure, distribution into tissues of concern, metabolism, and elimination of the parent compound
and/or metabolites in exhaled breath, blood, urine, and feces
Exhaled breath data can be related to total exposure
and  internal  target tissue  dose through the  use of
physiologically   based   pharmacokinetic   (PBPK)
modeling.   These physiologically relevant models are
powerful tools that can be used to simulate a chemical
exposure,   regardless  of  route,  and  estimate  the
amount of  the  compound or  its  metabolite at  a
particular internal target.  A PBPK model is based on
physiology  (tissue or organ volumes, blood flow rates,
breathing   rates,  gender-specific  physiology,  etc.),
chemical characteristics (partition coefficients, density,
binding characteristics, etc.), and biochemical factors
(enzyme-specific metabolic rates, etc.). These factors
are  either  available  in the  literature,  or  can  be
determined in focused in vitro or in vivo studies.

A  PBPK  model, such  as illustrated  in  Figure 1,
describes the body as a series of tissue compartments
representing  the  probable  route(s)  of exposure, the
metabolically  active  tissues,  target  organs,   and
excretion  pathways.   These  models  are   typically
developed   and   experimentally   validated   using
common laboratory animals,  then   extrapolated to
represent man.  A series of differential equations are
used to mathematically describe the absorption, tissue
distribution,   metabolism  and   elimination  of  a
compound   in  the   body.    Once   experimentally
validated,  a PBPK  model  will facilitate extrapolation
across different routes of exposure,  from high-to-low
doses, and  among animal species (Andersen  et  al.
1993).  Thus,  by  monitoring exhaled  breath  for  a
particular  compound,  the  estimated exposure and
target tissue dose can be determined.

Application in Dermal Bioavailability Studies

The  combination  of  real-time breath  analysis and
PBPK modeling provides an opportunity to follow the
changing  kinetics  of  the  uptake, distribution, and
elimination  phases  of a  compound  throughout  a
dermal  exposure.    The  sensitivity  of the  mass
spectrometer for  exhaled-breath  analysis  has been
pivotal in enabling studies wherein human volunteers
are exposed to  low  levels  of compounds  for short
periods  of  time.    For  example,  a  method  was
developed to determine dermal absorption of solvents
under  non-steady-state  conditions  by  monitoring
exhaled air using  real-time breath analysis in  rats,
monkeys,  and humans.  Dermal patch systems were
developed to expose experimental animals and human
volunteers  to volatile  chemicals  in soil  and  water
matrices under occluded and non-occluded conditions.
The occluded system consisted of a hand-blown glass
cell with  a  needle-hole opening  in the top to allow
addition of the dosing solution. The  needle  hole was
sealed using silicone  following  addition of the test
                                                    27

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                                                             Rat Kp = 0.25 cm/hr
                                            1.5   2.0   2.5   3.0    3.5    4.0    4.5    5.0
                                                                  Human Kp = 0.0063 cm/hr
Figure 2. Comparison of PBKB model predictions (lines) and exhaled breath (human) or chamber concentration (rat) following
dermal exposure to aqueous methyl chloroform. The human subject was a 29-year old Caucasian man, 95.3-kg body weight,
195.6-cm height, and 22.2-% body fat. Data are averaged after every 1-minute interval (Poet et al. 2000).
material.  For  non-occluded exposures, glass cells
were  constructed with  an upper chamber separated
from the matrix by a semi-permeable frit.  The upper
chamber was packed with activated charcoal to trap
volatilized test  materials.   Regardless  of exposure
system, the cells were attached to the forearms  of
human volunteers  (two per arm) or a clipper-shaved
area  on  the  lower  back of  the   rat  using  a
cyanoacrylate adhesive.

A number of comparisons of dermal absorption have
been  conducted,  including  occlusion  versus  non-
occlusion, soil  versus  water matrices,  and species
differences.  For example, Figure 2 compares rat and
human   dermal  exposures  to   aqueous  methyl
chloroform  under occluded  conditions.  Despite the
similarity  between  the  rat  and  human  exposure
concentrations  (0.1% and 0.12%,  respectively) and
exposure surface areas  (1.7%  rat and  3% human),
there  were clear differences  in both the  time to peak
exhalation and the amount of compound exhaled (area
under the curve)  between  rats  and  humans.   The
permeability constant  (Kp)  calculated  from  these
exhaled-breath  data  using  the PBPK model  was
roughly  40 times higher in the rat than that calculated
for the human (Kp = 0.25 cm/hr versus 0.0063 cm/hr).

Discussion

Exposure assessment  is a critical  component  of
industrial  hygiene   and  worker   health   protection
programs.  Given today's diverse work environments,
new  and  innovative  methodologies  for  exposure
assessment are needed  in order to fully understand
the potential health risks of the  individual worker.
Routine analysis of exhaled  breath  may be ideal for
tracking occupational exposures, particularly with the
advent     of     field-deployable      standardized
methodologies.   Exhaled  breath  analysis  offers  a
number of advantages,  including being  non-invasive,
is applicable to a number of compounds,  avoids the
handling of potentially infectious biological samples,
and  can be analyzed easily and  quickly using the
methodology   described  here.    Furthermore,  the
system  described here  goes well  beyond traditional
industrial     hygiene     exposure     assessment
methodologies  by   employing  PBPK   models  to
understand the relationship between exposure  and
internal, target tissue dose.

The  studies described  here  have focused  on the
utilization  of   exhaled   breath   analysis  following
controlled  dermal  exposures  to  understand  the
contribution of the dermal pathway on total exposure.
These studies  have  illustrated that the methodology is
sufficiently sensitive to successfully measure low-level
exposures,  or   exposures   to   poorly   absorbed
compounds in humans.  The field  portability  of the
system  has  the  potential  to  place the  exposure
assessment methodology in situations where it can be
used  to  conduct  occupational  and environmental
exposure assessments to volatile compounds.

References

Andersen, M.E., Krewski, D., and Withey, J.R. (1993).
Physiological   pharmacokinetics   and   cancer  risk
assessment. Cancer Letters. 69: 1-14.

Poet, T.S., Thrall, K.D., Corley, R.A., Hui, X., Maibach,
H.I.,  and  Wester,  R.C. (2000).  Utility  of real time
breath   analysis     and    physiologically    based
pharmacokinetic   modeling   to   determine   the
percutaneous absorption of methyl chloroform  in rats
                                                    28

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and humans. Toxicological Sciences. 54:42-51.               and  evaluation  of a   breath-analysis  system  for
                                                           biological monitoring of volatile compounds. American
Thrall, K.D., and Kenny, D.V. (1996).  Evaluation of a        Industrial Hygiene Association Journal. 62(1): 28-35.
carbon     tetrachloride     physiologically    based
pharmacokinetic model using real-time breath-analysis        The work described in this paper was not funded by the
monitoring in the rat.  Inhalation  Toxicology. 8(3): 251-        United  States   Environmental   Protection  Agency  and,
261                                                        therefore, the contents do not necessarily reflect the views of
                                                           the Agency and no official endorsement should be inferred.
Thrall, K.D., Callahan,  P.J.,  Weitz,  K.K., Edwards,
J.A., Brinkman,  M.C., and Kenny, D.V. (2001). Design
                                                      29

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                                           Appendix  D-2
    Odor,  Irritation, and Health Symptoms from Biosolids  Land Applications
Pamela Dalton, PhD, MPH
Monell Chemical Senses Center

Malodors  from  biosolids comprise  one  of  the  chief
complaints that  are  regularly   lodged  against  land
applications,  particularly  those   occurring  in   close
proximity  to residential  communities.   In addition to
generating annoyance  complaints,  however,  malodor
perception often  triggers reports  of health symptoms
among exposed  individuals.   Distinguishing  between
exposures to the volatiles from  land applications that
elicit ocular or  upper respiratory sensory irritation or
health  symptoms  from those that elicit only  odor-
mediated annoyance is a key component in establishing
and enforcing exposure guidelines that are protective of
residents  in nearby communities.  Laboratory studies
documenting  exposure  concentrations  and  multiple
levels of response  can  help to establish thresholds for
annoyance,  irritation and  health  symptoms.    Such
studies can also help differentiate  between the degree to
which  complaints  and  symptoms  are  mediated  by
psychological  rather   than   physiological   factors.
However, the successful application of these findings to
practice   requires  a   better  understanding   of  the
relationship between  actual community  exposures and
response  thresholds  for each  level.    This approach
necessitates an  increased focus  on   measuring  the
association between the dynamic profile (i.e.  intensity,
frequency and duration) for key odorants and irritants in
biosolids emissions and the perception and response of
exposed community residents.

This paper is divided  into two broad sections.  The first
section addresses  the factors which promote adverse
responses to the  volatile organic compounds associated
with biosolids land applications while the second section
outlines  some   key  components  which  should  be
included in a monitoring  plan to evaluate exposure and
impact of land applications on humans.

A workshop held  at Duke University in 1998 (Schiffman
et al., 2001) led to a consensus among attendees that
there  were three  paradigms or mechanisms by which
volatile  organic  compounds  (VOCs)  could generate
health symptoms and adverse responses: (1) The VOCs
emitted  from land  applications could individually or  in
combination  produce  ocular  and/or  upper   airway
irritation, (2) the  VOCs  could  produce objectionable
odors which themselves could elicit annoyance, stress
and perception of health  risks and (3) the VOCs  may
simply co-occur  with  bioaerosols  and/or particulates,
which themselves  are eliciting the symptoms.   This
paper will focus solely on the first two mechanisms for
VOC-induced complaints and examine the evidence for
each one in turn.

VOCs, Odor and Sensory Irritation

As odorant concentration increases, the sensory effects
experienced typically occur in a predictable sequence.
At low concentrations, the presence of an odor can be
detected against a clean air  background, although its
quality may not be apparent. The  recognition threshold
for  an odorant is  typically three  times  the detection
threshold.     With   increasing   concentrations,   an
undesirable  odor will  often  bring  about  annoyance
and/or  intolerance.     For   most  volatile   organic
compounds, direct physical  effects from an  odorant do
not occur until the  concentration increases to the point
beyond   trigeminal   or  sensory   irritant  activation.
Nevertheless, people  often report physical  symptoms
and  irritation at  concentrations much lower than the
irritant threshold.

Sensations of odor and upper airway irritation are often
experienced  as   a  unitary phenomenon,  principally
because most volatile chemicals have the potential to
activate   two  separate,   yet  interrelated,  sensory
pathways in the upper respiratory airways: the olfactory
nerve, which gives rise to  sensations of odor, and the
trigeminal, glossopharyngeal or vagal nerves which give
rise to temporary burning,  stinging, tingling or  painful
                                                   30

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sensations in the eyes and  upper airways  (Doty et al.,
2004). Chemical stimulation  of  the  trigeminal nerve
(known   as   chemesthesis)   often   combines   with
stimulation of the olfactory nerve to produce sensations
that  form an  overall perception  of a chemical.   For
example,  low concentrations  of  ammonia  produce  a
distinct odor; however, higher concentrations may also
elicit  a  mucosal  burning   or  tingling,  which  is  the
chemesthetic or irritant component of perception.  Most
chemicals at sufficiently high concentrations are capable
of eliciting upper respiratory tract  irritation in addition to
odor sensations.  Because these two sensory pathways
(olfaction  and  chemesthesis)  can  be activated  by  a
single chemical  stimulus, people  often experience and
report odor and irritation as a  unitary perception.   For
purposes  of  evaluating  the  irritant potential  of  a
chemical,   this  confusion   can  invalidate  self-report
methods,  such  as  symptom  questionnaires  or even
ratings of a chemical's irritancy.

The sensory detectability, irritant potential and quality of
land application  emissions can  be  determined in  a
variety of ways both  in the laboratory and the field.  For
example, in the laboratory, bagged samples of air taken
from various onsite and offsite locations can be fed into
air-dilution olfactometers and presented to panelists for
forced-choice detection of the presence  or absence of
odor.   To measure irritation  thresholds that  are  not
confounded by simple  odor detection, a method known
as nasal or ocular lateralization is  used. This technique
exploits the fact that the  presence of a pure  odorant
cannot be localized to the left or right nostril  or the left or
right  eye,  but  a   chemical  capable  of  stimulating
trigeminal receptors  will clearly provide a sensation in
the nasal or  ocular mucosa  which  can be  localized
(Wysocki et al., 1997; Doty et al., 2004).

For evaluation in the field, an  instrument known as the
Nasal Ranger™ can  be utilized to  actively dilute ambient
air samples in a series of known dilution ratios which are
then compared  with clean  filtered air (McGinley and
McGinely,  2004).    The  dilution  ratio  at  which  the
individual can reliably discriminate the  'stimulus' air from
the clean filtered air is the odor threshold.  In both cases,
this is reported as the dilutions to  threshold ratio for the
emission.     Although  not  explicitly designed  for this
purpose,   it  is  possible that a device  like the Nasal
Ranger™  could be adapted to do field  evaluations of the
lateralization  potential  (which nostril  or  eye  is  being
stimulated) of real-time emissions.  Such an adaptation
combines  the  advantage  of  onsite  testing  with  the
sophistication   of  the  lateralization  technique  for
determining whether emissions reach  the concentration
necessary to elicit sensory irritation.
Monitoring VOCs and Annoyance

Often the odors associated with  land  application  of
biosolids,   become   significant   community  issues.
Exposed residents may claim that the odors are making
them ill.  Research has shown that for some period of
time following land application,  malodors are typically
present at concentrations greater than those capable of
generating  odor  perception, but  at  offsite  locations,
frequently fall well below those concentrations capable
of generating sensory irritation  or other acute  health
effects.   The challenge  then is to identify the reasons
behind community reactions to odors and to understand
whether the  volatile odor chemicals (i.e. odorants)  elicit
health symptoms through direct physiological mediation
or through psychological or stress mechanisms.

One way to  think about the sensory determinants of
what makes  an  odor  annoying  is the acronym FIDO -
the  Frequency of an odor, the Intensity  at which it
occurs, its Duration and its Offensiveness. The first three
characteristics  can   be   measured  analytically  with
instruments. However, to understand Offensiveness, it is
necessary to  measure people's reactions. This requires
understanding not only the primary sensory attributes of
an odor, but non-sensory attributes, or the cognitive and
emotional factors that can produce  heightened  odor
awareness and annoyance.

Frequency.   Frequency of  exposure can by  itself alter
sensitivity.     While   it   is  well  acknowledged  that
continuous exposure  to a  chemical at a fairly steady
concentration  leads  to  a decreased  intensity  or
adaptation, most of the odors from land applications are
likely to  be  intermittent  in  nature  and   varying  in
concentration.   In studies that evaluated the impact of
intermittent exposure  to  low-level odors via threshold
testing,  it was found  that females of reproductive age
exhibited dramatically  increased sensitivity to the odor to
which they were  exposed,  while  males,  pre-pubescent
females and  post-menopausal females did not change
sensitivity (Diamond et al., 2005; Dalton et al., 2002).  In
the real world, intermittent exposure to VOCs from land
emissions, especially  at very low levels, may serve to
increase their detectability, even when emissions wane.

Intensity.  Measuring the number of dilutions to odorant
threshold is  important to establish the concentration at
which an odorant will become detectable, but provides
little information about how increasing concentration will
affect stimulus  intensity.     Although  many odorous
compounds  become  detectable at  approximately the
same  physical  concentration,   increases  in  physical
concentration for  one  odorant may result in  much larger
changes in   perceived  intensity than  do  the  same
increases in physical concentration for another odorant.
Thus, the shape of the psychophysical function and the
                                                     31

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slope are important indicators of how intensity  (and
perhaps how annoyance) will change with concentration,
and are not the same for all odorant chemicals that may
be found in land applications.

Duration.  The  duration  of exposure  is also  critically
important when evaluating  the  irritant  potential  of  a
volatile chemical.  Increased duration of nasal exposure
to ammonia,  for  example,  has  been  shown  to  also
increase the  perceived  irritant   intensity,  due  to  a
phenomenon known as temporal integration (Wise et al,
2005).  It is likely that many other irritants exhibit similar
patterns  of integration, thereby placing important limits
on the ability to extrapolate from  evaluation of irritants
for  very brief durations  (i.e., seconds) to  real-world
exposures that may last minutes or hours.

Which Odorous Components to Monitor?

It is also important to recognize that emissions from land
applications  comprise  a  complex mixture  of  many
different volatiles, some  of which  have the potential to
contribute to odor or irritant impact more than others.
For this reason, the  choice  of which volatiles to monitor
is critical in order to understand the sensory impact of
any emissions which migrate offsite.  The  most typical
compounds  measured  from biosolids emissions  are
hydrogen sulfide (H2S) and  ammonia; H2S has a readily
detectable  and  objectionable   odor   at  very   low
concentrations and  is often present at a high volume
while ammonia can  contribute to  the irritation impact of
emissions.  However, due to the highly heterogeneous
nature  of biosolids,  there  are  many other malodorous
compounds which may be  emitted during and after land
applications and which may contribute substantially to
the odor or irritant impact.  Both H2S and ammonia have
fairly high vapor pressure and while they readily diffuse
into air and may well comprise the majority of the early
sensory impact, they are also likely to dissipate more
rapidly  than  other  compounds  and  thus,  prolonged
sensory  impact may  well be   the  result   of  other
compounds (such as amines,  indoles) which volatilize
more slowly from the application site.

In the laboratory, characterizing the sensory impact from
biosolids via  methods  such  as  gas  chromatography/
olfactometry (Preti et al., 1993; Bazemore et al., 2000),
in which human reports  of odor qualities in a complex
mixture  are linked to the output of a gas chromatograph
column  separation and the  subsequent identification, is
important for understanding the sensory potency of any
biosolids mixture.  In the field, real-time  measurements
of other compounds with high  odor potential  should be
considered.  However, if emissions measurements are
confined to  sentinel compounds, such as  H2S  and
ammonia,  then  continuous  monitoring  should  be
augmented by  regular and frequent evaluation by human
detectors using Nasal Ranger™ technology, to  ensure
that compounds other than those being monitored are
not eliciting malodor perception offsite.

To summarize, the choice of when,  where and how
frequently to  measure and the selection  of which key
components to measure are important features of any
comprehensive  monitoring  plan  for  assessing  the
annoyance impact  of  biosolids land  applications on
nearby residents.

Cognitive Factors and Perception of Health Risk

Unfortunately,  measuring and finding ways to reduce the
sensory impact of any volatile compounds from land
applications may not always resolve the complaints from
people living or working near the land  application site.
The perception of even weak or transient  malodors can
also   elicit  adverse  responses via  psychogenically-
mediated mechanisms, such as annoyance and stress.
Although the presence of an odor is a signal of chemical
presence,  the potency or hedonic  nature  of an  odor
sensation  does  not  correlate  with  its toxic potential.
Because many  malodors  can  be  smelled  in  minute
concentrations, simply being able to smell the malodor
does  not  signify  that  it  is   present  in  a  harmful
concentration.

Research  in  our laboratory has shown  that people's
reaction to  odor and their beliefs about the effects from
odor are influenced by a diverse set of factors, including
personality traits,  personal  experience  and information
or social cues from the community and media.  These
factors  can increase, or in some  cases  decrease,  a
person's sensitivity and  awareness of environmental
odors. In a  series of studies, we have demonstrated how
the  misperception  of  the risk from  odors  actually
changes a person's sensory perception of odor levels
and their perception of well-being (Dalton  et al., 1997;
Dalton, 1999).

The  results from these studies indicate to  us that the
reaction that people have to odors  is not simply due to
their sensory impact but is also  shaped by the attitudes
and expectations that an  individual brings to an  odor
experience.  In  no way do we wish to  minimize the
importance  of remediating  the  sensory impact  of
emissions  as  the  first step in  reducing  the  level of
community annoyance and complaints.     However,
because even  small amounts of odorous molecules can
generate odors, reductions of  70-80 percent  in  odor
concentration can still result in  complaints if neighbors
can  detect even  weak and  transient  odors and are
concerned  about  the  health impact  of the emissions.
Thus,  working with communities   and  neighbors  to
provide  them  with  information  and  to  help  them
understand the nature of the odors, what they represent
                                                   32

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and their known effects can be a powerful tool to modify
the cognitive  factors that often  guide and influence
community reaction to odor emissions.

Conclusions

Monitoring programs for evaluating the effect of biosolids
land applications on  human  health  should  focus  on
closing the data gaps in the following three areas.  First,
simultaneous  documentation   of   actual   exposure
concentrations and  effects  at the  observer level  in
residential communities is a priority. At the  present time,
much  is  known  about  the  emissions  at  the  land
application site and at the fence line, but few  studies
have  undertaken  comprehensive  monitoring  at the
community level.   Second,  any such  program should
strive to identify the appropriate odorants  and potential
irritants for monitoring, which may well be more than just
H2S  and ammonia.  Finally, response  variation within
and across communities should be evaluated in light of
the composition  of the  biosolids, the temporal factors
(duration and frequency) of  exposure and  the attitudes
and  expectations of the exposed  community.  These
factors,  taken together, can do much  to identify and
illuminate  the actual  and perceived sensory  and health
concerns from biosolids land  applications.

References

Bazemore, R., Wysocki, C.J.,  Murray, S.,  Lawley, H.J.,
and   Preti,   G.   (2000).  Amelioration  of  odorous
components in spent mushroom compost.  Journal  of
Agricultural and Food Chemistry. 48: 3694-3697.

Dalton,  P.  (1999).  Cognitive  influences  on   health
symptoms  from  acute   chemical   exposure.  Health
Psychology. 18: 579-590.

Dalton,  P.,  Doolittle,  N.,  and  Breslin,  P.A.S.  (2002).
Gender-specific induction  of ultra-sensitivity  to odors.
Nature Neuroscience. 5: 199-200.

Dalton, P., Wysocki, C.J., Brody, M.J., and Lawley, H.J.
(1997). The influence of cognitive bias on the perceived
odor,  irritation  and  health  symptoms  from chemical
exposure. International  Archives  of Occupational and
Environmental Health. 69: 407-417.

Diamond, J., Dalton, P., Doolittle, N.,  and Breslin,  P.A.
(2005).    Gender-specific    Olfactory   Sensitization:
Hormonal and Cognitive  Influences. Chemical Senses.
SOSuppI 1:1224-1225.

Doty,  R.L.,  Cometto-Muniz,  J.E., Jalowayski,  A.A.,
Dalton,  P., Kendal-Reed, M., and Hodgson, M. (2004).
Assessment  of  upper respiratory tract  and  ocular
irritative effects of volatile chemicals in humans. Critical
Reviews in Toxicology. 34: 85-142.

McGinley, M.A. and McGinley, C.M. (2004).  Comparison
of field  olfactometers  in  a controlled chamber using
hydrogen sulfide as the test odorant. Water Science and
Technology. 50: 75-82.

Preti, G., Gittelman, T.S., Staudte, P.B., and Luitweiler,
P. (1993).  Letting the  nose lead  the  way.  Malodorous
components in drinking water. Analytical Chemistry. 65:
699A-702A.

Schiffman, S.S.,  Walker, J.M.,  Dalton, P.,  Lorig, T.S.,
Raymer, J.H.,  and Shusterman,  D.  (2001).  Potential
health  effects   of  odor   from   animal  operations,
wastewater treatment,  and  recycling of  byproducts.
Journal ofAgromedicine. 7: 7-81.

Wise,  P.M.,  Canty, T.M.,  and Wysocki,  C.J. (2005).
Temporal integration of nasal irritation  from  ammonia  at
threshold  and  supra-threshold  levels.  Toxicological
Sciences. 87: 223-231.

Wysocki, C.J., Dalton, P., Brody, M.J., and Lawley, H.J.
(1997).  Acetone odor and irritation thresholds  obtained
from acetone-exposed  factory workers and  from control
(occupationally   non-exposed)   subjects.   American
Industrial Hygiene Association Journal. 58: 704-712.
The work described in this paper was not funded by the United
States Environmental Protection Agency and, therefore,  the
contents do not necessarily reflect the views of the Agency and
no official endorsement should be inferred.
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                                          Appendix D-3
      Detection and Measurement of Odor, Ammonia, and Hydrogen Sulfide
   Emissions as part of USEPA Biosolids  Land  Application Research Project
Robert H. Forbes, Jr., P.E.
CH2M HILL, Inc.

Abstract

CH2M HILL participated  in a research  project  entitled
"Multimedia Sampling  at Biosolids  Land Application
Sites" as a consultant  to  Battelle Memorial Institute in
the fall of 2004. The overall goal of the research was to
advance  the  science  of air,  soil,  and water quality
measurements associated  with  land  application  of
biosolids to agricultural sites.  CH2M HILL's role was to
detect and characterize  odors from the  site,  and to
measure  the   concentrations  of two  volatile  gases
commonly associated  with  biosolids land application,
ammonia and  hydrogen sulfide.

Background odor  levels  prior to biosolids application
(Day -1) were measured as "trace"  by  the onsite field
receptors,  while the more sensitive, offsite odor panel
measured the  odor levels at 60-90 dilutions-to-threshold
(D/T).  This is indicative  of the sensitivity differences
between onsite and offsite odor measurements.  Odors
from  ground-level  flux  chambers   onsite  increased
substantially after biosolids application (Day 0) and the
following day  (Day  1) as measured  by  the offsite odor
panel. Odors measured in the field increased on Day 0
after application  as expected,  but  did  not increase
significantly above  Day  0 levels on  Day  1.   Odors
measured  by  the field crew began subsiding on Day 2
and  returned   to  background  levels at  all sampling
locations by Day 4.

Both ammonia and hydrogen sulfide were detected at
fairly  significant  concentrations  (15 ppmv  and 0.17
ppmv, respectively) from the flux chamber exhaust soon
after  biosolids application   on  Day  0,   but  their
concentrations at five feet above ground  level (where
most field measurements were taken) were much lower.
Their concentrations had subsided further at all locations
by Day 1 and Day 2, returning to background levels by
Day 4.

It  was  concluded  that  most odors  associated with
biosolids land  application are not  from  ammonia and
hydrogen sulfide,  but more  likely  are  from  reduced
organic  sulfur  compounds such  as methyl  mercaptan
and dimethyl sulfide, along with nitrogen-based  organic
compounds such as trimethyl amine, indole, and skatole.
Chemical measurements made by others during the field
study support these conclusions.

Introduction

CH2M HILL participated  in  a research project  entitled
"Multimedia  Sampling at Biosolids  Land Application
Sites" as a consultant to Battelle  Memorial  Institute in
the fall of 2004. The overall goal of the research was to
advance  the  science  of air,  soil,  and water quality
measurements  associated   with  land application  of
biosolids to agricultural sites.  CH2M HILL's role was to
detect and  characterize  odors from the site,  and  to
measure the  concentrations  of   two  volatile  gases
commonly  associated  biosolids   land   application,
ammonia and  hydrogen sulfide.  Field work began  on
the day before the biosolids application event (Sept. 29,
2004  =  Day -1)  and continued  through the  day  of
application (Sept. 30, 2004 = Day 0), followed by Days
1,2, and 4.

Materials and Methods

Odor measurements onsite were made on all five field-
sampling days using three  hand-held olfactometers
called Nasal Rangers™,  and the field odor levels were
compared with odor measurements made by an offsite
odor panel on three of the sampling  days (Day -1, Day 0,
and Day 1).   Field measurements of ammonia were
performed using chemical sensory  tubes manufactured
by  Drager™  coupled  with  a  calibrated  hand-held
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vacuum pump, also by Drager™.  Field measurements
of hydrogen sulfide were made using a Jerome™ type
gold-film  analyzer  as   manufactured   by   Arizona
Instruments,  with  an  internal  sample  pump  and a
detection  limit of  0.001  parts  per  million  by volume
(ppmv).

Three air samples were taken from onsite flux chambers
installed   by  other  researchers  (McConnell,  2005),
pumped  into  Tedlar  air-sample  bags,   and  shipped
overnight to St. Croix Sensory Laboratories in St. Croix,
MN, where a certified odor panel was used to conduct
offsite  odor  analyses  according  to a  standard  and
accepted procedure for measuring the intensity of odors.
In this  procedure, each odor panelist tries to distinguish
a diluted odor sample from two other samples that are
odor-free  blanks.   The sample  air is mixed with pure,
odor-free air at pre-set dilutions. The sample's dilution
level at which an odor is barely detected by the panelist
is called  the "detection threshold"  (DT)  of that  odor
sample.   The intensity of the odor  sample  itself is
measured in terms of the number  of dilutions that were
combined in the sample at which the DT was measured,
in units expressed as dilutions-to-threshold  (D/T).  A
seven  person panel  is used  in certified, offsite  odor
testing, and the measurements  by different panelists are
averaged to arrive  at the reported odor DT in odor units
of D/T.

Onsite  odor  measurements  taken  with  the Nasal
Ranger™  olfactometer   are   intended   to  mimic
measurements made  by  an offsite  odor panel,  with
results of both methods reported  in similar odor units
(D/T).  The offsite odor-panel measurements are usually
much more sensitive than onsite measurements, but the
relative differences among  odor samples should show
similar  trends  with   either  onsite  or  offsite  odor
measurements.

Results

Results of daily odor measurements were compared and
trended.  Background levels prior to biosolids application
(Day -1) were measured as "trace" by the  onsite field
receptors, while the more sensitive, offsite  odor panel
measured the odor levels at 60-90 dilutions-to-threshold
(D/T).  This is indicative of the sensitivity  differences
between onsite and offsite odor measurements.

Odors  from  ground-level  flux chambers  increased
substantially after biosolids application (Day 0) and the
following day (Day 1) as measured  by the offsite  odor
panel.  On the flux-chamber sample that was taken on
Day 0  immediately after the land-application event, the
offsite odor panel measured odor intensities in the range
of 500 to  1,000 dilutions-to-threshold (D/T).  The offsite
odor panel reported even  higher odor levels from flux-
chamber samples  taken  on Day 1  approximately 24
hours after land application, with their results ranging
from 2500 to 6100 D/T.

Offsite odor analyses by a certified odor panel are quite
expensive, however, so the three flux-chamber samples
taken on  Day -1,  Day 0, and Day 1 were the  only
samples to be shipped to St. Croix Sensory Laboratories
and analyzed there.

The  onsite odor panel measured odor levels  exceeding
30 D/T from  the flux chamber exhaust sample on Day 0
(about an  odor of magnitude  less than the 500 D/T  odor
measurements by  the offsite odor  panel). The onsite
odor panel also measured odor levels in the range of 15-
30  D/T  from  ambient  air onsite  and  immediately
downwind. At locations immediately upwind  of the site
on Day 0, the  onsite odor panelists  measured odors in
the range of 2-7 D/T.

On the day after biosolids application (Day 1)  the onsite
odor panelists once again measured odors from the flux
chamber  exceeding  30  D/T,  while  the  offsite panel
reported even higher odor levels. These results indicate
that  there may  be some acclimation and decreased
sensitivities to odor  levels by onsite  odor panels, as
compared with the more  sensitive  offsite odor panels.
Ambient odor levels measured  by the onsite field panel
were in the  range of 15  D/T  onsite  and immediately
downwind on Day 1, while the onsite panelists could not
detect any odors above background upwind  of the site
on Day 1.

Ambient odor levels  as measured  by  the onsite panel
began subsiding  on Day 2 to a level of 15 D/T from the
flux chamber exhaust.  Odors  were barely detected at
locations downwind  of the site  on Day  2,  and were
undetected at  all locations upwind  of the site.  Odor
levels as measured by the onsite panelists had returned
to background concentrations at all sampling locations
by Day 4.

Ammonia  was not detected  in any  of the background
measurements taken on Day -1.  Hydrogen sulfide was
detected  in the Day -1  background  measurements at
concentrations of 0.002 to 0.005 ppmv.  The hydrogen
sulfide measurements are at least partially attributed to
the  exhaust gases  of  the  biosolids-applicator  and
sampling vehicles, which were  running without biosolids
on the day prior to biosolids application  (Day -1).

Both ammonia and hydrogen sulfide were detected at
fairly significant  concentrations  (15  ppmv  and  0.17
ppmv,  respectively)  from the  flux chamber  exhaust
samples soon after biosolids  application on Day 0.  The
concentrations of ammonia and hydrogen  sulfide at five
feet   above  ground   level    (where    most   field
                                                   35

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measurements were taken) were much lower, however.
Their concentrations had subsided further at all locations
by  Day  1  and  Day 2,  and concentrations  of both
ammonia   and   hydrogen  sulfide  had  returned  to
background levels by Day 4.

At least one  other  researcher at the  experimental site
detected ammonia  using  the  open-path  integrated,
optical remote sensing techniques (Harris,  2005), which
utilizes  absorption  infrared  spectroscopy.   In  that
research, an ammonia plume  was measured during and
immediately after biosolids application,  but it  dropped
rapidly to  non-detect levels  within three  hours after
application.      The   results   of   those   ammonia
measurements  were  in  the  same  ranges  as results
obtained in this study using Drager™  chemical sensory
tubes.

Conclusions

It was concluded that ammonia is a source  of odors only
during  and immediately  after biosolids application, to
receptors that are either onsite or in close proximity to
the application site.  Ammonia is generally not detected
at any appreciable distance from the site because  it has
a high  detection threshold and it disperses very rapidly
upon volatilization.

Hydrogen  sulfide   may  be  detected  in   background
samples due  to  its  presence  in vehicle  exhaust, and it
also has sources in  livestock operations (chickens,  cattle
and  hogs,  for example)  that are often found in rural
areas.  Generally, the increase in hydrogen sulfide over
background levels due to biosolids application  is very
slight, and hydrogen sulfide is not generally a significant
contributor  to odors  from most biosolids  application
activities.
It was concluded, based on the results of this study, that
most  odors associated  with  biosolids  land  application
are not from ammonia and hydrogen  sulfide, but more
likely are from reduced organic sulfur compounds such
as methyl  mercaptan and  dimethyl sulfide,  along  with
nitrogen-based  organic  compounds such as trimethyl
amine,  indole, and skatole.  Chemical measurements
made by others during the field study (McConnell, 2005)
tend to support these conclusions.

References

McConnell,   L.L.  Preliminary  summary  of  odorant
chemical  analysis  in  headspace  and flux chamber
samples and Polybrominated Diphenyl Ether analysis.
USDA  presentation  to  Biosolids  Information  Sharing
Group, December 2005.

Harris, D.B. Open-path FTIR measurement of gaseous
emissions from  biosolids application to an  agricultural
field.   USEPA   NRMRL   presentation  to   Biosolids
Information Sharing Group, December 2005.

Although the work described in this paper was funded by the
United States Environmental Protection Agency under contract
number  68-C-00-159  to  Battelle,  the  contents  do  not
necessarily reflect the views  of the Agency and no official
endorsement should be inferred.

Mention  of  trade  names or commercial products does not
constitute endorsement or recommendation by EPA for use.
                                                    36

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                                           Appendix D-4
 Community and  Occupational Risk from Bioaerosols during Land Application
                                            of Biosolids
Ian L. Pepper
The University of Arizona

During land application of Class B biosolids there is the
potential   for  aerosolization   of  human   pathogenic
microorganisms that could  adversely  impact human
health  and welfare. Recently the University of Arizona
conducted a major study evaluating the community and
occupational   risk  from   bioaerosols  during   land
application of biosolids. In addition,  the incidence  of
aerosolized   endotoxin  was   also   evaluated.   For
community risk, aerosol samples were collected for two
years  from  land  application sites located  at various
locations  throughout the  U.S.,  representing  different
climatic conditions and different application practices.
Land  application  practices  involved the use  of liquid
biosolids  spray  and   "cake"   biosolids   applicators
depending  on location. Bioaerosols were collected via
the use of six SKC BioSamplers®, impinging air at a rate
of 12.5 L/min for  a total of 20  minutes.  Samples were
collected from both downwind  of land  application  and
background sites  from distances ranging between  2  m
and  70 m downwind.  Microbial  concentrations  were
measured     within   these    aerosols,    including:
heterotrophic plate  count  bacteria (HPC),  coliphage,
Clostridium perfringens, total conforms, Escherichia coli,
endotoxin  (lipopolysaccharide), enteroviruses, norovirus,
and Hepatitis A virus (HAV).  In addition a  model  was
developed to predict viral transport. Overall, the levels  of
aerosolized  indicator bacteria  and phage were at  or
below detection limits. Three samples were  positive for
the  presence  of norovirus  viral RNA via  reverse
transcriptase  polymerase chain  reaction, although  their
viability was unable to be determined  based on current
available techniques.
                                               and
                                               the
Community risk assessments were conducted using an
empirically  derived  transport  model.  (Brooks  et  al.,
2005).  The assumptions  used in the  risk  assessment
were:
  •  105 phage/g biosolid
  •  0.1 virus/g biosolid (assume Coxsackie)
  •  virus:phage = 1:1000000
  •  using the seeded water model for  phage
     adjusting  for biosolid  phage  concentration,
     estimated phage  number at 100 ft is 7.16 x 10~1
     phage/m3
  •  therefore estimated  virus concentration from  land
     application of biosolids at 1 00 ft = 7.1 6 x 1 0"7/m3
  •  assume 0.1 coxsackie virus/g biosolid
  •  distance from site = 1 00 ft
  •  exposure = 1 hr
  •  dose = 5.94 E-07 virus
  •  using one-hit exponential model (P = 1-erN)
  Risk = 1 .50 E-08
  Similar Risk for 8-hr exposure = 1 .2
                                   E-07
For a more conservative approach
  •   assume 100 viruses/g biosolid
  •   estimated   virus   concentration
                                       from
                                               land
     application of biosolids at 100 ft = 7.16 x io~4/m3
  •  assume 100 virus/g biosolid

One-hour Risk = 1.5 E-05
8-hour Risk = 1.2E-04

Figure 1 shows  risks determined assuming different viral
concentrations  within  biosolids  at  different  distances
from the land application site. Exposure times are also
varied.
                                                   37

-------
                                   One Time Risk  as per
                               Distance  from.  Point Source
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_§  I.ME-OI ^
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                                 29 40  «  89  100 120  140  ISO
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Figure 1.  Risk assessments generated  at set distances from the land application  site assuming different viral inputs and
exposures times
For occupational risk, three experiments were conducted
to characterize the  concentration of microorganisms  in
biosolids,  the  plume  of  aerosols created during land
application of biosolids  and the occupational  risk  of
infection  due  to pathogens aerosolized  during land
application of biosolids in the United States. In all, more
than  300  air  samples  were   collected  immediately
downwind  of  biosolids  applications  throughout the
United States using  liquid impingers, and more than 100
air-samplers  were  collected downwind of  microbially
seeded,  land  applied  water,   which  served  as  a
conservative model  system  of aerosol generation. The
novel model system made it possible to calculate the
flux of microorganisms through a virtual plane defined by
air samplers  in  vertical and horizontal arrays,  located
immediately downwind of a passing  spray  applicator.
The  rate  of  aerosolization  during land application  of
biosolids near Tucson, Arizona, was  calculated to  be
less  than  33 plaque forming units (PFU)  of coliphage
and  10 colony forming units (CPU) of coliform bacteria
per meter traveled by the  spray applicator (Tanner et al.,
2005). Rates of aerosolization from the model  system
were shown to be much greater. Exposure  duration was
shown to be brief and limited to the time when biosolids
were actually  in   the  air.  To  assess  the   risk   to
occupational health  from bioaerosols  generated  during
land   application   of   biosolids,   coliform   bacteria,
coliphages,  and  heterotrophic  plate  count   (HPC)
bacteria were  enumerated from air and  biosolids  at 10
land application sites throughout the nation. The method
of land application  strongly influenced aerosolization,
while relative  humidity,  temperature  and  wind  speed
                                 showed limited  correlation to concentrations  of fecal
                                 indicator microorganisms  in air. Occupational risks  of
                                 infection and illness from aerosolized  Salmonella and
                                 enteroviruses  were  calculated for a  variety  of  land
                                 application scenarios.

                                 The assumptions used in the risk assessment were:

                                   •   Worker on land application site 8 hours/work day
                                   •   251  work days/year
                                   •   Tractor cab  air filter 50%  efficient in removing
                                       aerosolized microorganisms
                                   •   Operator downwind of biosolids 50% of the time
                                   •   No inactivation of aerosolized microbe
                                   •   Concentration of pathogens  and indicators  in air
                                       will be a reflection of pathogens and indicators  in
                                       biosolids
                                   •   Therefore concentrations of conforms in air can be
                                       used to estimate concentration of Salmonella  in
                                       air, and
                                   •   Concentration  of phage in  air can be  used  to
                                       estimate human virus concentrations in  air
                                   •   Breathing rate = 10  m3/8 hours (light activity)
                                   •   100 to 1000- Salmonella and  0.1  to  10 human
                                       virus/g of biosolids
                                   •   10%  of  Salmonella  that  are   inhaled  are
                                       subsequently  ingested  (no   dose   response
                                       available for inhalation of Salmonella)
                                   •   Human  virus  is   Coxsackievirus  A21  (dose
                                       response for inhalation available)
                                                    38

-------
Where:
The probability of infection from ingestion of pathogenic
bacteria   was   calculated   using   the  Beta-Poisson
Distribution model.
       Here Pday = 1 - [1 + (d / N50) (21/a -1)]'°

       Pday is the probability of infection per workday

       d  is the number of pathogens ingested per day
       (10% of pathogens inhaled)

       N50   is the dose at which  half of subjects  are
       infected with a particular pathogen

       a     is  a   parameter  which describes   the
       distribution of infection
                                                        Summary
The  probability of infection  from the  inhalation  of virus
was   calculated   using  the   Single-hit   Exponential
Distribution Model
                       -rd
        Here, Pday = 1-e
Where,
        r  is a  parameter defining  the  probability  of  a
        single organism initiating infection

        d  is the number of pathogens inhaled per day

The annual risk of infection was calculated from the  daily
risk  of infection,  assuming  251   days per  year  of
occupational exposure, using the following formula:

        Here: Pyear=1-(1-Pday)251

Calculated occupational risks are shown in Tables 1 and
2 assuming different viral  and bacterial concentrations
within biosolids.
Overall risk of infection from bioaerosols resulting
from land application is low.
Duration of exposure during land application  is
very discrete.
Occupational risk  is greater than  community risk
due to enhanced exposure, but still low.
Community risk is insignificant.
The  greatest risk to occupational  workers occurs
during loading of biosolids.
For community and occupational risk, there is less
risk due to  bacteria than virus.
Application   method   influences   aerosolization
rates.
Environmental    factors    do   not    influence
aerosolization rates
                                                        References

                                                        Brooks, J.P., Tanner, B.D., Gerba, C.P., Haas, D.N., and
                                                        Pepper,  I.L.  (2005). Estimation  of  bioaerosol  risk of
                                                        infection to residents adjacent to a land applied biosolids
                                                        site  using  an  empirically  derived  transport  model.
                                                        Journal of Applied Microbiology. 98: 397-405.

                                                        Tanner, B.D., Brooks, J.P., Haas, C.N., Gerba, C.P., and
                                                        Pepper,  I.L. (2005).  Bioaerosol emission rate and plume
                                                        characteristics during land application of liquid Class B
                                                        biosolids. Environmental Science and Technology. 39:
                                                        1584-1590.

                                                        The work described in this paper was not funded by the United
                                                        States  Environmental Protection Agency and, therefore,  the
                                                        contents do not necessarily reflect the views of the Agency and
                                                        no official endorsement should be inferred.
Table 1: Occupational risk: annual risk of infection due to inhalation of coxsackievirus A-21
                                                         Enterovirus/g biosolids
                                         10
                                                                  1
                            0.1
      Loading

      Land Application
                                      1.9 x 10"
 2.1 x 10
                                                                      -2
                                      7.5x 10
                                             -2
 7.8 x 10
                                                                      -3
2.1 x 10
                                                                                               -3
7.8x 10
                                                                                               -4
Table 2: Occupational risk: annual risk of infection from non-typhi Salmonella assuming 10% of inhaled Salmonella are ingested
                                                        Salmonella/ g biosolids
                                                     1000
                                                                                     100
      Loading

      Spreader
                                                  1.3x 10
                                                          -3
                   1.3 x 10
                                                                                        -4
                                                  9.8 x 10
                                                          -5
                   9.8x 10
                                                                                        ,-6
                                                      39

-------
                                           Appendix D-5
  Lessons  Learned Regarding the Use of an Impinger for Collecting Airborne
                  Bacteria during  a Biosolids Application Field  Study
Edwin F. Earth, PhD, P.E., C.I.H.
USEPA-NRMRL

Abstract

The  National Risk Management Research  Laboratory
(NRMRL)  of the USEPA performed  laboratory wind
tunnel studies as well  as large field studies to evaluate
specific  bioaerosol   components  (bacteria,  fungi,
endotoxin,  p-d  glucan)  that  may be  associated  with
various  practices for  managing semi-solids  such as
biosolids or  sediments  (contaminated  by  combined
sewer  overflows).  The wind tunnel  studies  indicated
limitations involving the impingement collection method.
Modifications to common practices  for  impingement
collection  procedures,  gained  from  the  wind  tunnel
studies, were implemented   into the  design of  the
bioaerosol sampling collection plan for a large field study
involving the land application  of biosolids to increase the
likelihood that stress-sensitive bacteria (Gram-negative)
could be  collected  and detected.    These  modified
procedures  included:    covering the impinger  with
aluminum foil to  block ultra-violet radiation transmission
during  collection; periodic washing  of the  inlet neck
during collection to prevent drying of the biological mass
that may collect on the inlet neck during collection; and a
final  rinse step of the inlet neck and  jet  nozzles after
collection.

Introduction

Various sampling  methods   are  available  to  collect
airborne bacteria.  Interpreting data from  any of these
methods for use in  a human exposure assessment is
difficult since the flow  rate and mechanisms for particle
collection  of a  biological sampling   device  may  be
different from the physiology and particle  deposition of
the human pulmonary system.  Furthermore,  some of
these sampling devices are relatively large, making it
impractical to  collect  air samples within  the  personal
breathing zone of an individual.

The  survival of bacteria in air depends upon physical
and chemical factors of the particle such as the particle
size and membrane components. Environmental factors
also  play a  role  in survival in air such  as relative
humidity, evaporation rate, temperature, transport time,
and ultra-violet radiation. In addition,  an "open air factor"
also reduces viability (Cox et al.,  1973). In contradiction,
bacteria  have been  known to  travel long distances,
across oceans,  if ultra-violet radiation is blocked by
particulate matter (Griffin et al., 2002).

Traditional   bioaerosol  sampling   methods   include
filtration,  impingement, and  impaction.  The advantages
of the   impingement  method   for  collecting  viable
microorganisms  may  include the possibility of  longer
sampling times (depending  upon the collection fluid) to
more  accurately  assess  the  personal  exposure to
bioaerosols when a person spends a significant period
of time in a specific environment (Lin et al., 1999), less
susceptibility to collection medium overload, and an inlet
parallel to the  air stream.  In  addition, the collection of
the bacteria into a fluid  allows the flexibility to split the
collection fluid into several aliquots for various types of
analysis   other  than   bacteria   including  endotoxin
(Duchaine et al., 2001), viral  (Agranovski et al.,  2004),
and  molecular  methods   (Angenent  et  al.,  2005).
Disadvantages of this collection method include delicate
sampler device handling procedures, and more difficulty
in setting-up and utilizing  in the field  relative to other
bioaerosol sampling equipment devices.

Bacterial  enumeration   techniques  included  filtration/
plating of the  impinger fluid or a spread  plate method
used  in other bioaerosol impinger studies  (Lin  et al.,
1999).  Even with high collection efficiencies of airborne
                                                   40

-------
bacteria,  assays  for  individual  microorganisms  may
result in low  recovery.  Microbial ecology studies have
shown  the  culturability  of  microorganisms  is   low
compared  to actual  counts  in  many  environmental
settings  (Fabbian et  al.,  2004).    For example,  the
recovery efficiency for S. aureus seeded in a biosolids
sample  was  8.7% (Rusin  et al., 2003).   In  another
bioaerosol study,  less  than  10%  of the aerosolized
bacteria  were capable of forming visible  colonies with
culture techniques (Heidelberg et al., 1997).

Materials and Methods

Laboratory Wind Tunnel Studies
The evaluation of the impingement collection  procedure
consisted  of  several   laboratory  trials  that  were
completed prior  to a  large  field trial involving biosolids
application.  For the laboratory trials, a 0.3 m x 0.3 m x
3.6 m (1.0 ft  x 1.0 ft x 12.0 ft) wind tunnel was used to
evaluate various  bioaerosol sampling methods.  Three
BioSampler®  impingers (SKC Inc., Eighty Four, PA) were
located  0.9 m (3.0 ft) downwind of the source area in the
wind tunnel.  The source area contained various solution
concentrations of a stress-sensitive organism, E. coli, in
a 1000  ml beaker.  The bacteria were  aerosolized from
the beaker by bubble aeration and mechanical mixing.
The  velocity  of  the  filtered  air  in  the  wind  tunnel
approximated 0.75 m (2.5 ft)/ sec.

Three sets of trials (initial, continuation, threshold) using
E. coli  (ATCC 25922), a  fecal coliform  bacterium of
interest  for biologically contaminated  sediment, were
performed to develop an  efficient collection  procedure
for the impingers for collecting stress-sensitive  bacteria
in the field.

Two types of impingers were considered for the studies.
The  design  and  specifications  of the   BioSampler®
impinger  include an  inlet  neck,  collection body, and
collection vial. This more recent design is similar to the
All-Glass  Impinger (AGI-30),  which  has been available
for several years. A major difference between these two
impinger  types   is  that   particles   impinge  into  the
collection fluid at 90 degrees with the AGI-30, while the
combination  of impaction  and centrifugal  motion  may
result in  less re-aerosolization  of particles from  the
BioSampler®  (Lin et al., 2000).  Other studies with  the
BioSampler®   have  shown   nearly  100%  collection
efficiency for a  wide  particle (non-biological)  range,
decreasing to approximately  90% for  0.5  urn particles
and to 70% for 0.3 urn  particles (SKC, 2004; Lin et al.,
2000).  The collection body has a critical orifice in each
of the three  jet nozzles that result in a  flow rate of
approximately 12.5 Lpm through the nozzle jets (at sonic
flow) at  a pressure drop of 0.5 atmospheres  (15 in Hg)
or more across the sampler (SKC, 2004). From this
literature,  a  decision  was  made  to  only  use  the
BioSampler®  impinger design in  both the wind tunnel
and field studies.

The curved inlet neck of a similar impinger design, the
AGI-30, collects particles larger than  8 urn that can be
recovered with a  wash rinse  (ACGIH,  1995).   Non-
isokinetic  collection   conditions  involving  the AGI-30
sampler theoretically  result in  decreased   collection
efficiency  of particles larger than  1.0  urn (Grinshpun et
al., 1994).  A final rinse procedure, which consisted of
five separate  1.0  ml  rinses of a phosphate buffered
solution (PBS)  after  collection was used to wash down
any organisms  that may have aggregated and been in
contact with the inlet  neck or nozzles during collection.

Field Study
To prevent dry-out of organisms that might be collected
in  the  inlet neck,  as  well as  replace impinger fluid  that
may be lost to evaporation during  outdoor  collection
activities,  periodic rinses of distilled/de-ionized  water
were introduced into the sampler inlet neck during field
collection activities to increase the likelihood that stress-
sensitive organisms could be collected.  In addition, the
impingers were covered with aluminum foil to reduce the
chance of any collected bacteria being exposed to ultra-
violet  light radiation  during  the  collection  and sample
handling period, which  might have a detrimental effect
on bacteria.

For the field study, a sampling station layout for the  100
m  diameter circular study area (Figure 1) was designed
for flexibility in  sampler orientation.   Multiple sampling
zones containing multiple sampling stations were spread
apart to increase the size of the  collection area of the
sampling field,  as opposed  to  clustering  the samplers
within a narrow region of the field.  The sampling station
layout  included  three upwind  stations  (center  station
located 16 m  upwind  from the top edge of the application
area), three  stations  in  the first downwind zone  (center
station  located  16 m  downwind  of  the bottom of the
application area),  and three stations  in  the second
downwind zone (center station located  50 m from the
first downwind zone center station).   The stations in the
upwind zone  and first downwind zone were 75 m apart
within  each  zone.    The stations  within  the second
downwind zone were  approximately  90  m apart. This
station  layout would  allow physical movement of each
station (following an  arc pattern) because the samplers
were secured upon pull carts so that they could be re-
positioned with varying wind patterns.

The operational schedule involved periodic operation of
the samplers for the  period  of time when the spreader
passed through the application  area, with  an  allowance
time for reasonable particulate transport (two minutes) to
                                                     41

-------
            r
5C
UWA



^feHK.U' -Mid
,72T
*

/

/
J^^-
^X'
^r /
/
            60m
        DWA
                                                                                 Upwind
                                                                                  JUWO

                                                                               Downwind
                                                                                   (DW)
            50m
        OWB
           Key

      A   Sampling station containing two Biosamplers

      -A-  Mobile station containing twq Biosamplers

           r = v  ^ 0-25m  (distance varies per ambient conditions)

           A. B and C indicate sampling zone identification
                                                                         WOT TO SCALE
                           Figure 1.  Plan View of Sampling Station Layout
downwind stations, as opposed to continuous operation
of the samplers.  During the time the samplers were off,
distilled water was introduced  into the inlet neck.  This
intermittent  operation  and  rinsing  were  intended  to
reduce evaporative loss of impinger fluid associated with
outdoor sampling activities.
Results and Discussion

Laboratory Wind Tunnel Studies
Initial Trial Set (trials 1-4)
The  initial two trials indicated that the initial sampling
procedure using white mineral oil as a collection fluid in
the BioSamplers®, for a long time period (120 minutes),
                                                  42

-------
was not  effective  for  capturing  the  stress-sensitive
bacteria (£. coli) introduced into the tunnel, as no growth
was detected.  Various  reasons for not being able to
culture stress  sensitive  organisms from  the impinger
were considered. One reason may be that "clumping" of
the bacterial cells may be too large for the bend in the
impinger, collecting  in the inlet bend, possibly by charge
attraction, and remain sensitive to air drying (if collected
in  the sampler inlet). Another reason could be that the
cell viability  is  reduced due  to the  agitation in  the
impinger being too  severe. Another reason  is the  cell
viability may be  reduced during subsequent filtration,
prior to plating. A third trial employed a final inlet and jet
wash  after collection, and  (PBS) instead of  oil as the
collection medium in an  attempt to recover any bacteria.
However, to limit air drying, the trial was only  performed
for 10  minutes, raising  the  detection limit for this trial,
which did not result in  detection.   A fourth  trial using
Bacillus spores indicated that the impingers could collect
less stress-sensitive  organisms  over a  thirty  minute
collection period.

Continuation Trials (trials 5-7)
The continuation  trials  ultimately  indicated  that  the
impinger operational practice of using PBS, operated for
30  minutes,  employing  a  final  inlet/nozzle  wash was
satisfactory for collecting and culturing stress-sensitive
organisms, depending upon the analytical method used
for culturing the bacteria.

Threshold Trials (trials 8-11)
The last set of trials (trials 8, 9,  10 and  11) were used to
determine if  a concentration  threshold (initial culture)
was necessary to detect organisms in this experimental
set-up. These trials indicated that the culturable counts
from the  impingers were  dependent  upon  the initial
solution concentration counts, once above a threshold
(greater than 105 CFU/ml) for this particular wind tunnel
design.

Field Study
The impingers were operated during both a control  trial
and biosolids application trial following the sampling  and
analytical  plan.  The  re-positioning of the  sampling
stations due to changing wind patterns was  difficult to
implement because it was  difficult to communicate  and
coordinate  sampling  station  movement among   all
sampling personnel.  The  intermittent  operation of the
samplers and associated rinsing steps was able to be
implemented  during the  biosolids application  period,
using  pre-sterilized packets   of  water.    Detectable
bacteria concentrations were observed  for the impingers
at  each of the sampling stations.   Complete statistical
review of this data will be performed in the future.
Conclusions

Impingers may offer some advantages for collecting and
analyzing a large variety  of bioaerosol  components  as
compared to other bioaerosol collection methods which
typically   collect   only  a  single  biological  agent.
Operational  strategies  are  available  to   potentially
increase the  likelihood that stress-sensitive bacteria,
such as  Gram-negative bacteria, can be collected and
cultured  from air samples.    A  collection  procedure
involving  periodic operation and  rinsing  of  the foil
covered  impingers,  followed   by  a  final  rinse  step,
resulted in the collection of viable bacteria during a field
application study.

References

Agranovski, I., Safatov, A., Borodulin, A., Pyankov, O.,
Petrishchenko, V., Sergeev, A., Agafonov, A., Ignatiev,
G., Sergeev, A., and Agranovski, V. (2004). Inactivation
of Viruses in Bubbling Processes  Utilized for Personal
Bioaerosol  Monitoring.  Applied  and  Environmental
Microbiology. 70: 6963-6967.

ACGIH. (1995). Air  Sampling  Instruments. 8th Edition.
Cincinnati, OH

Angenent,  L, Kelley, S., St. Amand, A., Pace, N., and
Hernandez,  M.   (2005).   Molecular   Identification  of
Potential  Pathogens  in Water  and  Air of  a  Hospital
Therapy  Pool. Proceedings of the  National Academy of
Sciences. 102: 4860-4865.

Cox, C., Hood, M.,  and Baxter, J. (1973). Method for
Comparing  Concentrations of the  Open-Air  Factor.
Applied Microbiology. 26: 640-642.

Duchaine, C., Thorne,  P.,  Meriaux, A., Grimard, Y., and
Cormier, Y. (2001).  Comparison of Endotoxin Exposure
Assessment by Bioaerosol and Filter-Sampling Methods.
Applied and Environmental Microbiology. 67: 2775-2780.

Fabian, M., Miller, S., Reponen, T., and  Hernandez,  M.
(2005). Ambient Bioaerosol Indices for Indoor  Air Quality
Assessments of Flood Reclamation. Journal  of Aerosol
Science. 36: 763-783.

Griffin, D.,  Kellogg,  C., Garrison, V.,  and  Shinn,  E.
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This paper has been  reviewed in accordance with the United
States  Environmental   Protection  Agency's   peer  and
administrative review  policies and approved for presentation
and publication.

Mention  of trade  name or commercial products does not
constitute endorsement or recommendation by EPA for use.
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