CRCWQT A Series of Exposure Experiments

Transcription

The Cooperative Research
Centre for Water Quality and
Treatment is an unincorporated
joint venture between:
CRC for Water Quality and
Treatment
Private Mail Bag 3
Salisbury SOUTH AUSTRALIA 5108
Tel: (08) 8259 0351
Fax: (08) 8259 0228
E-mail: crc@sawater.com.au
Web: www.waterquality.crc.org.au
The CRC for Water Quality and Treatment is established
and supported under the Federal Government’s Cooperative
Research Centres Program.
Research Report 45
The Cooperative Research Centre (CRC) for Water Quality
and Treatment is Australia’s national drinking water research
centre. An unincorporated joint venture between 29 different
organisations from the Australian water industry, major
universities, CSIRO, and local and state governments, the CRC
combines expertise in water quality and public health.
• ACTEW Corporation
• Australian Water Quality Centre
• Australian Water Services Pty Ltd
• Brisbane City Council
• Centre for Appropriate
Technology Inc
• City West Water Limited
• CSIRO
• Curtin University of Technology
• Department of Human Services
Victoria
• Griffith University
• Melbourne Water Corporation
• Monash University
• Orica Australia Pty Ltd
• Power and Water Corporation
• Queensland Health Pathology &
Scientific Services
• RMIT University
• South Australian
Water Corporation
• South East Water Ltd
• Sydney Catchment Authority
• Sydney Water Corporation
• The University of Adelaide
• The University of
New South Wales
• The University of Queensland
• United Water International Pty Ltd
• University of South Australia
• University of Technology, Sydney
• Water Corporation
• Water Services Association
of Australia
• Yarra Valley Water Ltd
A Series of
Exposure
Experiments –
Recycled Water
and Alternative
Water Sources:
Part A - Aerosolsizing and Endotoxin
Experiments
Research Report
45
A Series of Exposure Experiments – Recycled
Water and Alternative Water Sources: Part A Aerosol-sizing and Endotoxin Experiments
Joanne O’Toole, Karin Leder, Martha Sinclair
Department of Epidemiology and Preventive Medicine,
Monash University
Research Report No 45
AEROSOL-SIZING AND ENDOTOXIN EXPERIMENTS
© CRC for Water Quality and Treatment 2008
DISCLAIMER
The Cooperative Research Centre for Water Quality and Treatment and individual contributors are not
responsible for the outcomes of any actions taken on the basis of information in this research report,
nor for any errors and omissions.
The Cooperative Research Centre for Water Quality and Treatment and individual contributors
disclaim all and any liability to any person in respect of anything, and the consequences of anything,
done or omitted to be done by a person in reliance upon the whole or any part of this research report.
The research report does not purport to be a comprehensive statement and analysis of its subject
matter, and if further expert advice is required the services of a competent professional should be
sought.
Cooperative Research Centre for Water Quality and Treatment
Private Mail Bag 3
Salisbury SA 5108
AUSTRALIA
Telephone:
Fax:
E-mail:
Web site:
+61 8 8259 0351
+61 8 8259 0228
crc@sawater.com.au
www.waterquality.crc.org.au
A Series of Exposure Experiments – Recycled Water and Alternative Water Sources. Part A - Aerosolsizing and Endotoxin Experiments. Research Report 45
ISBN 18766 16717
Published by the CRC for Water Quality and Treatment 2008
2
CRC FOR WATER QUALITY AND TREATMENT – RESEARCH REPORT 45
FOREWORD
A Series of Exposure Experiments – Recycled Water and Alternative Water Sources. Recycled Water
and Alternative Water Sources: Part A - Aerosol-sizing and Endotoxin Experiments.
Research Officers:
Ms Joanne O’Toole
Dr Martha Sinclair
Ms Naomi Cooke
Project Leader:
Dr Karin Leder
Research Nodes:
Department of Epidemiology and Preventive Medicine, Monash University
CSIRO
CRC for Water Quality and Treatment Project No. 2.0.1.1.1.0 – A Series of Exposure Experiments:
Recycled Water and Alternative Water Sources
3
EXECUTIVE SUMMARY
This is the first of two reports which describe the CRC for Water Quality and Treatment Project No.
2.0.1.1.1.0 – A series of exposure experiments: recycled water and alternative water sources. Two
experimental components of the project are described in this report and another two project
components are described in a second project report (Part B: Microbial transfer efficiency during
machine clothes washing and microbial survival turf-grass experiments. CRC Research Report 46).
The inhalation route of exposure to micro-organisms in recycled water is one about which there is a
paucity of information and one which is not addressed in current national guidelines. The Australian
Guidelines for Water Recycling (Phase 1) released in December 2006, provide guidance on how
recycling for uses such as agriculture, fire control, municipal, residential and commercial property and
industry can be safely employed and uses a risk management framework. However, as it is impractical
to set human health based targets for all micro-organisms that might be present in a source of
recycled water the guidelines focus on human health based targets for pathogens (enteric) that are
ingested. As transmission via inhalation, not ingestion is the more important route for some microorganisms (e.g. Legionella, Mycobacteria), biological components (e.g. endotoxin) and chemicals it is
important that information gaps about exposure to respirable aerosols during specific water-using
activities are addressed. This information is important to provide input to future iterations of national
recycled guidelines and for application to new recycled water schemes.
The two experimental components described in this report specifically assess exposure associated
with the use of water efficient devices (showers and high pressure hose devices) and the potential for
endotoxin exposure in recycled water.
Inhalation exposure during showering and car washing
The first objective of this experimental component was to characterise the size distribution of aerosols
(≤10 µm diameter), and droplets (>10 µm diameter) produced during showering and car washing by
Australian industry-approved, water-efficient devices and conventional (lower efficiency) devices using
drinking water. The second objective was to determine whether water-efficient devices produce
significantly higher inhalation exposure to respirable aerosols than conventional devices under typical
Australian usage conditions. Two types of showerhead were employed in experimentation, one
operated at 7.0 L/min (designated a ‘water efficient’ device equivalent to a WELS 3 ‘star’ rating) and
the other operated at 13 L/min (designated a ‘low efficiency’ device, equivalent to a WELS 1 ‘star’
rating). For the car washing scenario, a Gurney Tradesman high pressure spray unit was used as the
water efficient device and a standard garden hose in combination with a Pope hand spray nozzle was
used as the conventional (lower efficiency) device.
Overall, findings of the experiments allow a greater understanding of aerosol exposure to water-users
during typical domestic water-using activities relevant to an Australian context. In particular,
experimental results for water droplet and aerosol production show that complex particle
generation/removal processes occur within a shower stall when the shower is on. The operation of a
shower results in the removal of aerosols from the ambient air (i.e. the falling water scrubs ambient
aerosol from the shower stall) when high quality drinking water is used. At least 90% of total generated
aerosol mass for both showerhead designs at all tested flow rates was associated with particles with a
diameter greater than 6µm. Such particles are too large to be inhaled into the lungs, but may
contribute to ingestion exposure to waterborne contaminants.
Experimental results do not allow definitive conclusions to be drawn as to whether water-efficient
showerheads produce significantly higher exposure to respirable aerosols (<6 µm diameter) than
conventional (lower efficiency) showerheads under typical Australian flow conditions (WELS 3 star
versus 1 star ratings). However, results allow a tentative hypothesis to be formulated that conventional
showerheads (13 L/min) generate more particles >6 µm diameter (the size range where the ingestion
route of enteric pathogens and most chemicals is important) than do water-efficient showerheads
(7 L/min). The implications of this, if confirmed through additional experiments, are that the water
efficient showerhead (7 L/min) may result in reduced exposure to enteric pathogens and chemicals via
ingestion compared with the conventional showerhead (13 L/min).
4
Potentially significant higher exposure to respirable aerosols during car washing activities are
indicated for the high pressure device compared with the garden hose with trigger nozzle. Results for
the car washing module showed that the high pressure hose device almost exclusively gives rise to
aerosols in the respirable range (>96%) with lower percentages (59-95%) recorded for the garden
hose and trigger nozzle combination. These results indicate that the use of high pressure sprays and
garden hose with trigger nozzles are worthy of future investigation with respect to the total emission
source strengths of respirable aerosols. It is likely that there are significant differences (not seen in
these experiments due to the small number of replicates and variability in particle concentrations in
size bins) in inhalation exposure to respirable aerosols between the combination garden hose with
trigger nozzle and the high pressure device. Additional experimentation is required to verify and
quantify these differences.
This study provides an important precedent for the conduct of future studies and the refinement of
methodological aspects of experimental set-up and aerosol measuring instrumentation for aerosol
characterisation and measurement during typical domestic water-using activities. This is the first study
of water exposure to measure particles less than 1 µm and to use techniques which dry the aerosol
prior to measurement.
Australian endotoxin water survey
This experimental component comprised both methodological investigations and a survey of endotoxin
concentrations in a variety of Australian water types. Preliminary methodological investigations
included the study of the impact of the addition of sodium thiosulphate to sample bottles and the
storage of water samples at -80°C for extended periods (up to 22 weeks) in pyrogen-free sample
bottles prior to endotoxin analysis.
The primary objective of the preliminary survey of endotoxin concentrations was to determine the
concentration of endotoxin in a variety of Australian water types, including recycled water. A total of 48
sampling sites were surveyed, the majority on at least 2 separate occasions (total number of samples
analysed was 94). Recycled water containing less than 10 E. coli per 100mL, where the treatment train
did not include membrane filtration, gave rise to a mean measured endotoxin concentration of 2,030
Endotoxin Units (EU) per mL (N=7). For recycled water samples containing less than 10 E. coli per
100mL where membrane filtration was part of the treatment train (N=3), the mean endotoxin
concentration was 41 EU/mL. Measured endotoxin concentrations in drinking water varied from less
than 4 to 119 EU/mL.
Results obtained for various water types analysed as part of this Australian survey are in accord with
the concentrations reported for the same water types in the literature. Results indicate that those water
treatment processes that give rise to biomass removal, rather than oxidative disinfection processes,
lead to the greatest reduction in endotoxin levels. Study results showed that wastewater processes
where membrane filtration is employed produce finished water with endotoxin concentrations at least
as low as in existing drinking water supplies. This result indicates that membrane-treated waters do
not lead to an increased likelihood of adverse respiratory effects from endotoxin exposure compared
with the use of existing drinking water supplies. Observed higher levels of endotoxin in recycled
waters where membrane filtration was not employed indicate a potential increase in human health risk
from inhalation of endotoxin if these waters are used for activities which generate aerosols.
This study is an initial investigation and further monitoring of endotoxin levels in water throughout the
recycled water production process is required so that the fate of endotoxins in the treatment train is
better understood and judgments can be made as to whether or not LAL (Limulus Amoebocyte
Lysate) test results can be meaningfully used as a surrogate for recycled water treatment efficacy.
Validation of water sampling and preservation protocols and standardisation of endotoxin assay
methods are required in advance of such monitoring. For health effect determinations, further research
is required to quantify the aerosolised endotoxin concentrations arising from a variety of water-using
activities.
5
TABLE OF CONTENTS
Foreword ................................................................................................................................................ 3
Executive Summary .............................................................................................................................. 4
Abbreviations....................................................................................................................................... 10
Definitions ............................................................................................................................................ 11
1 Introduction....................................................................................................................................... 12
2 Aerosol sizing experiments............................................................................................................. 13
2.1 Background.................................................................................................................................. 14
2.1.1 Terminology........................................................................................................................... 14
2.1.2 Biological and non-biological particles size range ................................................................ 14
2.1.3 Particle (aerosol and water droplet) measuring instrumentation .......................................... 17
2.1.3.1 Aerosol measurement errors .......................................................................................... 18
2.1.4 Water-efficient devices and their rating................................................................................. 18
2.1.5 Research on inhalation aerosol exposure via water ............................................................. 18
2.2 Study objectives........................................................................................................................... 22
2.3 Materials and methods................................................................................................................. 22
2.3.1 Aerosol measurement instrumentation ................................................................................. 22
2.3.2 Water-efficient and conventional devices used in experimentation ...................................... 23
2.3.2.1 Showerhead ................................................................................................................... 24
2.3.2.2 High pressure device...................................................................................................... 25
2.3.3 Experimental conditions ........................................................................................................ 26
2.3.3.1 Clean room ..................................................................................................................... 26
2.3.3.2 Shower experiments....................................................................................................... 26
2.3.3.3 Car washing experiments ............................................................................................... 29
2.3.3.4 Other instrumentation and materials .............................................................................. 29
2.3.4 Experimental configurations and replication of runs ............................................................. 30
2.3.5 Data analysis......................................................................................................................... 31
2.3.5.1 Combining data from the APS, SMPS and PDPA.......................................................... 31
2.3.5.2 Data manipulation........................................................................................................... 31
2.4 Results ......................................................................................................................................... 31
2.4.1 Characteristics of aerosol and droplet distributions by water efficient and low efficiency
devices ........................................................................................................................................... 31
2.4.1.1 Showering module.......................................................................................................... 31
2.4.1.2 Car washing module....................................................................................................... 32
2.4.2 Comparison of water efficient and conventional devices...................................................... 33
2.4.2.1 Showering module.......................................................................................................... 33
2.4.2.2 Car washing module....................................................................................................... 37
2.5 Discussion.................................................................................................................................... 40
2.6 Conclusions ................................................................................................................................. 43
2.7 Future research directions ........................................................................................................... 44
3 Endotoxin .......................................................................................................................................... 46
3.1 Background.................................................................................................................................. 47
3.1.1 Definition and properties ....................................................................................................... 47
3.1.2 Health effects ........................................................................................................................ 48
3.1.2.1 Inhalation ........................................................................................................................ 49
3.1.2.2 Ingestion ......................................................................................................................... 50
6
3.1.3 Water analysis results ........................................................................................................... 51
3.1.4 Data gaps .............................................................................................................................. 55
3.2 Study objectives........................................................................................................................... 55
3.3 Methods ....................................................................................................................................... 55
3.3.1 Samples analysed................................................................................................................. 56
3.3.1.1 Preliminary methodological studies................................................................................ 56
3.3.1.2 Endotoxin survey ............................................................................................................ 56
3.3.2 Sample collection .................................................................................................................. 56
3.3.3 Sample storage and transportation....................................................................................... 57
3.3.3.1 Preliminary methodological study................................................................................... 57
3.3.3.2 Endotoxin survey ............................................................................................................ 57
3.3.4 Sample preparation............................................................................................................... 57
3.3.5 Endotoxin analyses ............................................................................................................... 57
3.3.6 Water quality parameters ...................................................................................................... 58
3.3.7 Analysis ................................................................................................................................. 58
3.4 Results ......................................................................................................................................... 59
3.4.1 Preliminary methodological study ......................................................................................... 59
3.4.1.1 Sodium thiosulphate addition to sample containers ....................................................... 59
3.4.1.2 Sample preservation....................................................................................................... 59
3.4.2 Survey results ....................................................................................................................... 60
3.4.2.1 Sample types .................................................................................................................. 60
3.5 Discussion.................................................................................................................................... 64
3.5.1 Methodological considerations.............................................................................................. 64
3.5.1.1 Sodium thiosulphate addition to sample containers ....................................................... 64
3.5.1.2 Sample storage .............................................................................................................. 64
3.5.1.3 Analytical method ........................................................................................................... 65
3.5.2 Relationship between endotoxin concentration and water type............................................ 66
3.5.3 Relationship between other water quality indicators and endotoxin concentration .............. 68
3.5.4 Health risk assessment: aerosolised endotoxin.................................................................... 68
3.6 Conclusions ................................................................................................................................. 68
3.7 Future research directions ........................................................................................................... 69
4 Acknowledgements.......................................................................................................................... 70
References ........................................................................................................................................... 71
Appendices .......................................................................................................................................... 75
Appendix 1: Major sources of biases that occur in droplet and aerosol measurement ..................... 75
Appendix 2: Summary results of research studies ............................................................................ 76
Appendix 3: Details of individual aerosol measuring instruments and limitations and uncertainties
associated with each.......................................................................................................................... 77
Appendix 4: Victorian classes of reclaimed (recycled) water ............................................................ 79
7
LIST OF FIGURES
Figure 2-1 Particle size diameters and the respirable µm range .......................................................... 15
Figure 2-2 Means of spread of microbial agents via aerosols/droplets................................................. 15
Figure 2-3 Particle deposition (unit density spheres) in the respiratory system.................................... 16
Figure 2-4 Interbath Watersaver series showerhead ............................................................................ 24
Figure 2-5 Bastow (Fjord) showerhead ................................................................................................. 24
Figure 2-6 Nylex hose with Pope spray nozzle and Gurney Tradesman high pressure spray unit ...... 25
Figure 2-7 ‘Clean room’ for aerosol experiments .................................................................................. 26
Figure 2-8 Photograph showing location of instrumentation relative to shower stall ............................ 27
Figure 2-9 Schematic of positioning of PDPA equipment in shower experiments ................................ 28
Figure 2-10 Aerosol sampling set-up .................................................................................................... 28
Figure 2-11 Garden Hose experiment setup ......................................................................................... 29
Figure 2-12 High pressure spray experiment setup .............................................................................. 29
Figure 2-13 Aerosol size distributions during showering determined by the SMPS and APS and
droplet size distributions determined by the PDPA for experiments carried out on 31 March 2006.
........................................................................................................................................................ 32
Figure 2-14 An example of aerosol size distributions during car-washing module determined by the
SMPS and APS for hose experiments carried out on 4 August 2006 in the tent enclosure. ......... 33
Figure 2-15 A simple schematic of the processes of aerosol and droplet removal and production
occurring in the shower .................................................................................................................. 41
Figure 3-1 Diagram of Gram negative bacterial cell membrane showing lipopolysaccharide
component...................................................................................................................................... 47
Figure 3-2 Diagram of lipopolysaccharide of Gram negative bacterium (adapted from Jakubowski and
Ericksen 1980) ............................................................................................................................... 48
Figure 3-3 Graph showing the effect of storage up to 22 weeks of 10 water samples in pyrogen-free
containers at -80°C on mean detectable endotoxin in the LAL assay expressed as a percentage
of initial detectable endotoxin concentration in samples stored at 4°C and analysed within 24
hours (0 weeks), standard error bars shown ................................................................................. 60
Figure 3-4 Box plots showing endotoxin concentration according to water type .................................. 61
Figure 3-5 Box plots showing endotoxin concentration for sewage impacted marine waters .............. 62
Figure 3-6 Box plots showing endotoxin concentration according to water type .................................. 63
8
LIST OF TABLES
Table 2-1 Water Efficiency Labelling and Standards (WELS) scheme rating specifications for
shower equipment .......................................................................................................................... 18
Table 2-2 Aerosol-measuring techniques employed to characterise aerosol production during
showering. ...................................................................................................................................... 20
Table 2-3 Experimental parameters used for characterising aerosols during showering and
research outputs............................................................................................................................. 21
Table 2-4 Size ranges of measurement for aerosol sizing instrumentation as configured
in experiments ................................................................................................................................ 23
Table 2-5 Summary of water efficient and control devices for shower experiments............................. 25
Table 2-6 Summary of water efficient and control devices for car experiments ................................... 25
Table 2-7 Number of replicate experiments and configurations............................................................ 30
Table 2-8 Aerosol and droplet concentrations ± standard deviations (aerosols or droplets /cm3)
measured in each size bin for the showerhead experiments carried out at 10 L/min.................... 34
Table 2-9 Aerosol mass ± standard deviations (aerosols µg/m3) measured in each size bin for
the showerhead experiments carried out at 10 L/min .................................................................... 35
Table 2-10 Aerosol and droplet concentrations measured in size bins for the showerhead
experiments for the Interbath showerhead at 7 L/min (3 ‘star’ rating) and the
Bastow showerhead at 13 L/min (1 ‘star’ rating equivalent) .......................................................... 36
Table 2-11 Aerosol mass ± standard deviations (aerosols µg/m3) measured in size bins for the
showerhead experiments carried out at 13 and 7 L/min. ............................................................... 37
Table 2-12 Concentration of aerosols and droplets (particles/ cm3) produced during hose
experiments as determined by the SMPS, APS and concentrations of droplets as determined
by the PDPA................................................................................................................................... 38
Table 2-13 Mass of aerosols (µg/m3) produced during hose experiments as determined by the
SMPS, APS and PDPA. ................................................................................................................. 39
Table 3-1 Endotoxin concentrations measured in different water types as reported in the scientific
literature ......................................................................................................................................... 53
Table 3-2 Classification of sewage treatment plant and recycled water samples................................. 58
Table 3-3 Mean, median, minimum and maximum endotoxin concentration according to water type . 61
Table 3-4 Mean, median minimum and maximum endotoxin concentration according to category
of recycled water ............................................................................................................................ 63
9
ABBREVIATIONS
APS
Aerodynamic Particle Sizer
cm
Centimetre (1/100 metre)
ºC
Degree Celsius
CSIRO
Commonwealth Scientific & Industrial Research Organisation
E. coli
Escherichia coli
EU
Endotoxin Unit
FEV
Forced Expiratory Volume
FFSSP
Fast Forward Scattering Spectrometer Probe
Hr(s)
Hour(s)
L
Litre
LAL
Limulus Amoebocyte Lysate
LPM
Litre per minute
LPS
Lipopolysaccharide
m
Metre
mL
millilitre (1/1000 litre)
ng
Nanogram (1/1.000,000,000 gram)
nm
Nanometre (1/1.000,000,000 metre)
NTU
Nephelometric Turbidity Unit
PDPA
Phase Doppler Particle Analyser
PM
Particulate Matter
PTFE
Polytetrafluoroethylene
QMRA
Quantitative Microbial Risk Assessment
RH
Relative Humidity
SMPS
Scanning Mobility Particle Sizer
WELS
Water Efficiency Labelling Standards
µg
Microgram (1/1,000,000 gram)
µm
Micrometer (1/1,000,000 metre)
10
DEFINITIONS
Accumulation mode
A mode in the atmospheric particle size distribution, formed primarily
by the coagulation of smaller particles.
Aerosol
An assembly of liquid or solid particles suspended in a gaseous
medium long enough to enable observation or measurement; particle
size is generally about 0.001-100 µm.
Aerodynamic diameter
Diameter of a unit-density sphere having the same gravitational
settling velocity as the particle in question (Reference to the
aerodynamic diameter of a particle is useful for describing particle
settling and inertial behaviour in the respiratory tract).
Bioaerosol
A suspension of particles of biological origin; viable or dead cells;
spores or pollen grains; fragments, products, or residues of
organisms.
Coarse particle mode
Large particle mode (>2 µm) in atmospheric particle size distributions,
consisting of particles generated by mechanical processes.
Class A reclaimed water
The degree to which recycled water is treated is often defined in
Classes; Class A being the ‘best’ or most highly treated. This
classification system differs from Australian state to state and has
other complications, as the water in some recycling schemes is
notably better than typical Class A. In this report from a
microbiological perspective Class A water contains less than 10 E.
coli organisms per 100 mL water.
Class B reclaimed water
In this report from a microbiological perspective Class B water
contains less than 100 E. coli organisms per 100 mL water.
Class C reclaimed water
In this report from a microbiological perspective Class C water
contains less than 1000 E. coli organisms per 100 mL water.
Cloud
An assembly of particles with an aerosol density that is more than 1%
higher than the density of gas alone.
Droplet
A particle of diameter greater than 10 µm with small solute
concentrations consisting mainly of water.
Droplet nuclei
The small residues of dried droplets which remain suspended in air
(usually <5 µm diameter).
Dual reticulation
Two separate pipelines that supply a building, one is a lilac coloured
pipe that delivers recycled water while the other pipe is reserved for
drinking water. Sometimes used interchangeably with the term ‘third
pipe’.
Evaporation time
The time taken for a water droplet to evaporate and become an
aerosol particle of diameter smaller than 10 µm.
Fog (mist)
Liquid particle aerosol. These can be formed by condensation of
super-saturated vapours or by physical shearing of liquids, such as
nebulisation, spraying or bubbling.
Non-potable
Water that should not be used for drinking, cooking, showering or
bathing.
Particle
A small, discrete object.
Potable
Water that is supplied for drinking and other uses.
Spray
Droplet aerosol formed by mechanical or electrostatic breakup of a
liquid.
11
1 INTRODUCTION
This report is the first of two which describe the CRC for Water Quality and Treatment Project No.
2.0.1.1.1.0 – A series of exposure experiments: recycled water and alternative water sources. Two
experimental components of the project are described in this report and another two project
components are described in a second project report (Part B: Microbial transfer efficiency during
machine clothes washing and microbial survival turf-grass experiments. CRC Research Report 46).
The inhalation route of exposure to micro-organisms in recycled water is one about which there is a
paucity of information and one which is not addressed in current Australian national recycled water
guidelines. The Australian Guidelines for Water Recycling (Phase 1) (NRMMC/EPHC/AHMC 2006)
released in December 2006 provide guidance on how recycling for uses such as agriculture, fire
control, municipal, residential and commercial property and industry can be safely employed and uses
a risk management framework. However, as it is impractical to set human health based targets for all
micro-organisms that might be present in a source of recycled water, guidelines focus on human
health based targets for pathogens (enteric) that are ingested. As transmission via inhalation, not
ingestion is the more important route for some micro-organisms (e.g. Legionella, Mycobacteria),
biological components (e.g. endotoxin) and chemicals it is important that information gaps about
exposure to respirable aerosols during specific water-using activities are addressed. This information
is important to provide input to future iterations of national recycled guidelines and for application to
new recycled water schemes.
The overall aim of the project was to address data gaps to facilitate the exposure assessment
component of the Quantitative Microbial Risk Assessment (QMRA) process to help set appropriate
water quality criteria to protect public health. The two experimental components described in this
report address information gaps relating to human health risk associated with the use of recycled and
alternative water sources. In addition they are inter-related in that they both address the inhalation
route of exposure that might occur during water-using activities. The two project components
described in this report specifically address exposure during the use of recycled water and alternative
water sources in water efficient devices (showers and high pressure hose devices) and the potential
for endotoxin exposure in recycled water.
Section 2 of this report describes the conduct and results of experimentation investigating aerosol
production during showering and car washing activities. Section 3 of this report describes a water
quality survey conducted to measure levels of endotoxin in a variety of Australian water types,
including recycled water. Relevant background literature, methods, results, discussion and
conclusions relating to each project component are presented and recommendations for potential
future research are made. Any mention of brand names or trademarks throughout the report does not
imply endorsement. They are cited for information only.
12
2 AEROSOL SIZING EXPERIMENTS
This section describes a series of experiments conducted to characterise the population of aerosols
and water droplets produced by Australian water industry-approved, water-efficient and conventional
(lower efficiency) devices during typical domestic water-using activities.
As described in Section 1, the over-arching aim of the research is to provide information that will assist
in assessing the human health risk associated with the use of recycled and alternative water sources
in a domestic and urban context. The experimental component described in this section fulfils this aim
by ‘overlaying’ another consideration related to the use of recycled and alternative water sources. This
consideration is the use of water-efficient devices in combination with recycled and alternative water
sources.
The sometimes concurrent, or at least overlapping, promotion of water-efficient devices and the use of
recycled and alternative water sources for domestic use has occurred as a means to address drinking
water shortages. This convergence leads to questions about the possibility that water-efficient devices
may have an additive effect in increasing the health threat to domestic water users from aerosol
exposure to micro-organisms and chemicals when used in combination with recycled and alternative
water sources. The inhalation pathway is an important transmission route for some microbial
pathogens and a significant exposure route for some chemicals. For example, micro-organisms and
compounds that may be inhaled during water-using activities and subsequently deposited in the lungs
include viruses, Legionella, Mycobacteria, volatile organic compounds and endotoxins. Enteric microorganisms also may be inhaled during water-using activities and enteric infections may be acquired
after inhalation and swallowing.
In terms of the numbers of aerosols generated during a water-using activity a key factor is the waterusing device employed. In this context, the terminology employed by Steinert and others, when
describing water-using devices associated with the transmission of Legionella bacteria, is useful
(Steinert et al. 2002). These investigators describe water-using devices such as showers, air
conditioning systems, cooling towers as ‘technical vectors’. The use of the term ‘vector’ serves to draw
an analogy with biological vectors and underscores that all vectors are not equally effective in the
transmission of disease. Thus, it is credible that certain ‘technical vectors’ (e.g. water efficient
showerheads, taps and other water-using equipment, such as fire hoses and cleaning equipment) may
lead to an increase in inhalation exposure above a critical threshold level or to an exposure that would
otherwise not occur (i.e. with a conventional device). The association of a particular technical vector
with disease transmission via inhalation will depend upon the effectiveness of the vector in generating
small size class aerosols.
In the past some investigators (Burrows et al. 1991), when considering the use of recycled water for
showers, have considered that it is preferable to bypass the question of aerosol inhalation by
specifying a low pressure shower head of such design that virtually no aerosols are generated, similar
to the head of a garden watering can. However, this suggestion when made some 16 years ago
included the caveat that only in the event that water conservation needs mandate the use of high
efficiency showerheads should the health significance of aerosol formation during showering be
investigated. The current Australia-wide drought and the imperative to conserve drinking water and to
use recycled and alternative water sources have now made such investigations pertinent. Even in the
event that recycled water not be considered for showering, rainwater tank water is an example of an
alternative water type where showering is currently considered to be a potential beneficial use. In
addition, water-efficient devices other than those used for showering are being promoted for purposes
where recycled water is in use, or its use is contemplated.
While established formulae and modelling approaches from the literature may be used to approximate
aerosol inhalation of water contaminants associated with water-using devices, results of prior
experiments, from which models are derived may not directly relate to Australian water-using devices
and/or conditions. To address this, a series of experiments were designed to characterise the
population of aerosols and water droplets, produced by Australian water industry-approved, waterefficient and conventional (lower efficiency) devices during typical domestic water-using activities and
comparing the numbers of respirable aerosols produced by each.
13
The Commonwealth Scientific and Industrial Research Organisation (CSIRO) was commissioned to
perform water droplet and aerosol measurements of both high-efficiency and conventional devices
used with drinking water during selected water-using activities. (Experiments were performed in March
– August 2006).
2.1 Background
Information about dermal and inhalation exposure routes is particularly important when considering
non-drinking uses of water as there are many water-using activities in the domestic and urban context
that do not involve drinking (unless it is inadvertent). These include showering, bathing, washing,
laundering, cleaning, toilet flushing, garden watering and irrigation of recreational areas.
To estimate inhalation exposure to contaminants in water, a number of parameters need to be
considered. These include temperature and agitation or aerosol dispersion of the water (as might
occur with a showerhead, in toilet flushing or with a high pressure spray device when cleaning
surfaces), air flows, frequency and duration of the activity and the fraction of the inspired dose that
reaches the alveolar region of the lungs. This information is required to supplement information about
the concentration of the microbial contaminant of interest in the water type under consideration.
2.1.1 Terminology
Different size ranges and definitions of aerosols and droplets are used in the literature. The definitions
of water droplet and aerosol, as stated by Pandis and Davidson, have been used (Pandis and
Davidson 1999). Aerosol is defined as an airborne particle sufficiently small (diameter<10 µm) that
does not rapidly settle out in the air. Water droplets are defined as being of diameter >10 µm (Pandis
and Davidson 1999).
The SI unit for aerosol mass concentration (the mass of particulate matter in a unit volume of gas) is
expressed in kg/m3. Because the amount of aerosol mass is generally low it is expressed in g/m3,
mg/m3, µg/m3 or ng/m3. Aerosol number concentrations are expressed in number/m3 or number/cm3.
2.1.2 Biological and non-biological particles size range
The dynamics of both biological and non-biological particles in the air is governed primarily by the
particle physical characteristics of which size is the most important. Particles in different size ranges
are governed by different physical laws. Particles only slightly larger than gas molecules are governed
by Brownian motion, while large, visible particles are affected primarily by gravitational and inertial
forces (Baron and Willeke 2001).
Figure 2-1 shows the relative size ranges of biological and non-biological particles in the size range up
to 1000 µm. This comparison is important as this information can be ‘overlaid’ onto the diameter size
range for aerosols and water droplets. As can be seen from this figure, aerosol particle sizes less than
0.01 µm diameter are too small to be of concern in relation to aerosol transmission of viruses, bacteria
and larger microorganisms. However, particles of less than 0.01 µm diameter may contain organic and
inorganic materials and thus may be of health concern in relation to aerosol transmission of chemical
contaminants present in water. Bioaerosols generated from water sources (such as during splashing
and wave action) are usually formed with a thin layer of moisture surrounding the micro-organisms and
consist of aggregates of several micro-organisms (Wickmann 1994 cited in (Pillai and Ricke 2002).
14
Source: Adapted from https:/.../imgs/particle_size_spectrum.gif and http://www.copa.co.uk/images/uploaded/mbr5.gif
Figure 2-1 Particle size diameters and the respirable µm range
In the spread of infectious disease by the airborne route, particles in the size range 0.1 - 60 µm are
particularly important (Sattar and Ijaz 1987). The theoretical means of spread of infectious microorganisms through droplets and aerosols is shown in Figure 2-2. In accord with the definition of
aerosols as being less than 10 µm diameter, this diagram shows aerosols as potentially associated
with inhalation exposure to micro-organisms and biological agents with their potential deposition in the
alveolar region of the lungs.
Figure 2-2 Means of spread of microbial agents via aerosols/droplets
However, this is not to say that the degree of deposition in the human lungs of all particles less than
10 µm diameter is constant. A variety of factors including particle size, density, shape,
hygroscopic/hydrophobic character and electrostatic charge play important roles in determining the
location and efficiency of deposition of inhaled droplets and aerosols in the lungs (Mercer 1999).
15
The human upper respiratory system filters essentially 100% of aerosols with a diameter greater than
approximately 7 µm (Wilkes 1999). However, particles less than 7 µm diameter are not filtered as
efficiently and therefore a significant fraction of the inhaled mass reaches the alveolar region (Wilkes
1999). Particles that reach the lower parts of the lungs are predominantly between 1 and 3 µm; below
0.4 µm, particles are diffused into the body (Figure 2-3) (Holmberg and Li 1998). Particles greater than
3 µm are deposited mainly in the upper respiratory system (Figure 2-3); with these particles
translocated by muciliary action and ingested (Slote 1976). Thus, they give rise to the potential spread
of certain infections of the gastrointestinal tract by the airborne route.
Particle size is not only important with respect to carriage of biological and other agents of health
concern but it is also an important criterion when considering the life span of aerosols and water
droplets in the atmosphere. For example, water droplets, due to their larger size, have a relatively
short lifetime and if they do not evaporate quickly they are removed in a few minutes mainly through
gravitational settling. Particles larger than 100 µm settle in less than 10 seconds. In contrast, a one µm
diameter aerosol settles at 0.003 cm sec-1 and thus may remain airborne for a substantial period of
time. Aerosol lifetimes in the indoor environment are of the order of hours (Pandis and Davidson
1999). Investigators also note that the time during which aerosol particles remain suspended and the
distance which they can travel from the point of their generation are greatly influenced by airflow and
turbulence (Sattar and Ijaz 1987).
Depending upon the level of relative humidity and the atmospheric temperature, most of the water
from aerosolised particles of small size evaporates almost immediately. The residual particle left
behind after evaporation may contain organic and inorganic materials as well as biological agents
(Sattar and Ijaz 1987). During droplet evaporation their liquid water content decreases and solute
concentration of non-volatile aerosol components (expressed as a ‘per volume of air’ basis) remains
constant. In contrast, volatile species dissolved in tap water are transferred to the gas phase during
droplet evaporation (Pandis and Davidson 1999).
Source: (Wickham, 1992 from (Holmberg and Li 1998)
Figure 2-3 Particle deposition (unit density spheres) in the respiratory system
The fraction of the inhaled aerosol mass deposited in each of the nose, airways and alveolar regions
depends upon the size particle distribution, upstream filtering and the respiration rate (Wilkes 1999).
For particles in the 1 to 5 µm range, the total respiratory tract deposition (upper respiratory +
conducting + gas exchange regions) efficiencies for particles are of the order of 20% (Mercer 1999).
16
Mathematical model-based estimates of alveolar deposition efficiency of inhaled 1 to 5 µm aerosol
particles have been estimated to be 5.2 and 17% respectively (Mercer 1999).
Water droplets, defined as having a diameter greater than 10 µm, are too large to be respirable. In
terms of inhalation exposure, water droplets are therefore important only as precursors of respirable
aerosols (Pandis and Davidson 1999). However, their importance as aerosol precursors is only
relevant for water droplets that are less than 200 µm diameter. This is because even if droplets larger
than 200 µm completely evaporate they will produce non-respirable (diameter greater than 10 µm) dry
particles (Owen 1992 as cited by (Anderson et al. 2007).
Large droplets which tend to settle out immediately, whilst not important to inhalation exposure to
infectious agents, may nonetheless be important in disease transmission of infectious disease via
direct contact with susceptible hosts or indirect contact via contaminated surfaces (Barker and
Bloomfield 2000; Carducci et al. 2000; Rusin et al. 2002).
2.1.3 Particle (aerosol and water droplet) measuring instrumentation
Particles may be characterised based upon a number of properties. These include: light-scattering
properties using an optical particle counter or nephelometer; elemental or chemical properties with
X-ray fluorescence or infrared spectroscopy; surface properties with a pycnometer or by an absorption
measurement; or dynamic behaviour from measurement of settling velocity or diffusion (Baron and
Willeke 2001).
There are two general approaches to particle measurement. These are collection and analysis and
real-time sensors. Real-time instruments generally separate or classify particles according to size but
require a particle sensor that responds quickly and efficiently to each particle. Real-time sensors can
give size distribution information, almost instantaneous results and allows many measurements to be
made over time (Baron and Heitbrink 2001).
For such real-time sensors, sampling may be performed in-situ using either extractive or external
sensing techniques. Extractive techniques require the particle to be brought to the instrument sensor,
while external sensing techniques measure the particle in its undisturbed state (Baron and Willeke
2001). The Phase Doppler Particle Analyser (PDPA) employs an external sampling technique and
‘interrogates’ a small sample volume. In contrast, the Aerodynamic Particle Sizer (APS) and the
Scanning Mobility Particle Sizer (SMPS) use an extractive sampling technique where air is drawn
through a tube to the sensor.
Most particle-sizing instruments effectively measure over a size range no larger than 1.5 orders of
magnitude. As such the largest measurable size may be about 50 times the smallest size for a given
instrument. Instruments measuring a cumulative value (e.g. total mass or volume) can cover a wider
size range (Baron and Willeke 2001).
A common in situ technique for particle sizing is the measurement of light scattered from the particles.
The amount of light scattered from individual particles is a complex function of particle parameters of
size, shape and refractive index as well as instrumental parameters such as the wavelength of light
and the scattering angle. For simple particle shapes such as spheres the amount of light scattered
may be calculated exactly (Baron and Willeke 2001). In the small particle size range, particle detection
by light scattering loses sensitivity, with a lower limit of about 0.1 µm under optimum conditions. The
Phase Doppler Particle Analyser (PDPA) and Aerodynamic Particle Sizer (APS) utilise optical particle
counters as the sensor, although the APS is not an in-situ method.
When particles are subjected to an external force, such as gravity or an electrical force the particles
will move in the force field. In an electrical field, a particle of known charge moves along a predictable
trajectory. The migration velocity in the force field is particle size dependent, a fact that is exploited by
most spectrophotometers for particle size discrimination. Particle motion in an electrical field can yield
high resolution measurements as well as separation of desired particle sizes (Baron and Willeke
2001). The Scanning Mobility Particle Sizer (SMPS) is an example of instrumentation based on
particle migration velocity in a force field.
17
2.1.3.1 Aerosol measurement errors
The major sources of biases that occur in droplet and aerosol measurement may be divided into 5
categories as follows: original droplet/aerosol, sampling efficiency, internal losses, sensor response
and data processing (Baron and Heitbrink 2001). These errors are shown in Appendix 1.
2.1.4 Water-efficient devices and their rating
The mandatory Australian Water Efficiency Labelling and Standards (WELS) scheme provides an
authoritative means of identification of water efficient devices for Australian consumers and water
industry practitioners. The Australian Standard Water efficient products - Rating and labelling (AS/NZS
2005) which underpins the WELS scheme, provides performance criteria for washing machines,
dishwashers, lavatory (toilet) equipment, showers, tap equipment and urinal equipment. In addition,
the standard requires that products that do not comply with specific performance requirements be
given a zero rating and labelled accordingly.
These rating specifications for showers are reproduced in Table 2-1. Of note in perusing the
information in Table 2-1 is that the highest water-efficiency rating possible for showers is currently ‘3
Stars’. The designation of ‘4 Stars’, ‘5 Stars’ and ‘6 Stars’ is pending a performance test which
includes a force of spray test especially for showers with low or very low flow rates (AS/NZS 2005).
Table 2-1 Water Efficiency Labelling and Standards (WELS) scheme rating specifications for shower
equipment
Product
type
Shower**
Water
consumption
unit
L/min
Rating
0 stars
(warning)
More than
16.0 or failing
performance
requirements
1 Star
2 Stars
3 Stars
(3**) 4
Stars
(3**) 5
Stars
(3**) 6
Stars
More
than 12
but not
more
than 16
More
than
9.0 but
not
more
than 12
More
than
7.5 but
not
more
than
9.0
More
than
6.0 but
not
more
than
7.5*
More
than
4.5 but
not
more
than
6.0*
More
than
4.5 but
not
more
than
6.0*
Source: (AS/NZS 6400:2005)
*includes compliance with force of spray requirements
** 3 Stars is maximum rating for showers
2.1.5 Research on inhalation aerosol exposure via water
The need for increased data about inhalation exposure during water-using activities has been
acknowledged for some time. In the late 1990s, a 15-member working group was convened by the
International Life Sciences Institute - Risk Science Institute under a cooperative agreement with the
United States Environment Protection Agency’s Office of Water, with the aim of summarising the state
of the science relating to contaminants in water and topical and inhalation exposure routes (Olin
1998). In particular, this group sought to present methodologies and data resources for characterising
population distributions of absorbed dose of water contaminants through the skin and by inhalation.
While this work concentrates on exposure to chemical contaminants in water, it represents an
invaluable resource when considering inhalation exposure to microbial contaminants in water. In
addition, it remains a resource on which more recent studies are based, or to which they refer. For
example, in a recent study, endotoxin inhalation from shower and humidifier use was estimated using
a modelling approach based upon established formulae described in this resource
(Anderson et al. 2007).
The use of modelling approaches and the dependence upon established formulae (often not exactly
applicable to the circumstance under investigation) to quantify inhalation exposure to contaminants
during specific water-using activities arises as a consequence of the difficulty in collecting primary data
18
through experimental means. For example, there are relatively few instances where aerosol emissions
in showers have been characterised.
Data arising from a study by Keating and McKone, which characterised the aerosol size distribution
present in the shower while in use (Keating and McKone 1993), and a simple linear model formulated
by Gunderson and Witham (Gunderson and Witham 1988) were both used in a case study estimating
exposure of a population group to water contaminants while showering (Wilkes 1999). The Gunderson
and Witham model has also been used for a recent estimation of inhalation health risk from
endotoxins during showering (Anderson et al. 2007).
A number of different droplet and aerosol measuring techniques have been employed for the
characterisation of aerosols produced during showering. Table 2-2 presents the aerosol-measuring
techniques employed by researchers in relevant studies (last 10 years) to characterise aerosol
production during showering. The water contaminant(s) under investigation in each of the studies and
the size range of aerosols measured is also given. As shown from the table, three (Xu and Weisel
2003; Cowen and Ollison 2006; Zhou et al. 2007) of the four studies employed optical measurement
techniques. In three of the studies (Keating et al. 1997; Xu and Weisel 2003; Cowen and Ollison
2006), water was seeded with chemical constituent(s) and in two studies (Keating et al. 1997; Xu and
Weisel 2003), assays for the chemical of interest were performed in addition to the measurement of
aerosols produced by the shower. In one study (Zhou et al. 2007), derived aerosol measurements
were combined with data from water quality reports to obtain deposition doses for several elements
present in the water supply.
19
Table 2-2 Aerosol-measuring techniques employed to characterise aerosol production during
showering.
Reference
Aerosol measuring technique
Water
contaminant
(Keating et al.
1997)
Model AIMS Droplet counter. This instrument utilises a hotwire anemometer probe which is cooled when droplets come
into contact with its thin platinum wire. An electronic signal
which is droplet size dependent is generated and converted to
one of 14 size categories between 1.2 and 586 µm in diameter
chloroform
(Xu and
Weisel 2003)
A Lasair model 1002 optical particle counter (Particle
Measuring Systems, Inc, Boulder, CO). The particle counter
measured particles from 0.1 to greater than 2 µm in eight size
bins
Haloacetic
acids and
haloketones
(Cowen and
Ollison 2006)
Climet monitors (Climet Instruments Co.) were used to
measure particle concentrations in six size fractions from 0.3
to greater than 10µm. These instruments use optical
techniques to size characterise sampled aerosol
Residential tap
water with and
without
injection of
salts
(Zhou et al.
2007)
DataRAM real-time particle monitor (Monitoring Instruments
for the Environment, Inc, Bedford, MA). The instrument is a
nephelometer-based instrument and measures particles in the
concentration range of 0.1µg/m3 – 400 mg/m3 for particles less
than 10 µm diameter
Aerodynamic particle sizer (APS 3310; TSI, Inc., Amherst,
MA) measured particle number and mass size distributions.
The particle size range by the APS was 1 -30 µm
Elemental
constituents of
water
In the conduct of experimental studies characterising aerosol distributions during showering,
experimental conditions employed by different researchers necessarily vary. Such experimental
variations include: showerheads, water pressure, flow rates, shower cubicle volumes and other factors
potentially influencing aerosol production rates and concentrations. Table 2-3 gives the experimental
parameters used by researchers when characterising aerosols during showering. Experiment outputs
of research are also detailed.
20
Table 2-3 Experimental parameters used for characterising aerosols during showering and research
outputs.
Experimental
conditions
(Keating et al.
1997)
(Xu and Weisel
2003)
(Cowen and
Ollison 2006)
(Zhou et al. 2007)
Cubicle
dimensions
1.53m3
1.18 (w) x 0.85 (d)
x 1.53 (h)
6m3
2.67 (w) x 0.94 (d)
x 2.39 (h)
Not applicable
2.0m3
0.92 (w) x 0.92 (d) x
2.41 (h)
7-10 L/min
5 & 8 L/min
5.1, 6.6, 9.0 L/min
36-38 °C
Hot and cold
43-44°C and 24-25 °C
Flow rate
Water
temperature
Details
35-45 °C
• Plexiglas® on
all sides
• Shower
curtain
• Full size
mannequin to
• Ventilation
provided by
exhaust fan
at top of
chamber
• No
mannequin
• Shower
located above
full size tub
within 11m3
bathroom
• No
mannequin
• No splash
back from
shower
cubicle sides
• Shower
curtain closed
when shower
was on and
open when
off
• With mannequin
1.70m high and
0.8m from
showerhead
• 0.35m
opening
between the
top of the
shower
curtain and
the ceiling
Position of
probe
Centre of
exhaust vent
At breathing
height 1.5m
through 1.75m
stainless steel
tube
At top of shower
curtain (2m) and
1m above floor
Breathing height 1.5m
inside and outside
shower
Nozzle
location
Centre top with
water sprayed
directly on the
floor (135cm
distance)
Top of shower
stall
Mannequin was
positioned in
shower spray
cone to simulate
splashing
Water showered on
mannequin at 30o angle
Time
Monitored every
2 minutes during
the first 15
minutes of the
simulation
Monitored for a
total of 42 min
with a 6 min
background
period, 10 min
shower on
period and 26
min shower off
period
Monitored 510min before
shower on, 10
min shower on
and 10 min after
shower off
APS completed one
measurement every
minute for each 10min
showering period. The
next test period began
when the room
temperature and
relative humidity were
back to baseline
Output of
experiment
Total aerosol
concentration
per cm3 for
elapsed time of
shower
Total aerosol
concentration
per cm3 for
elapsed time of
shower. Peak
aerosol number
and mass in
micron size bins
Total particle
concentrations
per cm3 for
elapsed time
period of shower
Shower particle size
distributions (mass
median diameter, count
median diameter, %
mass concentration in
micron size bins
21
Of note in the conduct of experiments is that two research groups in the most recent studies used a
mannequin in the shower cubicle to simulate the presence of a human (Cowen and Ollison 2006;
Zhou et al. 2007). The use of a mannequin in experimental studies was recommended in 1999 as
being necessary to supplement existing preliminary information about showering (Pandis and
Davidson 1999). The basis for this recommendation was the probable major contribution of respirable
particles arising from the collision of droplets with the body of the person in the shower.
Also notable with respect to studies detailed in Table 2-3 is the output of experiments. Of the four
studies only Zhou et al. (2007) present detailed shower particle size distribution data. In their paper
these investigators note that they found only one other publication that studied shower particle size
distribution. They cite the work of Giardino and Hageman (1996) but note that this study was
performed with a horizontally positioned showerhead, not in a shower stall. However the cited study
investigated radon volatilisation from showers and implications for dose. In fact it is an earlier study
(Giardino et al. 1992) where the role of drop size distribution was investigated and compared with
theoretical models.
Nonetheless some other shower particle size distribution data have been presented but this is only for
the peak aerosol number and mass just prior to the end of the shower (Xu and Weisel 2003). Also, as
discussed earlier, shower particle size distributions of Keating and McKone (Keating and McKone
1993) and Gunderson and Witham (Gunderson and Witham 1988) and have been used for estimates
of aerosol exposure in a recent study (Anderson et al. 2007).
Relevant available experiment outputs are presented in Appendix 2. Raw data from Gunderson and
Witham (Gunderson and Witham 1988) was unable to be accessed. The results of described studies
can thus be assumed to represent much of the current knowledge of shower particle size distributions.
2.2 Study objectives
The objectives of experimentation were:
•
To characterise the size distribution of aerosols (≤10 µm diameter), and droplets (>10 µm
diameter) produced by Australian industry approved water-efficient devices and conventional
(lower efficiency) devices.
•
To determine whether water-efficient devices produce significantly higher inhalation exposure
to respirable aerosols than conventional devices under typical Australian usage conditions.
2.3 Materials and methods
2.3.1 Aerosol measurement instrumentation
Instrumentation used to size and count aerosols comprised: an Aerodynamic Particle Sizer (APS); a
Scanning Mobility Particle Size (SMPS) and a Phase Doppler Particle Analyser (PDPA). The size
ranges of measurement for aerosol sizing instrumentation, as they were configured for the
performance of experiments, is given in Table 2-4.
22
Table 2-4 Size ranges of measurement for aerosol sizing instrumentation as configured in
experiments
Aerosol real-time instrument
Size range
Aerodynamic Particle Sizer (APS)
500 nm to 5 µm
Scanning Mobility Particle Sizer (SMPS)
15 nm to 700 nm
Phase Doppler Particle Analyser (PDPA)
500 nm to 250 µm
Details of the individual instruments and the limitations and uncertainties of aerosol measurements
using these instruments are given in Appendix 3.
2.3.2 Water-efficient and conventional devices used in experimentation
Water-using scenarios for experimentation and the types of devices selected for evaluation was based
on two or more of the following factors:
•
water-using activities potentially giving rise to significant aerosol production
•
the types of water-efficient devices actively promoted by Australian water authorities
•
water-using activities where the use of water-efficient devices potentially provide greatest
household water-savings.
The two scenarios/water-using devices selected for experimentation comprised:
•
Showering using water-efficient showerheads
•
Use of high pressure hoses for cleaning of cars.
For each scenario, a ‘control’ scenario was selected for the purpose of benchmarking. The basis for
the differentiation of water-efficient and control devices was the rating classification given in the
Australian/New Zealand Standard Water efficient products-Rating and labelling standard (AS/NZS
2005) described in section 2.1.4.
23
2.3.2.1 Showerhead
Two types of showerhead were employed in experimentation. Details of their WELS ‘star’ rating and a
photograph of each are shown in Figure 2-4 and Figure 2-5.
ELS registration No:
Nominal flow L/min
WELS ‘star’ rating
R000402A
7.3
3 star
(potential 4
star pending
force of spray
test)
Adjustable
spray settings
Figure 2-4 Interbath Watersaver series showerhead
WELS registration No:
Nominal flow L/min
WELS ‘star’ rating
R000506
9.0
3 star
(flow at cusp
of 2/3 star
category)
Non adjustable
spray
Figure 2-5 Bastow (Fjord) showerhead
Both showerhead design types are commonly employed in Australian households according to a
survey of hardware retailers (Bunnings pers comm. 2006) with the Bastow type showerhead the more
conventional showerhead design. As the objective of experimentation was to benchmark a waterefficient showerhead against a conventional (low efficiency) showerhead and both selected
showerheads were rated as being ‘3 star’ water efficient devices, the flow restrictor disks were
removed from each device and the flow rate controlled by the use of an external valve during the
experiments.
The removal of the flow restrictor for each device meant that each device could be ‘rendered’ a less
water efficient device than the WELS water efficiency rating. The Interbath water saver was
designated the water-efficient device and experiments were conducted at a flow rate of 7.0 L/min
(equivalent to a WELS 3 ‘star’ rating and the same rating category as per the WELS certificate for this
device). The Bastow (Fjord) showerhead was designated the conventional (low efficient) showerhead
with experiments conducted at 13 L/min (equivalent to a WELS 1 ‘star’ rating). Tests were also
performed with both showerheads at a flow rate of 10 L/min, equivalent to a WELS 2 ‘star’ rating.
This combination of flow rates used in experiments allowed the comparison of a 1 star (low efficiency)
and 3 star (high efficiency) rated showerhead to be made. Also, the performance of experimentation at
10 L/min for both devices allowed the impact of the showerhead design on aerosol production to be
compared at a constant flow rate, equivalent to a 2 star rating (see Table 2-5).
24
Table 2-5 Summary of water efficient and control devices for shower experiments
Parameter under test
Water-efficient device
Control device
Water efficiency showerhead
defined by flow rate L/min
Interbath (Watersaver) with
flow restrictor removed
7 L/min
Bastow (Fjord) with flow
restrictor removed
13 L/min
Showerhead design
Interbath (Watersaver) with
flow restrictor removed
10 L/min
Bastow (Fjord) with flow
restrictor removed
10 L/min
2.3.2.2 High pressure device
For the car washing scenario, a Gurney Tradesman high pressure spray unit was used as the water
efficient device (see Figure 2-6, right) and a standard garden hose (Nylex 13 mm diameter) in
combination with a Pope hand spray nozzle (see Figure 2-6, left) was used as the conventional (lower
efficiency) device.
Figure 2-6 Nylex hose with Pope spray nozzle (left) Gurney Tradesman high pressure spray unit
(right)
The Gurney Tradesman high pressure spray unit was employed in experiments using both a wide fan
spray and a jet spray setting. Table 2-6 summarises the control and water-efficient conditions/devices
used in experimentation.
Table 2-6 Summary of water efficient and control devices for car experiments
Parameter
under test
Cleaning device
for car washing /
pavement etc
Water-efficient device
Gurney Tradesman high pressure
spray unit used at 7.3 L/min (all
runs)
•
Wide spray
•
Jet spray
25
Control device
Nylex hose with Pope hand spray
nozzle
•
Wide spray (cone of 60o,
flow rate 15.4 L/min)
•
Jet spray (cone of 20 o, flow
rate 11.8 L/min)
2.3.3 Experimental conditions
All experiments were carried out in a large laboratory at CSIRO’s facilities at Highett. Two rounds of
experiments were performed for the showering module, with the first round of experiments conducted
in the general laboratory space and the second round in a ‘clean room’ within the general laboratory
space. All car washing experiments were conducted in the ‘clean room’.
2.3.3.1 Clean room
The clean-room consisted of a plastic tent which was fully seam-sealed and sealed to the floor (Figure
2-7). This gave an enclosure which could be positively pressurised. Compressed air was passed
through a PTFE backed quartz-fibre filter into the tent to remove most incoming particles. Several
small openings were created to allow the air to pass through and remove some of the existing particle
loading thus diluting the particle concentration in the clean-room.
Analysis of the particle count from inside and outside of the clean-room showed that filtration was
effective in reducing the particle numbers. Counts were reduced from up to 12,000/cm3 (depending on
size and day) outside the tent, to fewer than 500/cm3 inside the tent. The experimental rigs and
aerosol-measuring- instrumentation, including appropriate shielding for the laser, were able to be
placed inside the ‘clean room’ within the mounting frame for the performance of experiments.
Figure 2-7 ‘Clean room’ for aerosol experiments
2.3.3.2 Shower experiments
2.3.3.2.1 Shower rig and operating parameters
The shower enclosure (Figure 2-8) consisted of a 0.81m2 shower base with centre drain. Taps were
installed at 950 mm above the shower floor with the wall outlet height at 1700 mm. The shower walls
were 2100 mm high on two sides and a rail was supplied for the shower curtain at 2100 mm high. For
safe laser operation, and to represent an enclosed shower, black plastic was used to form the shower
curtain and a roof over the enclosure (black plastic curtain is not shown in the photograph).
26
When installed, the Bastow and Interbath showerhead were located at heights 1900 mm and
1880 mm respectively. For experiments using the mannequin, the showerhead was directed to spray
on the back of the neck of the mannequin. Figure 2-8 shows the relative position of instrumentation
and the shower stall with mannequin. The PDPA transmitter and receiver were mounted on the frame
and protruded through plastic sheet (removed for photographic purposes) with the SMPS and APS
instrumentation located outside the enclosure.
Figure 2-8 Photograph showing location of instrumentation relative to shower stall
The shower was run for up to 15 minutes before commencement of an experimental run to allow the
water temperature to stabilise. This also ensured that the air within the shower enclosure was
saturated (i.e. relative humidity at, or close to, 100%). Constant temperature was maintained by a
combination of the water pump (Davey Celsior SH30R5 that has an inbuilt heating element) and by
using hot water as the water supply. The water temperature was monitored in a reservoir throughout
the experimental runs and adjusted by varying the pump heater and flow of the hot water into the
reservoir. Pipes leading to the pump and to the shower were insulated using Armaflex expanded foam
insulation tubing. The positioning of the PDPA equipment in the shower experiments is shown in
Figure 2-9.
27
Figure 2-9 Schematic of positioning of PDPA equipment in shower experiments (left is side view; right
is top view)
Figure 2-10 shows a schematic of the sampling set-up for the APS and SMPS instrumentation.
Aerosols were sampled from near the shower stream via a copper tube (6.25 mm diameter and 40 cm
long). Sample air was dried from >99% RH to ~ 40% RH using a 40 cm long Nafion drier (comprised
of 20 tubes and sheath air dried using silica gel). The dried air then encountered a Tee connection, the
straight branch of which fed sample air to the APS (via 1 m of 12.5 mm diameter copper tubing with a
broad 90° bend). The sample flow rate through the inlet system was 5 L/min. The side-branch of the
Tee connection fed sample air to the SMPS via 1 m of 6.25 mm diameter conductive tubing at a flow
rate of 0.3 L/min.
Water was removed from the sample air because of the potential for water entering the SMPS to
promote electrical arcing within the instrument, causing significant damage. The sample air to the APS
was also dried so that the data measured by the two aerosol instruments were comparable.
Nafion drier
shower
RH = 99%
RH = 20-40%
APS
SMPS
Figure 2-10 Aerosol sampling set-up
28
2.3.3.3 Car washing experiments
A car door from a Ford Falcon XD was mounted within the Unistrut frame 320 mm off the ground and
with the top of door at 1350 mm. The water impact point on the glass section was 1170 mm above the
ground and the impact point on the metal section was 850 mm above the ground. The PDPA sample
space was set up to be 1600 mm above the ground and 150 mm in front of the mannequin. Under the
premise that when washing a car the tendency is to avoid being wet by the back spray; the nozzle was
set up such that it did not directly splash back through the sample space.
A standard garden hose arrangement consisting of a Nylex 13 mm hose and a Pope hand spray
nozzle (Figure 2-11) were set up to spray on the metal and the glass of the car door. The nozzle was
tested at a fine spray as would be used for rinsing the car, and as a narrow jet as would be used for
dislodging mud or road grime. Water flow through the nozzle was measured during the experiments
using a Rosemount magnetic flow meter. Relative humidity within the tent during the hose tests was
measured as varying between 92 and 95.5%.
The hose nozzle was then replaced with a Gurney Tradesman high-pressure spray unit (Figure 2-12)
and the experiments repeated, using both a wide fan spray and a jet spray setting. Data was collected
for each condition over a 5-minute period.
Figure 2-11 Garden Hose experiment setup
Figure 2-12 High pressure spray experiment
setup
2.3.3.4 Other instrumentation and materials
A Rosemount 15 mm Magnetic Flow tube (model 8711) was used to measure the flow rate of water
through all the devices under test. The uncertainty associated with the flow rate measurement is 0.5%.
29
Relative humidity was monitored during the first set of shower experiments using Vaisala 500 Humitter
probes calibrated using saturated salt solutions (sodium chloride and sodium bromide). The
uncertainty associated with the Humitter probes is 3%. During the car washing and second round
shower experiments a Fluke 971 temperature and humidity probe was also used to monitor the
humidity within the tent enclosure. The uncertainty associated with the Fluke probe between 10% to
90% Relative Humidity at 23°C (73.4°F) is (±2.5%Relative Humidity) for (<10%, > 90%Relative
Humidity at 23°C (73.4°F) the uncertainty is (±5.0%Relative Humidity).
2.3.4 Experimental configurations and replication of runs
Table 2-7 summarises the number of replicates for each experimental condition.
Table 2-7 Number of replicate experiments and configurations
Experiment
Shower
Conditions
Water efficient flow 7 L/min (3 star)
Low efficiency flow 13 L/min (1 star)
Water-saving showerhead design
10 L/min (2 star)
Conventional design 10 L/min (2 star)
Car washing
High pressure device, Jet setting
Garden hose, Jet setting
High pressure device, spray narrow
High pressure device, spray wide
Garden hose, spray setting
30
Number of
experiment
replicates
38°C with mannequin
3
38°C no mannequin
2
3
42°C no mannequin
3
4
38°C no mannequin
4
3
42°C mannequin
3
3
38°C no mannequin
3
3
42°C no mannequin
3
3
38°C no mannequin
2
3
42°C no mannequin
2
Metal
3
Glass
4
Metal
3
Glass
3
Glass
3
Metal
3
Glass
3
Metal
3
Glass
3
Metal
3
2.3.5 Data analysis
2.3.5.1 Combining data from the APS, SMPS and PDPA
The methodology for combining data from the APS, SMPS and PDPA is described in detail in
Keywood et al. (2007). In summary, the method employed takes into account the growth of the
particles determined by the APS and SMPS to the relative humidity experienced in the shower (recall
that a Nafion drier was used to dry the sample air to 40% Relative Humidity to prevent water
interfering with the aerosol measurement instruments) and calculation of the PDPA sample volume.
2.3.5.2 Data manipulation
For the car washing experiments the particle size distribution of the semi-enclosed tent (clean room)
before each set of experiments was subtracted from the experimental results to determine the change
in concentration and size distribution caused by the experiment. This was because the rate at which
the particle-free air was entering the tent was not sufficient to remove droplets and aerosols produced
by the equipment (showerhead, spray device) being tested.
For the graphical presentation of the aerosol size distributions, modes were fitted with log normal
distributions (dNlogdP).
The mass distribution of the aerosols in each size range was calculated assuming spherical shaped
aerosol with a density of 1.8 g/cm3. This is the density of ammonium sulphate and one which
characterises typical indoor atmospheric particle density.
The mid point of the size range was used as the diameter to calculate the sphere volume.
2.4 Results
2.4.1 Characteristics of aerosol and droplet distributions by water
efficient and low efficiency devices
2.4.1.1 Showering module
Figure 2-13 shows a typical aerosol and droplet size distribution measured during the series of shower
experiments. Experimental conditions were the Bastow showerhead and the absence of a mannequin
at flow rates of 7, 10 and 13 L/min in first round tests conducted in the general laboratory space. Size
modes in the aerosol/droplet distributions shown have been fitted with representative log-normal
distribution functions. These results show typical aerosol and droplet size distributions during shower
experiments to have the following characteristics:
•
The aerosol distributions (derived from the APS and SMPS) show a relatively consistent
decrease in concentration with increasing particle size
•
A size mode at around 200 nm diameter represents the accumulation mode (shown in red)
where the particles generally evolve through coagulation of smaller particles and by
heterogenous condensation of gas vapour onto existing aerosol particles
•
A size mode at around 1000 nm represents a coarse particle mode (shown in blue)
•
PDPA data shows a strong aerosol mode at around 10,000 nm diameter representing a
cloud/fog mode (shown in green) where water vapour condenses to form a fog or cloud
distribution
•
PDPA data shows a positive sloping line that extends between 1 µm and 100 µm mode
indicative of a spray mode (shown in pink) where droplets escape the main shower stream
•
The size distributions for the room air (shown in black) as determined by the APS and SMPS
show higher concentrations below 2 µm than for experimental size distributions.
31
Source: (Keywood et al. 2007)
Figure 2-13 Aerosol size distributions during showering determined by the SMPS and APS and
droplet size distributions determined by the PDPA for experiments carried out on 31 March 2006.
Results obtained for round two shower experiments conducted in the ‘clean room’ (not shown)
confirmed the removal rather than the production of particles below 2 µm by the shower with similar
aerosol size distributions as shown in Figure 2-13.
2.4.1.2 Car washing module
Figure 2-14 shows a typical aerosol and droplet size distribution measured during the series of car
washing module experiments carried out in the ‘clean room’. Size modes in the aerosol/droplet
distributions shown have been fitted with representative log-normal distribution functions. These
results show typical aerosol and droplet size distributions during car washing experiments to have the
following characteristics:
•
The aerosol distributions (derived from the APS and SMPS) show a relatively consistent
decrease in concentration with increasing particle size, from around 100-200 nm diameter
•
Accumulation and coarse modes are present in the aerosol
•
A spray can also be observed in the PDPA data
•
A cloud mode is absent.
32
10000
tent air
jet against glass
spray against glass
spray against metal
accumulation mode
coarse mode
spray mode
-3
concentration dN/dlogdD (number cm )
1000
100
10
1
0.1
0.01
10
100
1000
diam
10000
100000
Figure 2-14 An example of aerosol size distributions during car-washing module determined by the
SMPS and APS for hose experiments carried out on 4 August 2006 in the tent enclosure.
2.4.2 Comparison of water efficient and conventional devices
2.4.2.1 Showering module
2.4.2.1.1 Showerhead design
Table 2-8 summarises particle concentration data for the showerheads operated at 10 L/min, so that
any differences are due to showerhead design. Listed are the bin diameter range and mid-points, the
mean and standard deviation of concentration (number of aerosols or droplets per cubic cm), and the
number of replicates. Where results for paired Bastow and Interbath showerheads are bolded this
indicates that mean counts are significantly different at a 0.05 significance level based on a paired ttest. Table 2-9 summarises mean particle mass (µg/m3 + standard deviation) for each diameter bin
size range for each device operated at 10 L/min.
33
Table 2-8 Aerosol and droplet concentrations ± standard deviations (aerosols or droplets /cm3)
measured in each size bin for the showerhead experiments carried out at 10 L/min
Aerosol and droplet concentrations/cm3 (standard deviation)
according to diameter size range (mid point) µm)
Experimental
conditions
Device
#
Man
Temp
°C
0.2-1 µm
(0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
6-10 µm
(8 µm)
10-20 µm
(15 µm)
B
Y
38
0.17
(0.29)
0.90
(0.75)
1.6
(1.25)
4.5
(3.8)
4.3
(4.1)
14
(3.6)
3
I
Y
38
16
(4.9)
36
(1.06)
31
(1.8)
98
(11)
116
(26)
58
(21)
3
B
Y
42
7.6
(2.5)
23
(9.0)
22
(2.2)
92
(30)
131
(52)
55
(20)
3
I
Y
42
25
(20)
79
(48)
57
(36)
115
(77)
58
(38)
20
(9.3)
3
B
N
38
6.9
(1.47)
22
(4.2)
19
(2.2)
61
(5.1)
197
(0.05)
248
(24)
2
I
N
38
3.8
(1.28)
12
(4.5)
11
(5.5)
66
(26)
185
(84)
150
(51)
3
B
N
42
2.1
(0.20)
6.6
(1.24)
8.5
(6.3)
42
(28)
198
(96)
200
(11)
2
I
N
42
494
(113)
680
(124)
3
6.5
(2.6)
# = number of replicate experiments
I = Interbath showerhead design
Y= yes mannequin
15
13
92
(6.0)
(2.5)
(20)
B = Bastow showerhead design
Man = mannequin
N = no mannequin
Acknowledging the limitations in comparing pair-wise results for Bastow and Interbath showerheads
and the variability in replicate experiments as shown by the standard deviations, some preliminary
observations about the data overall can be made. Initial perusal of results suggests that the Interbath
showerhead design generally (except for experimental conditions without mannequin at 38°C)
produces more aerosol particles in the respirable size range (<6 µm) than the Bastow showerhead
design when both are operated at 10 L/min (Table 2-8).
When statistical tests are applied (t-test) to ascertain whether the mean number of droplets and
aerosol produced by different showerhead designs in each size bin are significantly different, results
show a significant difference in numbers of particles <6 µm diameter produced for experimental
conditions with the mannequin at 38°C, but not for any other experimental conditions.
Statistical analysis of mean particle counts >6 µm diameter (mid point 8 µm) generated by each of the
showerheads shows that the Interbath showerhead generates significantly higher numbers of particles
under experimental conditions with the mannequin at 38°C and for particles >10 µm for experimental
conditions without mannequin at 42°C. The failure to detect statistical differences between mean
counts for particles generated by each of the showerheads for diameter size bins greater than 6 µm
for other experimental conditions is notable, as is the fact that the number of particles in size bins >
6 µm for some experimental conditions was higher for the Bastow showerhead design.
Calculation of particle mass in each size bin (Table 2-9) for both showerheads shows that >90%
(>95% for all except Interbath with mannequin at 42°C) is greater than 6 µm (mid point 8 µm)
diameter.
34
Table 2-9 Aerosol mass ± standard deviations (aerosols µg/m3) measured in each size bin for the
showerhead experiments carried out at 10 L/min
Experimental
conditions
Aerosol mass µg /m3 (standard deviation) according to
diameter size range (mid point) µm)
Device
M
Temp
°C
0.2-1 µm
(0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
6-10 µm
(8 µm)
10-20 µm
(15 µm)
B
Y
38
<0.1
(<0.1)
2.9
(2.4)
23.6
(19.2)
386.5
(326.4)
2075.2
(1978.7)
44537.9
(11452.6)
3
I
Y
38
3.3
(1.0)
114.5
(3.4)
456.6
(26.5)
8417.7
(944.8)
55982.9
(12547.9)
184514.0
(66806.8)
3
B
Y
42
1.6
(0.5)
73.2
(28.6)
374.0
(32.4)
7902.3
(2576.8)
63222.1
(25095.8)
174970.1
(63625.5)
3
I
Y
42
5.1
(4.1)
251.3
(152.7)
839.5
(530.2)
9877.9
(6613.9)
27991.5
(18339.2)
63625.5
(29585.9)
3
B
N
38
1.4
(0.3)
70.0
(13.4)
279.8
(32.4)
5239.6
(438.1)
95074.4
(24.1)
788956.2
(76350.6)
2
I
N
38
0.8
(0.3)
38.2
(14.3)
162.0
(81.0)
5669.0
(2233.3)
89283.1
(40539.3)
477191.3
(162245.0)
3
B
N
42
0.43
(<0.1)
21.0
(3.9)
125.2
(92.8)
3607.6
(2405.0)
95557.0
(46330.7)
636255.0
(34994.0)
2
I
N
42
47.7
191.5
7902.3
238409.9
(19.1)
(36.8)
(1717.9) (54535.1)
M = mannequin
N = no mannequin
2163267.0
(394478.1)
3
1.3
(0.5)
Y= yes mannequin
#
2.4.2.1.2 Showerhead operating flow rates (water-efficiency)
Table 2-10 summarises data for the Bastow and Interbath showerheads operated at 13 and 7 L/min
respectively. Any observed differences in results are therefore due to showerhead design and/or the
operating flow rates, which define the ‘star’ water efficiency rating. Listed are the bin diameter range
and mid-points, the mean and standard deviation of concentration (number of aerosols or droplets per
cubic cm), and the number of replicates. Where results for paired Bastow and Interbath showerheads
are bolded this indicates that mean counts are significantly different at a 0.05 significance level based
on a paired t-test. Table 2–11 summarises mean particle mass (µg/m3 + standard deviation) for each
diameter bin size range for the Interbath showerhead operated at 7 L/min and the Bastow showerhead
operated at 13 L/min.
35
Table 2-10 Aerosol and droplet concentrations measured in size bins for the showerhead experiments
for the Interbath showerhead at 7 L/min (3 ‘star’ rating) and the Bastow showerhead at 13 L/min
(1 ‘star’ rating equivalent)
Aerosol and droplet concentrations / cm3 (standard deviation)
Experimental
conditions
Device
#
Man
Temp
°C
Flow
LPM
0.2-1 µm
(0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
6-10 µm
(8 µm)
10-20 µm
(15 µm)
B
Y
38
13
0.05
(0.11)
0.9
(1.3)
1.0
(0.9)
6.5
(0.5)
16
(5.8)
32
(5.4)
4
I
Y
38
7
4.4
(2.0)
11
(3.7)
15
(4.8)
56
(7.8)
71
(13)
32
(7.4)
3
B
Y
42
13
15
(5)
43
(13)
41
(7.0)
273
(98)
509
(161)
182
(45)
3
I
Y
42
7
28
(18)
85
(53)
73
(52)
151
(84)
90
(13)
30
(3.2)
3
B
N
38
13
6.7
(2.2)
15
(4.9)
16
(5.3)
88
(18)
439
(144)
591
(206)
4
I
N
38
7
8.5
(5.6)
17
(11)
14
(7.1)
38
(12)
49
(5.6)
42
(21)
2
B
N
42
13
12
(2.1)
31
(3.6)
24
(2.2)
147
(25)
505
(96)
344
(56)
3
I
N
42
7
20
7
20
66
(4.6)
(1.1)
(2.8)
(9.5)
Y= mannequin
N = no mannequin
222
(45)
273
(45)
3
Pair-wise analysis of particle concentrations for each of the size bins for Interbath and Bastow
showerheads (Table 2–10) does not show a consistent pattern indicating a greater production of
higher numbers of particles in the respirable particle diameter range for one flow rate, showerhead
design combination. Statistical analysis of results (t-test) for paired observations (7 vs 13 L/min)
replicate the observation of statistically higher numbers of particles in the < 6µm size range produced
by the Interbath showerhead (at 38°C with a mannequin), as observed at 10 L/min. However, this
observation of statistically higher numbers of respirable particles by the Interbath (7 L/min), as
compared with the Bastow (13 L/min) showerhead, is not replicated for any other experimental
condition. In fact, statistical testing (t-test) showed that for all other pair-wise comparisons showing
statistical significance the difference was in the opposite direction, with the Bastow producing
significantly higher numbers of particles in the < 6µm size range than the Interbath showerhead. Non statistically significant results showed higher particle counts in the respirable size range by the
Interbath, as compared with the Bastow showerhead, for the majority of size bins and experimental
conditions.
Results for the Bastow and Interbath showerheads operated at 13 L/min and 7 L/min respectively are
similar to those at 10 L/min in terms of the particle mass produced in different particle size diameter
bins (Table 2–11). Results show for all experimental conditions except one (Interbath, 7 L/min, no
mannequin, 42 °C) that >90% particle mass generated by the showerheads is in the diameter size
range greater than 6 µm.
36
Table 2-11 Aerosol mass ± standard deviations (aerosols µg/m3) measured in size bins for the
showerhead experiments carried out at 13 and 7 L/min.
Experimental conditions
Aerosol mass µg /m3 (standard deviation) according to
Device
M
Temp
°C
Flow
LPM
0.2-1 µm
(0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
6-10 µm
(8 µm)
10-20 µm
(15 µm)
B
Y
38
13
<0.1
(<0.1)
2.9
(4.1)
14.7
(13.3)
558.3
(43.0)
7721.8
(2799.1)
101800.8
(17178.9)
4
I
Y
38
7
0.9
(0.4)
35.0
(11.8)
220.9
(70.7)
4810.1
(670.0)
34265.4
(6274.0)
101800.8
(23541.4)
3
B
Y
42
13
3.1
(1.0)
136.8
(41.4)
603.9
(103.1)
23449.2
(8417.7)
245649.1
(77700.4)
578992.1
(143157.4)
3
I
Y
42
7
5.7
(3.7)
270.4
(168.6)
1075.2
(765.9)
12970.1
(7215.1)
43435.0
(6274.0)
95438.3
(10180.1)
3
B
N
38
13
1.4
(0.5)
47.8
(15.6)
235.7
(78.1)
7558.7
(1546.1)
211866.3
(69496.0)
1880133.5
(655342.7)
4
I
N
38
7
1.7
(1.1)
54.1
(35.0)
206.2
(104.6)
3264.0
(1030.7)
23648.0
(2702.6)
133613.6
(66806.8)
2
B
N
42
13
2.4
(0.4)
98.6
(11.5)
353.5
(32.4)
12626.5
(2147.4)
243718.7
(46330.7)
1094358.6
(178151.4)
3
I
N
42
7
1.4
63.6
294.6
5669.0 107139.7
(0.2)
(14.6)
(41.2)
(816.0) (21717.5)
M=Mannequin
Y= mannequin
N = no mannequin
868488.1
(143157.4)
3
#
2.4.2.2 Car washing module
Table 2-12 summarises data obtained during car washing module experiments for a variety of
experimental conditions. Table 2-13 summarises mean particle mass (µg/ m3 + standard deviation) for
each diameter bin size range for the garden hose with trigger nozzle and the high pressure device
against glass and metal surfaces.
Whilst a high degree of variability in aerosol concentrations in each size bin does not allow statistical
differences between experimental conditions to be detected, trends in the concentration of aerosols
generated for specific water-using combinations are evident. Results (Table 2-12) show that the high
pressure hose produced more particles less than 2 µm in diameter than the garden hose on both glass
and metal surfaces, while the garden hose produced more particles greater than 2 µm for both glass
and metal surfaces. The spray produced more large particles than the hard jet and the jet produced
more small particles than the spray for both glass and metal surfaces.
Mean results for the generated mass of aerosols (Table 2-13) show that for the garden hose with
trigger nozzle that between 59-95% aerosol mass is associated with particles of size diameter less
than 6 µm, with the jet setting giving rise to a smaller percentage (69 and 59%) than the spray setting
(79 and 95%). This contrasts with the high pressure hose device where in excess of 96% of generated
aerosol mass is associated with particles less than 6 µm. These results indicate that the use of high
pressure sprays and garden hose with trigger nozzles are worthy of future investigation with respect to
the total emission source strengths of respirable aerosols. The variability in aerosol emissions
observed for set conditions however precluded the detection of statistically significant differences in
emissions associated with device type, spray setting or the surface (glass/metal) being washed.
37
Table 2-12 Concentration of aerosols and droplets (particles/ cm3) produced during hose experiments
as determined by the SMPS, APS and concentrations of droplets as determined by the PDPA.
Experimental conditions
Device
Aerosol and droplet concentrations / cm3 (standard deviation)
Setting W/N Glass/
Metal
0.06-0.2 µm
(0.13 µm)
0.2-1 µm
(0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
#
6-10 µm 10-20 µm
(8 µm)
(15 µm)
G
J
glass
28
(39)
347
(182)
292
(80)
152
(61)
78
(30)
8.3
(7.8)
0.12
(0.21)
3
HPH
J
glass
245
(304)
1639
(442)
545
(275)
116
(51)
6.8
(4.4)
0.3
(0.3)
0.01
(0.01)
4
G
J
metal
15
(31)
-4.4
(144)
132
(38)
74
(27)
55
(19)
7.7
(5.9)
0.15
(0.30)
3
HPH
J
metal
252
(311)
2269
(830)
429
(279)
70
(38)
0.5
(0.7)
-0.1
(0.1)
0.00
(0.01)
3
G
S
glass
29
(59)
744
(449)
303
(82)
119
(47)
36
(16)
2.7
(3.1)
0.06
(0.15)
4
HPH
S
glass
150
(211)
1256
(440)
400
(211)
85
(43)
1.2
(1.2)
-0.1
(0.1)
0.00
(0.01)
3
HPH
S
N
glass
161
(247)
1537
(636)
293
(198)
44
(27)
0.3
(0.5)
-0.1
(0.1)
0.00
(0.01)
3
G
S
W
metal
25
(40)
327
(183)
319
(89)
149
(51)
25
(16)
0.3
(1.0)
0.03
(0.15)
3
HPH
S
N
metal
220
(341)
1858
(720)
405
(244)
62
(36)
0.2
(0.5)
-0.2
(0.1)
0.00
(0.01)
3
HPH
S
W
metal
130
898
(169)
(303)
G= Garden Hose
HPH = High Pressure Hose
S = Spray setting
N = Narrow Spray
# = Number of experimental replicates
247
(157)
61
0.8
(31)
(0.7)
J = Jet setting
W wide spray
0.0
(0.1)
0.00
(0.01)
3
38
Table 2-13 Mass of aerosols (µg/m3) produced during hose experiments as determined by the SMPS,
APS and PDPA.
3
Experimental conditions Aerosol mass µg /m (standard deviation) according to
Device
Setting W/N Glass/ 0.06-0.2 µm 0.2-1 µm
Metal (0.13 µm) (0.6 µm)
1-2 µm
(1.5 µm)
2-3 µm
(2.5 µm)
3-6 µm
(4.5 µm)
6-10 µm
(8 µm)
#
10-20 µm
(15 µm)
G
J
glass
0.1
(0.1)
70.6
(37.1)
928.9 2238.7 6699.8 4005.7 381.8
(254.5) (898.4) (2576.8) (3764.4) (668.1)
3
HPH
J
glass
0.5
(0.6)
333.7
(90.0)
1733.8 1708.5 584.1
144.8
(874.9) (751.1) (377.9) (144.8)
31.8
(31.8)
4
G
J
metal
<0.1
(0.1)
-0.9
(29.3)
419.9 1089.9 4724.2 3716.1 477.2
(120.9) (397.7) (1632.0) (2847.4) (954.4)
3
HPH
J
metal
0.5
(0.6)
462.0 1364.8 1031.0
(169.0) (887.6) (559.7)
G
S
glass
0.1
(0.1)
HPH
S
glass
HPH
S
N
G
42.9
(60.1)
-48.3
(48.3)
-7.5
(31.8)
3
151.5
(91.4)
965.1 1753.1 3085.3 1322.2 178.1
(260.9) (692.2) (1374.3) (1496.1) (477.2)
4
0.3
(0.4)
255.8
(89.6)
1273.6 1252.2 102.4
(671.2) (633.3) (103.1)
glass
0.3
(0.5)
312.9 932.9
654.6
(129.5) (629.9) (397.7)
S
W metal
0.1
(0.1)
66.6
(37.3)
HPH
S
N
metal
0.5
(0.7)
378.3 1288.9 916.5
(146.6) (776.2) (530.2)
21.2
(42.9)
-74.6
(48.3)
-10.3
(31.8)
3
HPH
S
W metal
0.3
(0.3)
182.8
(61.7)
72.6
(60.1)
-14.8
(48.3)
-7.8
(31.6)
3
-24.2
(48.3)
0.1
(31.8)
3
-59.5
(48.3)
-9.4
(31.8)
3
1013.5 2199.5 2163.3 157.1
102.4
(283.1) (751.1) (1374.3) (482.6) (477.2)
3
785.9
901.8
(499.5) (456.6)
G= Garden Hose
HPH = High Pressure Hose
S = Spray setting
N = Narrow Spray
# = Number of experimental replicates
39
23.6
(42.9)
J = Jet setting
W wide spray
2.5 Discussion
Inhalation of bioaerosols is a route of exposure to microbial pathogens or components that may give
rise to adverse health outcomes. Exposure to microbial components during water-using activities
depends upon the level of microbial component(s) in the water, the numbers and size distribution of
aerosols generated during the water-using activity, the duration of the activity and the
longevity/survival of the aerosolised microbe/component.
As this study was tailored to compare devices used in an Australian context, specific conditions and
parameters were assessed. For example, for the shower experiments flow rates of 7 L/min (WELS 3
star rating), 10 L/min (WELS 2 star rating) and 13 L/min (WELS 1 star rating) were used. A flow rate
as high as 13 L/min, designated the low efficiency flow rate in this study, has not been used by other
investigators.
This study also distinguishes itself in the breadth of aerosol particle sizes measured. No single
instrument is currently available to cover the wide size range of aerosol particles, hence a combination
of instruments (SMPS, APS and PDPA) was used in this series of experiments. The theoretical size
range of particles measured using these three pieces of instrumentation, as they were configured for
these experiments, was 15 nm to 250 µm. Measurement of aerosols over this size range is
exceptional and other relevant studies ((Keating et al. 1997; Xu and Weisel 2003; Cowen and Ollison
2006; Zhou et al. 2007) have only measured aerosols over a lesser size range using only one, or at
most, two different pieces of instrumentation. In addition, in all of these other studies, the
methodologies reported did not include drying of the sample air before particle measurement. Hence
the data reported in the scientific literature are comparable to the PDPA data, rather than the reported
dried aerosol data derived from the SMPS and APS measurements in this study.
Another notable difference between this study and relevant published studies conducted prior to this
series of experiments (Keating et al. 1997; Xu and Weisel 2003) is that water was not seeded with
chemical constituent(s) prior to measurement of particles produced by the shower. This difference is
pertinent as addition of chemical constituents to the water being aerosolised will provide a ‘seed
kernel’ on which water condensation and subsequent droplet formation may occur. In this study
Melbourne drinking water was used for all experiments. This is a high physico-chemical quality
drinking water. The decision to use drinking water in experiments, rather than another water type, was
made because the focus of experimentation was on conventional versus water-efficient devices, as
they are currently used. Following this series of experiments, future experimentation was anticipated
to overlay the impact of water quality (such as recycled water) onto the use of water-using devices for
the production of respirable aerosols.
Preliminary shower experiments conducted in the general laboratory area, showed high levels of
background particles present in the ambient air. Background particle levels (maximum concentration
and mode diameter) measured by both the APS and SMPS in shower experiments were observed to
vary over the course of the experimental day. Both the maximum concentration and mode diameter
showed more variation in small particle data determined with the SMPS than the large particle data
determined by the APS. As shown in Figure 2-13, the room air size distribution below 2 µm (as
determined by the APS and SMPS) showed higher concentrations than the experimental size
distributions suggesting that particles less than 2 µm in diameter are removed from the aerosol
population by the shower action.
To understand whether the net effect of particle loss and production, or simply particle loss was being
observed, second round experiments were carried out in which the room air particles were removed
from the shower area before the experiments commenced. Modified experimental conditions
comprised very low background particle concentrations, a constant aerosol size distribution between
tests and a significant reduction in the concentration of particles in ambient air across all size ranges.
The reduction, rather than production of particles below 2 µm diameter was verified under these
conditions.
Potential mechanisms for the loss of these particles below 2 µm diameter can be proposed, including
the venturi effect of the warm water flowing through the shower space resulting in drawing of the
particles down and out of the shower area and the diffusion, impaction and interception of particles to
and by the water droplets as the droplets pass the particles. Losses will be due not only to the shower
40
spray droplets but also, importantly, to any cloud/fog droplets formed. These are all processes that
occur naturally in the atmosphere in cloud and rain (Keywood et al. 2007). A simple schematic of the
processes of aerosol and droplet removal and production occurring in the shower is shown in
Figure 2–15.
O
O
O
H
H
O
H
O
O
O
H
O
H
O
O
Spray
airflow
Rain out (scrubbing)
Cloud formation
Temperature gradient
Room aerosols and ~100% RH
Source (Keywood et al. 2007)
Figure 2-15 A simple schematic of the processes of aerosol and droplet removal and production
occurring in the shower
The complexity of aerosol interactions within the shower chamber observed in this experimentation is
also in accord with those observed in work published in 2006 and thereafter (Cowen and Ollison 2006;
Zhou et al. 2007). This information however was unavailable prior to the design and execution of this
series of experiments. In accord with the processes shown diagrammatically in Figure 2-15, these
investigators have also noted the competing processes of: particle formation from evaporation of
satellite droplets; removal by air exchange; deposition and shower cone interactions. Specifically, the
following processes are described: formation of micrometer-sized satellite droplets; spray droplet
formation from the showerhead, from splashing on the mannequin and from splashing against the
shower chamber walls and floors; chimney-like convection flow within the stall created by the hot water
heating the stall air and convective stratification giving rise to the concentration of water shower
particle-rich air into the upper half of the room volume, including the breathing area (Zhou et al. 2007).
41
Of note is the observation of particle scrubbing observed in this series of experiments, not described in
the published scientific literature prior to 2006 (but has since been alluded to by others (Cowen and
Ollison 2006)). These investigators suggested that additional particle loss processes may occur during
showering relative to those occurring in the absence of shower spray. Of relevance is that these
investigators observed this particle loss with experiments performed with drinking water, parallelling
the situation in this series of experiments. Their findings also provide evidence that the phenomenon
of particle scrubbing during showering with high quality drinking water might not be replicated when
water with higher total dissolved solids content, such as recycled water, is used in experiments. Their
study of particle emissions during showering was conducted not only with residential tap water but
with the injection of salt solutions into the source water during some tests to assess the effects of total
dissolved solids on particle emission rates. Results showed that injection of salt solutions into the
source water increased particle formation rates for size fractions <10 µm, with 70-75% of the particle
mass concentration occurring in the particulate matter PM10-2.5 size range. Little apparent change
occurred above 10 µm under these conditions (Cowen and Ollison 2006). These observations imply
that it is possible that recycled water, containing a significantly higher concentration of chemical
constituents than drinking water may result in the production, rather than scrubbing, of particles during
showering due to the presence of ‘seed kernels’ providing sites for condensation. This requires further
investigation.
For the shower experiments performed both within and outside the clean room, the relevant
‘background’ aerosol size distribution was subtracted from the size distribution for each experiment to
determine the change in aerosol distribution caused by the shower and only PDPA data was able to
be employed to compare aerosol production by water-efficient and conventional devices.
Examination of available PDPA data for the different showerhead designs at 10 L/min shows that at
least 90% (and possibly greater than 95%) of total generated aerosol mass for both showerhead
designs is associated with diameter particles >6 µm. Results for the Bastow and Interbath
showerheads operated at 13 L/min (WELS 1 star rating) and 7 L/min (WELS 3 star rating),
respectively were similar to those at 10 L/min in terms of the mass of aerosol produced in different
particle size diameters. Looking at a comparison of the water efficiency of showerheads (Interbath at
7 L/min versus Bastow at 13 L/min) for particles <6 µm, results showed non-statistically significant
higher particle counts in the respirable size range by the Interbath, as compared with the Bastow
showerhead for the majority of size bins and experimental conditions. These results are difficult to
interpret and future experiments are required with more replicates to elucidate whether there is a
significant difference in the numbers of aerosols in the respirable <6 µm size range produced by these
showerhead flow rate combinations.
Whilst no conclusions are able to be drawn about the relative exposure to contaminants of primary
relevance to the inhalation route (e.g. Legionella, Mycobacteria, bacterial endotoxin) for showerheads
operated under high (WELS 3 star) and reduced (WELS 1 star) water-efficiency, results for particles
generated in the >6 µm size range by each of the showerhead flow rate combinations appear to be
more straightforward and consistent. For all experimental conditions, except with mannequin at 38°C,
the number of generated particles of >6 µm diameter was significantly higher for the Bastow
showerhead at 13 L/min as compared with the Interbath at 7 L/min. For the experimental condition
with mannequin at 38°C, for particle sizes 10 µm and above, results were equivalent. These results,
although requiring confirmation through the performance of additional experiments and more
replicates, indicate that the Bastow showerhead operated at 13 L/min potentially leads to increased
exposure levels of contaminants where the ingestion route (i.e. >6 µm particles) is important (enteric
pathogens and most chemicals) as compared with the Interbath showerhead operated at 7 L/min.
When results for the Bastow and Interbath showerheads operated at 10 L/min are overlayed onto
these results, it appears that this effect is not associated with the showerhead design but rather, the
difference in operating flow rates 13 L/min vs 7 L/min.
The conduct of car washing module experiments in the enclosed clean room had a significant impact
on particle concentration and the ability of instrumentation to measure generated aerosols and/or
water droplets. For example, results for the car washing module experiments showed the production
of particles by the device under test to exceed the rate that particle free air was entering the clean
room. Consequently, as the replacement rate of particles exceeded that of their removal from the
clean room, this required that the particle size distribution of the enclosed clean room was subtracted
from each set of experimental results. In this way the change in concentration and size distribution of
42
aerosols resulting from experimental conditions under test were able to be determined for the APS
and SMPS. Of greater significance however is the impact that the clean room environment had on
water droplet measurement during the car washing module. During the experiments, particularly the
high pressure hose experiments, a visible fog formed in the tent and persisted for several minutes
after the high pressure hose was turned off. The PDPA failed to count any droplets when the fog
formed, suggesting that the fog droplets were below the minimum (500 nm) size that the PDPA
instrument could measure. Because particles below this size were measurable with the APS and
SMPS, it is likely that the fog droplets were hydrated aerosols (Keywood et al. 2007). As a
consequence, no PDPA aerosol-sizing results were obtained for the car washing experiments.
The inability to obtain droplet counts in the clean room using PDPA has implications for the conduct of
subsequent experiments relating to the ventilation within the experimental area. Ventilation has been
variously addressed by other researchers in experiments where droplets and aerosols have been
measured. Approaches taken in relation to shower experiments have been to control ventilation using
the building’s ventilation system (Zhou et al. 2007); the use of a fan exhausting air through a 9 cm hole
in the top of the chamber (Keating et al. 1997) and use of a ceiling exhaust fan, an air vent connected
to the house heating, ventilation and air conditioning (HVAC) system and a muffin fan placed near the
floor to help with vertical mixing during test runs (Cowen and Ollison 2006). While enhanced
ventilation and removal of ambient aerosols and droplets may produce experimental results which are
more clear cut and easy to interpret, they do not reflect the real life situations in which water use
occurs. Therefore, it is recommended that future car module experiments be performed in an
unenclosed space and that the background concentration of particles is not too low so as to result in
lack of sites for water condensation and subsequent droplet formation.
Results for the APS and SMPS for the car washing module show that the high pressure hose device
almost exclusively gives rise to aerosols in the respirable range (>96%) with lower percentages (5995%) recorded for the garden hose and trigger nozzle combination. These results indicate that the use
of high pressure sprays and garden hose with trigger nozzles are worthy of future investigation with
respect to the total emission source strengths of respirable aerosols.
The variability in aerosol emissions observed for set conditions in the series of car washing
experiments precludes the detection of statistically significant differences in emissions associated with
device type, spray setting or the surface (glass/metal) being washed. As for future shower module
experiments, future car module experiments should focus on the performance of greater numbers of
replicate experiments and these are best performed with the testing of a limited number of parameter
variables (spray setting, surface being cleaned etc).
2.6 Conclusions
Study objectives to characterise the size distribution of aerosols and droplets produced by Australian
industry-approved, water-efficient devices and conventional devices and to determine whether
inhalation exposure to respirable aerosols is higher for water-efficient devices under typical usage
conditions were only partially met. Characterisation of the aerosol and water droplet size distributions
of water-efficient and conventional devices was performed for two water-using activities (showering
and car washing). However, a comparison of inhalation exposure with respirable aerosols by waterefficient and conventional devices was only able to be performed in a limited manner. This was
because of the wide variability in measured water droplet and aerosol concentrations for individual
size bins for replicate (generally 3) experiments and other methodological limitations.
In addition, findings from this suite of experiments provide data about the sub-micrometre aerosol
distribution produced during various water-using activities that are generally absent from prior studies.
This is the first study to measure particles less than 1 µm and to dry the aerosol prior to measurement.
43
The following conclusions are derived from experimental findings:
Showering
•
Complex particle generation/removal processes occur within a shower stall when the shower
is on. The operation of a shower results in the removal of aerosols from the ambient air (i.e the
falling water scrubs ambient aerosol from the shower stall) when high quality drinking water is
used
•
At least 90% of total generated aerosol mass for both showerhead designs and all flow rates
(10 L/min and 7 L/min or 13 L/min) is associated with particles with a diameter greater than
6 µm
•
Experimental results do not allow definitive conclusions to be drawn as to whether waterefficient showerheads produce significantly higher exposure to respirable aerosols (<6 µm
diameter) than conventional (lower efficiency) showerheads under typical Australian flow
conditions (WELS 3 star versus 1 star ratings)
•
Results allow a tentative hypothesis to be formulated that conventional showerheads
(13 L/min) generate more particles >6 µm diameter (the size range where the ingestion route
of enteric pathogens and most chemicals is important) than do water-efficient showerheads
(7 L/min). The implications of this, if confirmed through additional experiments, is that the
water efficient showerhead (7 L/min) may result in reduced exposure to enteric pathogens and
chemicals via ingestion compared with the conventional showerhead (13 L/min)
•
Experimental results indicate that differences in particle production between the Interbath
showerhead at 7 L/min flow rate and the Bastow showerhead at 13 L/min are most likely
primarily associated with the different operating flow rates rather than the showerhead design.
Additional experimentation is required to verify this
Car washing
•
Results for the APS and SMPS for the car washing module show that the high pressure hose
device almost exclusively gives rise to aerosols in the respirable range (>96%) with lower
percentages (59-95%) recorded for the garden hose and trigger nozzle combination. These
results indicate that the use of high pressure sprays and garden hose with trigger nozzles are
worthy of future investigation with respect to the total emission source strengths of respirable
aerosols. It is likely that there are significant differences (not shown in these experiments due
to the small number of replicates and variability in particle concentrations in size bins) in
inhalation exposure to respirable aerosols between the combination garden hose with trigger
nozzle and the high pressure device. Additional experimentation is required to verify and
quantify these differences.
2.7 Future research directions
Additional research of aerosol production during typical urban water-using activities is required to
supplement data from this and other research and to reduce the uncertainty surrounding aerosol
production estimates. This is because much of the available information is based on a limited number
of experiments. Due to the variability in particle counts between experimental runs, future experiments
should focus on the performance of greater number of replicate experiments and these are best
performed with the testing of a limited number of parameter variables (spray setting, water-using
device, temperature, surface being cleaned etc).
As part of the performance of additional experiments, future research also needs to address
methodological aspects of aerosol measurement and of the experimental set-up. From a
methodological viewpoint future investigations of aerosol production using the APS should comprise
sampling at two relative humidities so that the growth rate of the aerosol as a function of relative
humidity can be determined. In addition, where the PDPA is used, the volume of air sampled by
instrumentation should be more accurately determined either using active flow mode or an equivalent
instrument such as the Fast Forward Scattering Spectrometer Probe (FFSSP).
44
In terms of experimental environmental conditions, experiments investigating aerosol production
during showering should be carried out in a very clean room with negligible background aerosol
concentration, with continuous measurement of room air aerosol concentrations. Experimental space
ventilation should also be optimised. For evaluation of aerosol production during activities such as carwashing, future experiments, where measurements are made using the PDPA, should be carried out
in an unenclosed space to prevent the formation of fog particles. Additionally, background particle
counts should be high enough to provide sites for water condensation and subsequent droplet
formation.
From a methodological perspective, future experimentation of aerosol production using a variety of
devices during water-using activities might more easily be performed using water ‘seeded’ with
chemicals (e.g. salts). This together with the use of appropriate experimental environmental conditions
would enable aerosols produced during the water-using activity to be clearly separated from any
ambient particles. For example, water could be seeded with salt at levels typical of recycled water to
reflect aerosolisation of recycled water by water-using devices. Whilst this bypasses the question of
the impact of water-using devices on aerosol production using drinking water, it simplifies the conduct
of experiments and results interpretation and directly answers the question of the combined effect of
water-efficient devices used in combination with recycled water.
For the purposes of health risk estimation, in addition to information characterising aerosol production
during typical water-using activities, future research is required focusing on collecting time/activity data
for relevant activities. Collection of information about contemporary behaviours is important as many
parameters, such as characteristics of household appliances, housing characteristics and behaviours
vary over time. Water use data specific to a population group (e.g. by age; by occupations such as
fire-fighters or by household type such as dual reticulation households etc) are also needed.
45
3 ENDOTOXIN
The contemplated use of alternative water sources, including recycled water, to alleviate domestic
water shortages leads to questions about the public health significance of biological components
present in such waters. One such biological component is endotoxin. This section describes a
descriptive study undertaken to determine the concentration of endotoxin in a variety of Australian
water types, including recycled water.
The current relevance of endotoxin as a potential contaminant of concern in recycled water arises as a
consequence of multiple factors. Firstly, the contemplated expansion of non-drinking end uses of
recycled water within households means that health concerns associated with biological components
in water should be thoroughly investigated. Secondly, the nature of recycled water itself, including the
origin of recycled water (raw sewage) and the wastewater treatment train commonly employed to
produce recycled water leads to questions about endotoxin levels in recycled water relative to those in
drinking water.
Thirdly, reports of health issues such as respiratory symptoms, fever and tiredness by workers
exposed to airborne Gram negative enteric bacteria and endotoxin at industrial and municipal
wastewater treatment plants (Melbostad et al. 1994; Rylander 1999; Thorn et al. 2002a) add relevance
to enquiries about the health significance of bacterial endotoxin in recycled water derived from sewage
effluent. These reports are particularly pertinent when considering that the contemplated expanded
uses for which alternative waters may be employed in the urban context include those where water
aerosols are generated, such as toilet flushing, garden irrigation, irrigation of recreational playing
fields, fire fighting and humidifier use.
46
3.1 Background
3.1.1 Definition and properties
Endotoxin is a component of the lipopolysaccharide complexes that make up a part of the outer layer
of the cell wall of most Gram negative bacteria (Figure 3-1 ) and some cyanobacteria (Burger et al.
1989; Crook 1996; Anderson et al. 2003a). The term endotoxin is used to describe the toxic activities
associated with bacterial envelope components. Endotoxins are part of the structure of the bacterium
and are usually released as ‘blebs’ of outer membrane that are heavily impregnated with
lipopolysaccharide (Walker 1998). Small amounts of endotoxin are released in a soluble form
especially by young cultures. However, for the most part, endotoxin remains associated with a cell wall
until disintegration of bacteria.
http://www.horseshoecrab.org/med/med.htm
Figure 3-1 Diagram of Gram negative bacterial cell membrane showing lipopolysaccharide
component.
Lipopolysaccharide complexes are macromolecules composed of 3 main regions: Lipid A, core
polysaccharide and O antigens. The O antigen polysaccharide is exposed on the exterior of the cell,
whereas the lipid A faces the interior. These components are shown diagrammatically in Figure 3-2 .
The lipid A component is critical for all biological responses to endotoxin. The biological activity of the
endotoxin has been reported to be dependent upon the structure of lipid A and it is known that the
biological properties of lipopolysaccharide from different species of bacteria may vary qualitatively
(Jakubowski and Ericksen 1980; Park et al. 2004). Lipid A appears to be a complex array of lipid
residues rather than a single molecular structure (Anderson et al. 2002).
Responses to endotoxin can be elicited by the lipopolysaccharide fragment of Gram negative bacteria
alone or by the intact organism. However, it is not known whether the released endotoxin is a more
potent stimulus for target cells than is the bacterially bound endotoxin (Anderson et al. 2002). Also,
humans are more sensitive to the pyrogenic (fever producing) action of endotoxin than any other
tested animal (Anderson et al. 2002). Although the terms endotoxin and lipopolysaccharide are often
used synonymously, endotoxin refers to the toxin as present on the bacterial cell wall or its fragments.
Lipopolysaccharide, in contrast, implies a chemically purified endotoxin with an absence of, or with
only trace amounts of, cell wall proteins (Thorn 2001a).
47
Core
Oligosaccharide
Lipid A
•
Hydrophobic
endotoxin
•
Long chain fatty
acids
Faces interior of
cell
Highly conserved
•
•
•
Handle for
variable O
Antigen
O specific
polysaccharide
•
Structurally diverse
repeating units
•
Exposed on exterior of
cell
Figure 3-2 Diagram of lipopolysaccharide of Gram negative bacterium (adapted from Jakubowski and
Ericksen 1980)
Individual monomeric lipopolysaccharide molecules range from 0.002 to 0.02 µm but are often found
aggregated in vesicles ranging in size from 0.1 to 1.0 µm (Williams 2001 cited in Anderson et al.
2002).
3.1.2 Health effects
Symptoms of endotoxin exposure in humans are general and include fever, diarrhoea, and vomiting.
Other symptoms such as hypotension, shock, intravascular coagulation, and even death are possible
at higher dosages (Anderson et al. 2003a; Anderson et al. 2003b), with such exposures occurring
through contamination of intravenous therapeutic solutions and equipment with Gram negative
bacteria and endotoxin. In addition many occupational studies have shown positive associations
between endotoxin exposure and health effects including both reversible (asthma) and chronic airway
obstruction, respiratory symptoms and increased airway responsiveness (Douwes et al. 2003).
Endotoxin can cause profound inflammation of any tissue exposed, including lung tissue. They
provoke the release of agents called cytokines, which cause swelling and seepage from blood
vessels. Endotoxin also stimulates host cells to release proteins known as endogenous pyrogens,
which affect temperature-regulation. In order to elicit the pyrogenic response, the endotoxin must enter
the bloodstream. Entry to the bloodstream may occur via intravenous injection and dialysis using
water contaminated with endotoxin or may be associated with infections in which Gram negative
bacteria occur in the blood. Another potential mode of entry of endotoxin into the bloodstream,
associated with water usage, is via the lungs following inhalation of aerosolised water droplets.
Assuming that most humans range in weight from 1 to 140 kg and that the minimum pyrogenic dose
(to achieve a 1.9°C increase in body temp) is 0.002 µg/kg body weight, the range of minimum
endotoxin concentrations needed to induce fever would be 0.002-0.280 µg/individual (via intravenous
exposure). This information is relevant, because it indicates how much endotoxin would have to pass
into the bloodstream during haemodialysis or by inhalation of aerosolised water droplets to induce
fever (Anderson et al. 2002).
Potential endotoxin exposure routes of relevance to recycled water include exposure through
ingestion, dermal abrasions or inhalation of water vapour. Of these three exposure routes most
information is available for inhalation exposure, with multiple studies reporting an association between
inhalation of moisture-laden air and observed ill health. The health risks associated with both
48
endotoxin ingestion and entry through dermal abrasions are not well quantified and more research is
required in these areas (Douwes et al. 2003).
It is also important to note that water is not the only potential source of endotoxin. Environmental
endotoxin is ubiquitous and is detected in settled house dust and home air (Park et al. 2004). During
normal breathing each individual is exposed to at least low levels of endotoxin associated with dust
particles and ultra fine water vapour droplets (Steckel and Furkert 2004). The development of fever
after exposure to aerosol is a consistent observation in varying circumstances not involving water,
including exposure to grain dust, cotton and fibre processing, metal fumes, polymer fumes and fungal
spores (Anderson et al. 1996). Airborne endotoxin has been found in several occupational
environments such as sewage treatment plants (Kowal and Pahren 1981; Rylander 1999; Thorn and
Kerekes 2001; Thorn et al. 2002b), industrial environments (Castellan et al. 1987) and waste
processing (Thorn 2001b; Heldal et al. 2003).
3.1.2.1 Inhalation
Some investigators propose that exposure to endotoxin in raw sewage aerosols at sewage treatment
plants is a possible cause of observed ill health among exposed sewage workers (Thorn and Kerekes
2001). Of note however is that many sewage workers do not suffer ill-health effects. A possible
explanation for this is that aerosols generated are too large to be respirable and/or exposure is too low
to cause an effect (Prazmo et al. 2003). This is supported by research that has shown that workers at
sewage treatment plants are exposed to changeable quantities of endotoxin depending upon the type
and capacity of the facility, performed activities/locality within the plant and weather conditions
(Lundholm and Rylander 1983; Melbostad et al. 1994; Rylander 1999). For example, one study
reported that inhaled bacterial endotoxin was responsible for general symptoms such as fatigue and
diarrhoea in sewage plant workers. They also reported that atmospheric endotoxin levels in the
treatment site ranged between 2 and 32,000 ng/m3 and at the control sites, levels were between 3 and
39 ng/m3 air (Rylander 1999).
Inhalation fever after exposure to aerosols containing endotoxin has also been reported (Muittari et al.
1980). An outbreak of chronic hypersensitivity pneumonitis has been linked to recurrent exposure to
bacterial endotoxin in aerosolised swimming pool water (Rose et al. 1998). In addition, work-related
inhalational fever has been reported associated with exposure to endotoxin-contaminated aerosol
generated from biologically contaminated water (Anderson et al. 1996). Of note is that all reported
incidents associated with inhalation of moisture-laden air containing endotoxin also report bacterial
contamination and/or the presence of high levels of aqueous endotoxin and an effective delivery
vector (Anderson et al. 2007).
In humans there is considerable variability between individuals in the amplitude of both the clinical and
inflammatory responses to purified lipopolysaccharide by inhalation or by intravenous administration
(Michel et al. 2001). Controlled human exposure trials have established that a dose-response
relationship exists between the inhalation of purified endotoxin compounds and health effects (Michel
et al. 1997). For example, a dose of as little as 0.5 µg of lipopolysaccharide (LPS) from E. coli in a
water solution delivered via a dosimeter (which produces a calibrated aerosol) induced a significant
change in blood inflammatory markers.
A systematic and comprehensive review of water-associated endotoxin, its sources, and potential
3
health effects (Anderson et al. 2002) has reported that inhaled endotoxin doses as low as 9ng/m of
air can cause a drop in forced expiratory volume (FEV1). In addition, these authors cite research that
suggests that an airborne endotoxin concentration as low as 100-200 ng/m3 of air may be the critical
level for a decrease in lung function. They also caution that care must be exercised when
extrapolating data from studies related to inhalation of particles other than aerosolised water droplets
(in particular, cotton dust). Whilst it is reasonable to assume that a similar relationship would exist
between endotoxin in aerosolised water and absorption in the lungs as for other endotoxin sources
and lung absorption, quantification of absorption of endotoxin associated with aerosolised water is
more complex. This is due to the changing size distribution of the droplets (which must be in a specific
range to be absorbed), rates of droplet per aerosol production (mass of droplet per unit of time per
mass of water), and other activity-related parameters (e.g. flux of water, aperture size of water-using
device, etc).
49
Information about the endotoxin dose response relationship can also be gleaned from reported water
associated endotoxin incidents. Fever was observed in subjects who had received a calculated
inhaled dose of 0.01-0.03 µg of endotoxin per kg of body weight from contaminated drinking water in
Finland (assuming 50% retention of endotoxin). Endotoxin content in the contaminated water system
ranged from 0.2 to 10 µg/mL. Following the outbreak, four of the previously affected subjects were
given 2 x 2 mL (inhalation challenge) of contaminated tap water and within four hours developed a fall
in single breath lung diffusion capacity and experienced fever with coughing and shortness of breath
(Muittari et al. 1980).
Whilst the acute effects of endotoxin are well-documented in several inhalation experiments in man
(Michel et al. 1997; Thorn and Rylander 1998), the effects after chronic exposure are less well known.
Some have suggested that permissible endotoxin levels established in other environments should be
adopted for the wastewater environment (Mulloy 2001). For example, an 8 hour time weighted mean
(TWA) recommended exposure limit of 30 ng/m3 of air based on a 10 fold safety factor and a median
exposure level of approximately 300 ng/m3 associated with a 5% decrease in FEV1 has been
suggested (Mulloy 2001).
No regulation or guideline limits for endotoxin in air or water exist in Australia. A maximal admissible
concentration in the environmental air at the work place of 50 Endotoxin Units (EU)/m3 in the
Netherlands was briefly proposed (Douwes et al. 2003). Experimental and epidemiological studies
describing endotoxin dose response curves based on acute and chronic effects have shown no-effect
levels of 90-1,800 EU/m3 in air (Steckel and Furkert 2004). In order to relate endotoxin concentrations
in air to endotoxin levels in water, it is necessary to quantify aerosolised endotoxin densities arising
from typical water-using activities. To this end, theoretical assessment has recently been undertaken
of the potential for adverse health impacts following inhalation of endotoxin while showering and in
humidifier-treated environments (Anderson et al. 2007). These investigators reported that endotoxin
levels in water greater than 1,000 EU/mL are likely to induce symptoms such as chills and fever if
used in humidifiers. For showering even the highest predicted endotoxin concentration in air (based on
levels in drinking water of 38,000 EU/mL) were not deemed to result in health outcomes that would
cause more than temporary discomfort. For these assessments only those conditions resulting in
acute health outcomes were considered (Anderson et al. 2007).
3.1.2.2 Ingestion
While ingestion is perhaps the most obvious route of exposure via water for humans, ingestion of
endotoxin has not been conclusively demonstrated as posing a health risk (Anderson et al. 2002).
Some researchers (Schwebach et al. 1988) have proposed the ingestion route as a possible cause of
health risk associated with endotoxin in water but this is speculative and evidence for this is scant.
Investigators have estimated the amount of endogenous endotoxin produced daily in the intestine
assuming that 1 mg endotoxin can be obtained from 1011 bacteria and that one gram of faeces
contains 2 x 108 enterobacteriaceae. Based on 150 g as the total daily faecal output of an adult, it is
estimated that 300,000 ng of endogenous endotoxin is produced daily in the intestine (Jakubowski and
Ericksen 1980). This estimate is considered to be somewhat low as it only takes into consideration
bound endotoxin and not endotoxin that is released during growth or death of enterobacteriaceae.
Nonetheless it serves to highlight that high levels of endotoxin may be present in the intestine and not
give rise to adverse health effects.
Human endotoxin feeding studies are described in the literature with authors concluding that results of
such studies support the contention that little or no endotoxin is absorbed from the gastrointestinal
tract and that observed symptoms may be due to a localised reaction of toxicant within the
gastrointestinal tract (Jakubowski and Ericksen 1980). A more recent review of endotoxin literature
(Anderson et al. 2002), whilst expected to provide more information on ingestion as an exposure
route, given some 20 years between literature reviews, states that the ingestion risks of endotoxin in
drinking water remain unquantified.
A prospective cohort epidemiological study that investigated the gastrointestinal effects of employing
recycled water as an irrigation source for urban public parks implicated endotoxin as a possible cause
of gastrointestinal symptoms (Durand and Schwebach 1989). In this study, subjects active in parks
irrigated with potable water, non-potable water of wastewater origin and non-potable water of runoff
50
origin were studied. The study found that exposure to wet grass was associated with greater reporting
of gastrointestinal symptoms regardless of the type of irrigation water used. Toxic exposure to
pesticides, herbicides, fertilizers and viruses were ruled out as the operative causal factors. The
authors hypothesised that symptoms were related to exposure to endotoxin from living or dead
enterobacteriaceae that were already present on the grass, had survived sun and dryness and could
be dissolved in moisture to make them more readily available to man (Schwebach et al. 1988). To
date, no further data have been forthcoming in the scientific literature to support this hypothesis.
3.1.3 Water analysis results
The presence and concentration of endotoxin can be determined using aqueous extracts of blood cells
(amoebocytes) of the horseshoe crab (Limulus polyphemus). This test is known as the Limulus
Amoebocyte Lysate (LAL) assay. The basis of the test is that endotoxin causes an opacity and
gelation in LAL, based on an enzymatic reaction.
A number of investigators have employed the LAL for the determination of endotoxin in water and
sewage samples. Eight studies of endotoxin levels in drinking water were identified (Anderson et al.
2002). All eight studies used the LAL assay method to determine endotoxin concentrations. Closer
reading of scientific papers indicates that a variety of versions of the kit have been employed with the
earlier studies employing a gelation end point. More recent studies have employed chromogenic end
point and kinetic chromogenic methods. The kinetic chromogenic method uses the kinetics of
endotoxin activation of the LAL enzymatic cascade (measured as absorbance at 405-410nm).
In terms of the target of the LAL assay, the literature contains reports that have shown that the
Limulus lysate reacts with the lipopolysaccharide as well as the lipid A fraction of the endotoxin (Di
Luzio and Friedman 1973). Thus, the LAL assay can measure both solubilised (free) endotoxin and
endotoxin associated with cell walls (bound) of Gram negative bacteria. A centrifugation or filtration
procedure is used to separate these two components and some investigators have sought to assay
both bound and free endotoxin in water (Haas et al. 1983; Korsholm and Sogaard 1988). The
concentration of bound endotoxin is obtained by subtracting the amount of endotoxin in the
centrifuged/filtered sample (free endotoxin) from that obtained from an uncentrifuged/unfiltered sample
(total endotoxin). For example, some investigators (Korsholm and Sogaard 1988) undertook endotoxin
analysis using LAL assay and determined the level of free lipopolysaccharide by testing the
supernatant obtained after centrifuging for either 30 minutes at 6000 g or 10 minutes at 15,000 g. In a
review article, investigators Anderson and others provide further comment in relation to the
measurement of bound endotoxin and state that it is unclear whether bound endotoxin is a structural
component of the lipopolysaccharide that is solubilised by the LAL reagent or is endotoxin that has
been released and is stuck to the cell surface (Anderson et al. 2002).
In terms of the specificity of the LAL test for endotoxin from Gram negative bacteria some researchers
state that the LAL assay is very specific for the Gram negative bacterial product lipopolysaccharide
and it will not detect any other pyrogenic substance present in water (Wichmann et al. 2004). This
statement is seemingly contradicted by results of earlier studies where the LAL endotoxin
determination has been found to quantify endotoxin from algal cells as well as bacterial populations
(Sykora et al. 1980). However, whilst cyanobacteria may elicit a positive response to the LAL,
associated with a cell wall lipopolysaccharide component, Gram negative bacteria are by far the most
reactive with the LAL (Korsholm and Sogaard 1988). This discrepancy may also be accounted for by
considering more recent findings that have shown that while high endotoxin concentrations may be
detected in cyanobacterial water blooms, the increased endotoxin concentration is probably due to the
presence of Gram negative bacteria in the blooms as cyanobacteria themselves show little or no
endotoxin activity (Rapala et al. 2002).
Earlier surveys of endotoxin in air or water report endotoxin concentrations in units of weight, typically
3
as ng/m or ng/mL, however more recent surveys report endotoxin concentrations (or more accurately,
endotoxin activity) in endotoxin units (EU). In order to accurately convert weight units to endotoxin
units it is important to use the relevant conversion factor. This conversion factor depends upon the
genus of bacteria and, within species, on the specific lot or batch from which the reference toxin is
isolated (Anderson et al. 2002). Where the specific conversion factor is not known a default factor of
one nanogram = 10 EU is commonly used.
51
Table 3-1 gives typical endotoxin levels in various water types obtained using LAL testing and is
based on summary results presented by Rapala and others (Rapala et al. 2002). However, this table
now includes additional available water monitoring data from the scientific literature and some results
have been omitted. Omission of results is based upon the view of Anderson and others (Anderson et
al. 2002) that it is prudent to consider levels reported by Di Luzio and Friedman (Di Luzio and
Friedman 1973) with scepticism due to the fact that similar high endotoxin levels have not been
reported since 1974. In addition, for ease of comparison of results, all endotoxin concentrations
reported for individual studies in micro grams per litre or nano grams per millilitre have been converted
to endotoxin units per mL. Where the conversion factor from weight units to endotoxin units has been
specified in individual study reports it has been used. Otherwise, it has been assumed that one
nanogram of endotoxin standard from E. coli corresponds to 10 EU.
52
Table 3-1 Endotoxin concentrations measured in different water types as reported in the scientific
literature
Site (reference)
USA 10 water (drinking)
works in 9 states and 6
pilot advanced waste
treatment plants
(Jorgensen et al. 1976))
Drinking water
Advanced wastewater
treatment plant effluent
USA: five water
(drinking) works in PA
(Sykora et al. 1980)
Raw water
Treated water
Drinking water
USA: two water
(drinking) works in New
England (Haas et al.
1983)
Raw water
Treated water
Drinking water
Denmark: six water
(drinking) processing
works (Korsholm and
Sogaard 1988)
Distribution system
(ground water derived
water supplies)
Canada: pilot plant
(drinking water) (Huck et
al 1998 as cited in
(Anderson et al. 2002)
River water
Throughout plant
South Africa and
Namibia: two waterworks
(Burger et al. 1989)
Raw water (maturation
pond effluent)
Treated water
No. of
samples
Endotoxin
concentration
as reported
Conversion
factor
Endotoxin
concentration
EU/mL
10
<0.625-500ng/mL
12.5
<7.8-6,250
6
<0.313-1250ng/mL
62
57
41
0.3-3200 ng/mL
0.3-3.6 ng/mL
0.3-5.4 ng/mL
2.5
0.75-8000
0.75-9
0.75-13.5
Nr
Nr
Nr
8.8-12.2 µg/L
3.7-11 µg/L
4.6-11.4 µg/L
10ng/mL*
88-122
37-110
46-114
60
2-30 EU/mL
Not given
2-30
2.91 ng/mL
0.33-0.95 ng/mL
10ng/mL*
29.1
3.3-9.5
>30
~1050-1350 EU/mL
5ng/mL
~1050-1350
>36
~5-71 EU/mL
Nr
Nr
Finland: nine water
works (Rapala et al.
2002)
Raw water
12
18-434 EU/mL
Treated water
9
3-15 EU/mL
Distribution system
5
14-32 EU/mL
* default of 1ng endotoxin standard = 10 EU assumed
Nr – not recorded
53
<3.9–15,600
~5-71
Not given
18-434
3-15
14-32
Available endotoxin data in the scientific literature for raw waters (including sewage effluent) and
following water treatment processes, although limited, provides some evidence about the removal
efficiency of some water treatment processes used in the production of recycled water for urban uses.
The series of treatment processes typically used for production of recycled water have been
demonstrated to reduce both the concentration and potency of endotoxin to some extent.
Wastewater treatment generally comprises a sequence of processes comprising preliminary treatment
(grit removal); primary treatment (gravity sedimentation to remove suspended solids), secondary
treatment (biological treatment to reduce biochemical oxygen demand (BOD), and remove suspended
solids) and in some cases, tertiary treatment (biological and chemical processes to reduce
phosphorous and nitrogen) (Pillai and Ricke 2002). Following review of the scientific literature,
Anderson and others report that coagulation, flocculation and sedimentation provide substantial
removals of endotoxin, with limited data suggesting that removals may be of the order of 60-90%
(Anderson et al. 2002).
Measured endotoxin concentrations at nine different full-scale drinking water treatment plants in
Finland showed that water treatment processes, which varied in complexity, were found to remove
from 59 to 97% of the endotoxin present in the raw water. Furthermore, the flocculation-coagulationfiltration process was the most significant process in terms endotoxin reduction (Rapala et al. 2002).
These results are consistent with the general observation that highly treated recycled water showed
reduction in endotoxin levels of between 91 and 99% (Anderson et al. 2002). The overall high
reduction in endotoxin levels by wastewater treatment processes is not surprising on the basis that the
bulk biological material, including the biomass of Gram negative bacteria, present in sewage is
primarily targeted for removal in waste water treatment (Deere and Davison 2004).
Limited data exists regarding the impact of water disinfection processes on endotoxin levels but
findings are nonetheless consistent. Experimentation investigating the inactivation of endotoxin by
commonly employed disinfectants (chlorine, ozone, monochloramine and potassium permanganate)
has shown that these processes result in little or no reduction in endotoxin levels (Rapala et al. 2002;
Anderson et al. 2003b). For example, in one study ozonation only decreased endotoxin levels by 8%
and chlorination did not decrease the levels at all (Rapala et al. 2002). A comparison of water
treatment oxidants, chlorine, monochloramine and potassium permanganate in controlled bench scale
experiments found that even the highest inactivation rates were relatively slow (Anderson et al.
2003b).
These results highlight that disinfection processes may be used as a ‘finishing’ process but not as a
primary removal process for endotoxin. Researchers comment that water disinfection processes
cannot be relied upon to effectively reduce endotoxin levels in circumstances of a high challenge (5005000 EU/mL range) when considering contact times typically available (Anderson et al. 2003b). Given
that high levels of endotoxin are present in raw sewage, it is clear that chlorination in the absence of
coagulation-flocculation-filtration processes is not an adequate measure to achieve levels of endotoxin
in recycled water comparable to those found in drinking water.
A study investigating the inactivation of endotoxin by medium pressure UV lamps found that when deionised water was spiked with various concentrations of endotoxin and exposed to UV lamps that the
inactivation was proportional to the UV dose under the conditions examined (Anderson et al. 2003a).
The inactivation rate was determined to be approximately 0.55 EU/mL per mJ per square cm of
delivered irradiation (Anderson et al. 2003a). The rationale for these researchers to conduct this
evaluation was that UV irradiation is increasingly being employed to augment water treatment trains
associated with the difficulty inactivating Cryptosporidium oocysts with chemical disinfectants. Of note
is that where endotoxin concentrations are in the range 1 to 50 EU/mL, typically found in untreated
2
surface waters, UV disinfection treatment at doses of 40 -100 mJ/cm has the potential to completely
remove or substantially reduce endotoxin levels. Removal ranging from 11 to 55% is expected where
endotoxin levels are in the range 50 to 100 EU/mL. Researchers conclude that typical endotoxin
concentrations in drinking water could be completely inactivated by applying fluences of up to
500 mJ/cm2 (Anderson et al. 2003a). These fluences are well in excess of recommended levels for
water treatment (generally 50-100 mJ/cm2) and highlight in relation to recycled water that UV
irradiation at generally recommended doses may be used as a ‘finishing’ process to inactivate
endotoxin (conditional upon economic feasibility) where levels have been previously reduced to levels
in the range 1 to 50 EU/mL but not where endotoxin levels are in excess of this.
54
Biological filtration represents a particular case with respect to impact on endotoxin levels in that such
processes rely upon biomass for their functioning. As a consequence of this, endotoxin levels may
increase following these processes. In one study endotoxin levels were measured following biological
filtration for drinking water treatment (Huck et al. 1998). Raw water had higher endotoxin
concentrations than settled water (p= 0.0002), settled water had higher concentrations than water
following post-sedimentation ozone (p= 0.0049), and the non-disinfected anthracite-sand filter had
higher concentrations than the chlorinated water anthracite-sand filter (p= 0.0010). The authors
concluded that ‘routine’ drinking water treatment (including GAC or anthracite-sand filters) generally
did not produce higher endotoxin levels than those reported for “conventional” treatment processes.
However, following a 24-hr shutdown period of the biologically active filter simulation models,
endotoxin levels had increased markedly (Huck et al. 1998).
In summary, available endotoxin data relating to the treatment trains relevant to recycled water
indicates that a least one log removal of endotoxin may be achieved by processes that remove
particulates and that subsequent water treatment disinfection processes including UV irradiation and
chlorination result in minimal reduction in endotoxin levels at dosage rates and contact times
commonly available.
3.1.4 Data gaps
Review of the scientific literature points to a number of research data gaps in relation to endotoxin and
their presence in water used for urban purposes. Of particular pertinence are the research priorities
that are highlighted in the more recent (2002 and later) scientific literature.
Investigators, (Rapala et al. 2002) report that information on the occurrence of endotoxins and their
removal during drinking water treatment is so poor that no guidelines can be set. Further, they note
that most of the studies that measured endotoxin concentrations and their removal during drinking
water treatment have been conducted at a relatively small number of plants. They also highlight that
most studies have been conducted in North America at waterworks using simple water purification
processes dating back to times when there existed no uniform methods or endotoxin standard. They
conclude that studies should be directed to determine safe endotoxin levels in drinking water. Clearly,
once safe endotoxin levels are set for drinking water, the same levels can also be applied to other
water types such as recycled and alternative water sources, proposed as substitutes for drinking water
supplies.
Other researchers, who have reviewed the scientific literature make recommendations for future
research that include the following research priorities: additional endotoxin surveys to determine
typical endotoxin levels in raw water and following treatment and distribution of drinking water;
confirmation of the beneficial effects of drinking water treatment physical removal processes and the
quantification of the inhalation and ingestion risks to humans (Anderson et al. 2002).
One review of bioaerosol health effects and exposure assessment, although not addressing water
supply specifically, highlighted that more research is needed to establish better exposure assessment
tools and validate newly developed methods (Douwes et al. 2003). They note that even the more
established methods such as the LAL assay are only poorly validated. They highlight that
interpretation of exposure results is impossible without detailed information about the sampling and
analytical procedures.
3.2 Study objectives
The primary objective of this study was to perform a descriptive preliminary survey to determine the
concentration of bacterial endotoxin in a variety of Australian water types, including recycled water. A
secondary objective was to investigate whether current wastewater treatment processes lead to a
reduction of endotoxin levels similar to levels present in existing drinking water supplies.
3.3 Methods
Prior to performing the survey of endotoxin concentrations in a variety of water types, preliminary
investigations were performed to assess the impact of sample preservation on detected endotoxin
55
concentrations. These included: i) investigation of the impact on measured endotoxin concentrations
by the addition of sodium thiosulphate to sample bottles and, ii) investigation of the storage of water
samples at -80°C for extended periods (up to 22 weeks) in pyrogen-free sample bottles prior to
endotoxin analysis. Methods employed in the preliminary investigations, where they differ from those
used in the endotoxin survey, are identified in the relevant method sections.
3.3.1 Samples analysed
3.3.1.1 Preliminary methodological studies
The impact of the addition of sodium thiosulphate to sample bottles on measured endotoxin levels was
performed using 12 samples (4 x secondary effluent; 3 x drinking water, 2 x sewage impacted marine
water, 2 x surface (reservoir) water, 1 x drinking water sample seeded with endotoxin). Sodium
thiosulphate is commonly used to neutralise chlorine present in disinfected water samples.
The influence of storage conditions on the detectable endotoxin concentration was performed using a
variety of environmental samples (N=10) over a period of 22 weeks. Endotoxin levels in stored (frozen
at -80°C) samples were compared with those in never frozen samples maintained at 4°C for 24 hours.
Samples evaluated were: secondary treated sewage (4), sewage impacted marine water (2), surface
(reservoir) water (2) and drinking water (2).
3.3.1.2 Endotoxin survey
Water samples from a total of 48 different systems/sites were surveyed, with the majority of samples
analysed on more than one occasion (total number of samples analysed was 94). Samples surveyed
included: marine water receiving secondary treated effluent (6), drinking water samples (12), drinking
water reservoir samples (4), wastewater samples (11) and recycled water samples (15) for uses
ranging from irrigation to urban domestic uses (e.g. toilet flushing).
A number of means were employed to obtain samples for endotoxin monitoring. Marine (6) and
associated sewage treatment plant and discharge samples (4) were collected on three occasions at
the same time as routine microbiological samples. Likewise, drinking water reservoir samples (4) and
some drinking water samples (3) were collected as part of existing water quality monitoring programs.
Some samples, each monitored on one occasion, were part of an Australia-wide survey (8 potable
water samples and 7 recycled water samples). The remainder of samples (16) were collected
independently of any existing water quality survey and consisted of ten different systems each
sampled on a single occasion (10 samples) and two systems each sampled on three separate
occasions (6 samples).
3.3.2 Sample collection
For the evaluation of the impact of sodium thiosulphate addition to sample containers, two samples
were collected at each site. One sample was collected in sterile pyrogen-free sample containers
(50 mL) containing sodium thiosulphate solution (0.01g/mL) and the other in sterile pyrogen free
containers without sodium thiosulphate solution. Sampling was performed using aseptic technique and
samples were placed in a refrigerated container and transported to the laboratory for processing.
For sample storage evaluations and the endotoxin survey, water samples were collected in sterile
pyrogen free sample containers (50 mL) containing sodium thiosulphate solution (0.01 g/L). Sampling
was performed using aseptic technique and samples were placed in a refrigerated container and
transported to the laboratory for processing.
Free and total residual chorine readings were measured on site for chlorinated samples. In general for
the endotoxin survey, chlorine residual readings were only taken at sites monitored as part of an
existing water quality monitoring program. For preliminary methodological investigations, residual
chlorine readings were determined for all chlorinated samples.
56
3.3.3 Sample storage and transportation
3.3.3.1 Preliminary methodological studies
For the evaluation of the impact of sodium thiosulphate addition to sample containers, samples were
stored at 4°C upon reception at the laboratory. All samples were analysed for endotoxin within 48hrs
of sample collection.
For the evaluation of sample storage conditions, samples were sub-sampled into sterile pyrogen-free
containers in 10 mL aliquots upon reception at the laboratory, giving rise to multiple aliquots for each
sample. Four 10 mL aliquots of each sample were frozen at -80°C. The remaining aliquot was
maintained at 4°C prior to endotoxin analysis which was performed within 24hrs of sample collection.
This sample represented endotoxin concentration of the sample at T=0.
At a minimum of 4 weekly intervals, frozen samples were removed from storage at -80°C and thawed
at room temperature prior to analysis. Samples were thawed and analysed at 4 weeks (T=1), 8 weeks
(T=2), 15 weeks (T=3) and 22 weeks (T=4). In all instances except T=1, analysis of thawed samples
was performed within 1-2hrs of thawing. At T=1, thawed samples were maintained at 4°C for 48hrs
prior to sample analysis.
3.3.3.2 Endotoxin survey
For the endotoxin survey, samples were stored at 4°C upon reception at the laboratory. All samples
were analysed for endotoxin within 48hrs of sample collection.
3.3.4 Sample preparation
Dilution of samples was performed using pyrogen-free water containing 0.05% Tween-20 as this
medium has been reported as markedly enhancing endotoxin extraction efficiency in filtered air
samples compared with pyrogen-free water without changing the kinetics of the Limulus Amoebocyte
Lysate (LAL) test (Douwes et al. 1995).
For the preliminary methodological study (storage evaluation), at T=0, multiple dilutions of each
sample were analysed to ascertain whether there were any inhibition or enhancement effects
associated with the sample matrix. At T=1 up to T=4, only one serial dilution of the sample was
prepared. All sample analyses for the preliminary methodological study were performed in triplicate or
quadruplicate.
For the endotoxin survey, two to three serial dilutions (1:10 to 1:10000) of each sample were analysed
based on expected endotoxin concentrations. All sample analyses were performed in duplicate.
3.3.5 Endotoxin analyses
All glassware employed for endotoxin assays was rendered pyrogen-free by heating at 200-220°C for
2 – 3 hours. Other products such as tips for the micropipettors, deionised water and micropipettor
trays were purchased pyrogen-free.
Biological active endotoxin was assayed using a chromogenic LAL assay (Kinetic QCL, Cambrex,
Walkerville, MD, USA Lot 4L6500, Lot 4L0580, and Lot 4L8360). All standard curves were made by
reconstituting the endotoxin standard E. coli strain 055:B2 or 055:B5 with sterile non-pyrogenic water
containing 0.05% Tween-20. The endotoxin potency of the endotoxin from E. coli 055:B2 was 15
endotoxin units (EU)/ng and from E. coli 055:B5 was 11 endotoxin units (EU)/ng and 9 endotoxin units
(EU)/ng for lot numbers 3L5430 and 2L6370 respectively. Reagent blanks and at least a 5-point
standard curve using control standard endotoxin in duplicate or triplicate were assayed on the same
micro-titre plate in the same manner as samples. The absorbance was measured in a micro-plate
reader (Bio-Tek Instruments Inc., Winooski, Vermont ELx808IU) at 405 nm every 30 seconds for
50 minutes.
57
Endotoxin determinations were based on the maximum slope of absorbance versus time plot of each
micro-plate well compared with the standard curve. The standard curve ranged from 0.01 to
100 EU/mL. Sample concentrations were reported as endotoxin units (EU) per mL of sample and were
generally derived from the mean concentration of the highest sample dilution.
Negative controls were present in each set of samples assayed. As inhibition or enhancement of the
LAL assay may occur associated with compounds in the sample, positive product controls consisting
of spike concentrations of 1.562 -25 EU/mL were added to multiple samples, or dilutions of samples,
chosen at random to find an appropriate dilution.
3.3.6 Water quality parameters
For sample sites that constituted a part of a routine monitoring program or a special research survey
the determination of other water quality parameters was also performed. These included E. coli
(66 sites), total coliforms (18 sites), heterotrophic plate count (14 sites), electrical conductivity
(22 sites) and turbidity (10 sites).
3.3.7 Analysis
Endotoxin results were summarised according to the sample type and classes of wastewater and
recycled water. At the time the endotoxin monitoring program was devised the Australian Guidelines
for Water Recycling were not finalised (Phase 1 guidelines were released December 2006), hence
Victorian reclaimed water classifications were used to classify recycled water samples. The Classes of
reclaimed water and corresponding standards for biological treatment (EPA Victoria 2003) are given in
Appendix 4.
The following broad classifications employed in the summary and analysis of sewage treatment plant
and recycled water sample results are given in Table 3-2
Table 3-2 Classification of sewage treatment plant and recycled water samples
Class
Treatment processes included in this category
Number of
samples
(sample sites)
Primary
treatment
• Screening and sedimentation to remove solids
3 (1)
Lagoon
• Clay lined effluent lagoons
• Oxidation ditch extended aeration, maturation lagoon
6 (4)
Secondary
treatment
• Primary treatment, activated sludge to produce clarified
effluent, micro screens and chlorination
• Lagoons, activated sludge plant, maturation ponds
• Activated sludge plant
14 (6)
Class C
• Secondary treatment with additional chlorination
12 (6)
Class A
• Secondary treatment, chlorination, UV
• Secondary treatment, supplementary chlorination
8 (6)
Class A+
membrane
treatment
• Lagoon system, trickling filter, Zenon membrane,
chlorination
• Primary treatment, activated sludge to produce clarified
effluent, micro screens, chlorination and ultrafiltration
(with and without storage post ultrafiltration)
3 (3)
46 (26)
Total
58
The statistical significance of the differences in measured endotoxin levels obtained with and without
the addition of sodium thiosulphate to sample containers was analysed by Student’s t-test (Microsoft
Office 2003 Excel®).
For the analysis of endotoxin survey results, mean, median, maximum and minimum endotoxin values
were calculated (STATA™ Stata Corporation, Texas USA). In addition, the spread of results for each
type of water and class of recycled water was represented as a box plot (STATA™ Texas USA).The
middle of the box represents the median value. The end of the box represents the 25th and 75th
percentile value. The whiskers represent the 75th percentile plus 1.5 times the difference in these
percentiles and the 25th percentile minus 1.5 times the difference in these percentiles (upper and lower
adjacent values respectively). Outlier values are represented as dots. In order to allow the relative
endotoxin concentrations to be shown for all water types and classes, results were converted to log10
endotoxin concentrations.
3.4 Results
3.4.1 Preliminary methodological study
3.4.1.1 Sodium thiosulphate addition to sample containers
Comparison of endotoxin results showed no significant difference (N=12, p= 0.16) in the mean
endotoxin concentration for samples collected in sample containers with sodium thiosulphate as
compared with sample containers without sodium thiosulphate.
3.4.1.2 Sample preservation
In the sample preservation investigation free chlorine readings for chlorinated samples ranged from
0.01-0.91 mg/L. Total chlorine residuals ranged from 0.03-1.03 mg/L.
Results showed that freezing of a variety of water samples (N=10) for 4 weeks and thawing leads to
significant percentage decline (mean 44%, range 25-58%) in the concentration of detectable
endotoxin in the LAL assay when compared with the water samples stored at 4°C and analysed within
24hrs of sample collection. Results also showed that the decline in detectable endotoxin continued to
occur with extended storage beyond 4 weeks at -80°C. After 22 weeks storage at -80°C the mean
percentage recovery of detectable endotoxin was 12% (range 6%-21%) compared with the initial
endotoxin concentration.
The amount of residual endotoxin in water samples (N=10) stored at -80°C for time periods up to 22
weeks, expressed as a percentage of initial endotoxin levels in samples stored at 4°C and analysed
within 24hrs, is represented diagrammatically in Figure 3-3.
59
Endotoxin concentration EU/mL
90%
70%
56%
50%
23%
30%
21%
12%
0weeks
4weeks
8weeks
15 weeks
22weeks
10%
Residual endotoxin %
110%
100%
-10%
Storage time
Figure 3-3 Graph showing the effect of storage up to 22 weeks of 10 water samples in pyrogen-free
containers at -80°C on mean detectable endotoxin in the LAL assay expressed as a percentage of
initial detectable endotoxin concentration in samples stored at 4°C and analysed within 24 hours (0
weeks), standard error bars shown
3.4.2 Survey results
3.4.2.1 Sample types
Mean, median, minimum and maximum endotoxin concentrations for the different sample types
analysed in the survey are presented in Table 3–3. Highest mean and median endotoxin levels were
obtained for the sample type ‘sewage treatment plant’ with a two log10 range in counts. The lowest
mean and median concentrations were obtained for potable water and storage reservoir (potable)
water samples. Of note for the recycled water samples is the difference between the mean and
median values. The 3-4 log10 range in endotoxin concentrations reflects a high degree of variability for
this sample type.
There were 2 samples where measured endotoxin levels were below the level of detection of the
method. For data manipulation purposes, the detection limit for these samples was used in the
calculation of mean, median, minimum and maximum values.
The spread of counts for each water type is seen visually in the box plots presented in Figure 3-4. This
figure shows that the greatest spread of results was obtained for recycled water samples. A relatively
large, but reduced spread of results, was also observed for sewage treatment plant samples and for
marine samples. Of note are the two outlier results shown as ‘dots’ on the box plots. The outlier result
for potable water samples pertains to the two potable water sample derived from a bore supply.
Drinking water samples derived from bore (groundwater) supplies (N=2) gave rise to endotoxin values
below the level of detection (<4 EU/mL). The outlier result for sewage treatment plant (STP) samples
pertains to the only primary treated effluent sample (all other samples in this group were at least
lagoon/secondary treated).
60
Table 3–3 Mean, median, minimum and maximum endotoxin concentration according to water type
No.
observations
Mean
Median
Minimum
Maximum
Marine
18
961
362
124
4,258
Potable
18
66
69
4
119
Recycled
15
4,724
347
7
20,149
Water type
Reservoir
12
62
58
43
83
Sewage
treatment
plant
27
19,867
6,290
1,674
182,550
Total
90
1
Log10 Endotoxin EU/mL
2
3
4
5
Endotoxin concentrations
Marine
Potable
Recycled
Reservoir
STP
Figure 3-4 Box plots showing endotoxin concentration according to water type
Results for sewage-impacted marine samples are shown graphically in Figure 3-5. Endotoxin counts
have not been log10 transformed for this presentation of data as it was possible to represent all sample
types in the one figure. Sample sites 1 and 5, closest to the treated sewage outfall recorded the
highest endotoxin concentrations. Sites 2, 3, 4 and 6, further from the discharge site and located
closer to the shoreline, not unexpectedly recorded lower endotoxin levels. Each site was sampled on
three occasions hence the displayed readings in Figure 3-5 represent the median, minimum and
maximum values. The greater range in endotoxin concentrations at sites 1 and 5 is not unexpected
given greater temporal variations possible at these sites associated with prevailing tidal movement.
61
0
1,000
Endotoxin EU/mL
2,000
3,000
4,000
Site1
Site 2
Site 3
Site 4
Site 5
site 6
Figure 3-5 Box plots showing endotoxin concentration for sewage impacted marine waters
Mean, median, minimum and maximum endotoxin concentrations for the different classes of sewage
treatment plant and recycled water samples are presented in Table 3–4. These results show highest
mean endotoxin concentrations for primary treated sewage and lowest concentrations for Class A
recycled water with membrane treatment.
Survey results showed 91% reduction in endotoxin concentrations between primary (mean
122,573 EU/mL, N=3) and secondary (mean 11,038 EU/mL, N=3) treated wastewater samples
sampled from the same wastewater treatment plant on 3 separate occasions. A mean value of
5,463 EU/mL (N=12) was obtained for secondary treated wastewater samples overall.
Class C recycled waters, with or without membrane treatment defined microbiologically in Victorian
regulations (refer Appendix 4), as having less than 1000 E. coli per 100 mL, recorded mean measured
endotoxin concentrations of the same magnitude as for secondary treated sewage samples (mean
9,392 EU/mL, N=11).
Class A recycled waters without membrane treatment (N=7) gave rise to a mean endotoxin
concentration of 2,031 EU/mL. For recycled water Class A samples where membrane filtration was
part of the treatment train (N=3), endotoxin concentrations were less than 80 EU/mL.
62
Table 3–4 Mean, median minimum and maximum endotoxin concentration according to category of
recycled water
Recycled
water class
Primary
treated
No.
observations
Mean
Median
Minimum
Maximum
3
122,573
130,582
54,587
182,550
Lagoon
6
9,388
8,910
303
20,149
Secondary
treated
12
5,463
4,230
1,674
14,699
Class C
11
9,392
9,569
185
19,731
Class A (no
membrane
treatment)
7
2,031
872
296
5,047
Class A with
membrane
treatment
3
41
40
7
77
Total
42
The spread of counts for each class of recycled water is shown in the box plots presented in Figure
3-6. This figure shows visually the relationship between endotoxin levels throughout the wastewater
treatment train and their relativity to endotoxin concentrations in potable water supplies. A log10 scale
is used in this representation. Results show that levels of endotoxin in Class A recycled water
produced using membrane filtration are as low as those for potable water. This is not true for Class A
waters where membrane treatment has not been employed. Results for Class C recycled water show
that some waters in this class recorded endotoxin levels higher than that for waters in the secondary
effluent class. Outlier results for both the lagoon and Class C waters are noted indicating that there is
some heterogeneity in these classes. The figure shows a one log reduction in endotoxin levels
between primary and secondary treatment of wastewater.
1
Log10 Endotoxin EU/mL
2
3
4
5
Potable
Primary
Lagoon Secondary Class C
Class A
Class A+
Figure 3-6 Box plots showing endotoxin concentration according to water type
63
3.5 Discussion
3.5.1 Methodological considerations
3.5.1.1 Sodium thiosulphate addition to sample containers
As this was the first Australian survey of a variety of water types for endotoxin, a series of preliminary
experiments were designed to investigate the impact of sample preservation and storage protocols on
detected endotoxin levels. Because the analysis of disinfected recycled and drinking waters for
endotoxin was of particular interest, the first consideration in relation to the proposed sampling
protocols for the Australian endotoxin survey was whether the addition of sodium thiosulphate to
sample containers would impact on measured endotoxin levels.
Sodium thiosulphate is added to sample containers prior to sampling, or to the water sample
immediately following sampling, to neutralise oxidants that may be present in the sampled water. As
residual chlorine is generally present in both recycled and drinking waters the addition of sodium
thiosulphate to these waters is relevant. If sodium thiosulphate is not added to water samples
containing oxidant at sampling, the oxidant will continue to act during the period between sample
collection and analysis. This means that the number of viable micro-organisms will be reduced during
transit and sample storage, as compared with the circumstance where the disinfectant is neutralised at
the time of sampling. This reduction in the numbers of viable bacteria potentially impacts on the
relative amounts of free and bound endotoxin in the water sample when analysed. While total
endotoxin was being measured in the Australian endotoxin survey, it was considered that it was
important to standardise the sampling protocol to include addition of sodium thiosulphate to water
samples at the time of sample collection. This standardisation is pertinent given varying levels of
oxidant in individual water samples and the differences in sample transportation times to the
laboratory.
While the potential impact of the addition of sodium thiosulphate to water samples for endotoxin
analysis is acknowledged in the scientific literature, there are few published data directly quantifying
the impact of sodium thiosulphate addition on measured endotoxin. For example, some investigators
(Anderson et al. 2003b), when performing experiments to investigate endotoxin inactivation by
selected drinking water treatment oxidants, added sodium thiosulphate to flasks following the specified
contact time with oxidants to destroy any remaining oxidant residual. In fact these investigators
specifically report that sodium thiosulphate was added at double the theoretical equi-molar
requirement to provide a safety factor. Work by others (Jorgensen et al. 1976; Haas et al. 1983) was
cited by these authors to support the absence of any interference of sodium thiosulphate with the LAL
assay. Although these referenced authors have inferred minimal effect of sodium thiosulphate, details
presented by them are scant (results only given for 7 samples), hence verification was considered
important.
Results obtained for this preliminary study showed that sodium thiosulphate addition to sample
container did not significantly impact on endotoxin levels in analysed water samples (N=12, p= 0.16)
and supports reports in the scientific literature. Thus, all subsequent samples for endotoxin analysis
were collected in sample containers with added sterile sodium thiosulphate.
3.5.1.2 Sample storage
The need to explore extended sample storage arose as a consequence of a number of factors. The
first of these was that some samples for endotoxin analysis were to be ‘opportunistically’ collected in
tandem with existing water quality monitoring schedules, limiting endotoxin program flexibility in terms
of sample collection dates. As monitoring dates were to be aligned with more than one existing water
quality (including interstate) programs, problems of coordination of sample receipt at the laboratory
were to be exacerbated. The other factor motivating investigation of extended storage conditions was
the underlying cost of analysis of samples for endotoxin using the LAL assay. Due to the limited shelf
life of the LAL lysate (once hydrated) and the requirement to prepare a standard curve of endotoxin
concentrations each time samples are analysed, it is preferable to batch process samples to maximise
operational (cost and labour-saving) advantages.
64
Perusal of the scientific literature showed that as an alternative to sample storage at 4°C, some
investigators have reported the freezing of water samples at -20°C upon arrival at the laboratory
(Rapala et al. 2002) or freezing at -80°C following filter sterilisation (Wichmann et al. 2004) to extend
the sample storage time prior to endotoxin analysis. However, no validation data for water sample
preservation practices has been presented in the literature.
Selection of -80°C as the water sample storage temperature was based on the availability of
laboratory equipment and reports in the literature (Laitinen 1999) of the diminishment of endotoxin
concentrations with time when extracted (aqueous) air samples (no data available for water samples)
are stored in a deep freeze (-20°C), and subsequently thawed, compared with samples stored at 4°C.
This investigator concluded that aqueous extracted samples should be stored in a refrigerator rather
than in a deep freeze (-20°C) because freezing and thawing decrease the concentration of detectable
endotoxin in the LAL assay.
Results of the preliminary study of extended storage of water samples performed prior to the
Australian endotoxin survey showed that freezing of water samples at -80°C for extended periods
(from 4 weeks up to 22 weeks) then thawing, gave rise to a significant decline in detectable endotoxin
concentration (see Figure 3-3).
Potential reasons for the decreased endotoxin activity include the denaturation of the functional
structure of the endotoxin associated with the freezing and thawing process; enhancement of specific
interactions between endotoxin and the constituents of the water sample during storage and
irreversible binding of endotoxin to the container material during storage. Irreversible binding to the
sample container is the least likely reason based on the observed continuing decline of detected
endotoxin with extended storage time; the variability in endotoxin loss between individual samples and
the use of the surfactant Tween-20 to enhance the extraction of endotoxin.
As a consequence of the observed decline in endotoxin concentration following storage, a decision
was made that water samples for endotoxin assay in the Australian survey would be stored in the
refrigerator (4°C) and analysed within 48hrs. As there are no other reports in the literature of the
impact of water sample storage at -80°C on measured endotoxin, this constitutes an area of potential
future research endeavour. It is possible that storage of water samples at -80°C for periods less than 4
weeks may not give rise to significant decreases in the concentration of detectable endotoxin in the
LAL assay.
Even though reasons for the decline in measured endotoxin following storage at -80°C were not
investigated, work performed as part of the preliminary survey served to highlight that water sample
preservation practices are an important determinant in the concentration of endotoxin detected using
the LAL endotoxin assay and a critical factor in obtaining accurate quantitation estimates. This
observation melds with the call for standardisation of endotoxin methods for air samples. In the
context of standardisation of endotoxin methods, researchers (Douwes et al. 2003) report that
international round robin testing of the draft protocol for measurement of endotoxin of the European
Standardization Organisation (CEN) in air showed good correlations between laboratories but
significant differences in absolute levels. This observation led these investigators to call for further
validation and standardisation of sampling, extraction and analytical procedures. As for analysis of air
samples for endotoxin, this same imperative applies equally to the analysis of water samples for
endotoxin. In particular, specification of sample preservation and storage of water prior to analysis, as
well as performance of analytical procedures, is required if endotoxin determinations are to be
included as part of existing or future water quality monitoring programs.
3.5.1.3 Analytical method
Preliminary investigations performed prior to the Australian endotoxin water survey also allowed the
appropriate sample dilutions for different water types to be determined. As samples may contain
compounds that interfere with the LAL assay and cause inhibition or enhancement (Rapala et al.
2002), sample dilution in pyrogen free water provides a means to overcome such effects. Fortunately
those water samples which contain highest levels of endotoxin are also generally those which are
more likely to contain higher levels of interfering substances. This means that even for those samples
requiring significant dilution to reduce sample interference with the LAL assay, endotoxin is present in
detectable levels in the higher sample dilutions. Results for the Australian endotoxin survey showed
65
that interference with the LAL assay, mostly observed for primary and secondary treated sewage
samples if present at all, was one of inhibition rather than enhancement. For such samples, the higher
sample dilution gave rise to a higher assayed endotoxin concentration than that obtained for the lower
sample dilution(s).
Prediction of sample dilutions containing detectable levels of endotoxin was largely successful,
however there were 4 samples out of 94 where endotoxin concentrations were unable to be quantified
due to insufficient dilution of samples. In part this was due to the variability in endotoxin levels for that
water type.
Of note in relation to the method employed throughout the endotoxin survey was the employment of a
broad standard endotoxin curve range (0.01 to 100 EU/mL). This broad range is a characteristic of the
kinetic chromogenic LAL procedure where, with the help of a microplate reader equipped with kinetic
software, it is possible to measure over a three log range. This is done by continuously registering the
absorbance during the assay and specifying the time to reach a specified absorbance at 405 nm for
each well in the microplate (Dunér 1993).
As expected levels of endotoxin in various water types were largely unknown prior to the survey, it
was advantageous to have an endotoxin standard over a broad range. A broad range allowed a
variety of water types, each with two serial (ten fold) dilutions and containing different levels of
endotoxin, to be assayed concurrently, with the high likelihood that the range would allow endotoxin
concentrations for all samples to be obtained from the curve. However it is entirely possible that in
future water surveys endotoxin concentration of samples will be able to be better predicted. Thus,
samples could be grouped appropriately such that they require the same dilution set with
concentrations able to be read off an endotoxin standard curve over a small range. This would allow
endotoxin analysis to be performed using end point LAL methods that do not require the use of
microplate readers and kinetic software. For end-point methods the linearity of the standard curve is
restricted to endotoxin concentrations extending over only one logarithm (Dunér 1993).
Some investigators have successfully used the end-point method in endotoxin inactivation studies but
have specifically noted that the absorbance at 410 nm is linear only in the concentration range 0.1 to
approximately 1.0 EU/mL (Anderson et al. 2003b). The use of a small range for the standard curve is
also employed in monitoring pharmaceutical products for endotoxin where the allowable levels of
endotoxin is set and products are being evaluated against this standard. If an endotoxin level is
eventually set for drinking waters (or waters used for domestic non-potable purposes), as is the case
for testing of pharmaceutical products, this simplifies testing further in that it is a simple pass/fail
determination, in which case the use of a standard curve over only one logarithm is possible.
3.5.2 Relationship between endotoxin concentration and water type
Results obtained for various water types analysed as part of the Australian survey are in accord with
the concentrations found for the same water types in the literature.
In this survey, water samples were grouped generically according to the (waste) water treatment
applied. Not unexpectedly, reservoir (drinking water) samples gave concentrations within a relatively
small concentration range. This is because samples were taken at 4 locations only, each on 3
occasions. These samples were taken from an unfiltered drinking water supply reservoir, both before
(2 samples) and immediately after chlorination (2 samples). The levels of endotoxin detected (range
43-83 EU/mL are within the ranges reported by others (Haas et al. 1983; Rapala et al. 2002).
Results for potable water distribution samples were likewise relatively consistent despite being
collected from 12 different sites throughout Australia. Drinking waters included distribution water from
unfiltered surface, filtered surface and groundwater supplies. Measured endotoxin concentrations are
also in accord with results reported elsewhere (Haas et al., 1983; Burger et al., 1989; Rapala et al.,
2002). Of particular note is the low levels of endotoxin (<4 EU/mL) in drinking water samples derived
from bore supplies. This is consistent with low endotoxin levels recorded for groundwater drinking
water supplies elsewhere (Korsholm and Sogaard 1988).
66
A 91% reduction was observed in endotoxin concentrations between primary (mean 122,573 EU/mL,
N=3) and secondary (mean 11,038 EU/mL, N=3) treated wastewater samples from the same
wastewater treatment plant, sampled on 3 separate occasions. This reduction is in accord with
removal efficiencies quoted in the literature (Anderson et al., 2002; Rapala et al; 2002). A mean value
of 5.463 EU/mL (N=12) was obtained for secondary treated wastewater samples overall, with the
range in endotoxin concentrations over almost 1 log10. Likewise, water samples in the lagoon sample
category gave a large range of endotoxin results (over almost 2 log10). As this was a preliminary study,
reasons for the persistence of elevated endotoxin concentrations (treatment train variations, treatment
reliability, plant capacity versus demand etc) in some secondary treated effluent samples, but not
others, were not investigated.
Measured endotoxin concentrations in marine water impacted by treated sewage ranged from 124 –
4258 EU/mL with measured concentrations in accord with endotoxin levels in the sewage outfall
discharge, the distance of the sample location from the discharge point and dilution effects.
Results showed mean endotoxin levels in secondary treated sewage and Class C (<1.000 E. coli
/100 mL) recycled waters to be of the same order of magnitude. As additional chlorination is often the
only treatment process separating the designation of secondary treated effluent and Class C and A
recycled waters, endotoxin survey results support findings reported in the literature that chlorination as
a water treatment process has a relatively small impact on reducing endotoxin levels (Anderson et al.,
2003). Whilst previous investigations have focused on drinking water treatment, reported findings also
apply to wastewater treatment. Anderson and co-investigators comment that if a large amount of
endotoxin (in the 500-5,000 EU/mL range) were to pass through a water treatment plant, free chlorine
and monocholoramine would be relatively ineffective in reducing endotoxin levels at distribution
residence times typically available (Anderson et al., 2003). Results for this survey showed that
endotoxin levels for secondary treated sewage were within, or in excess of, this range. Hence it is not
surprising that, if additional chlorination only is used to render secondary treated sewage water to
Class C or Class A quality, mean endotoxin levels are not significantly lower than for secondary
treated sewage.
Results obtained for Class A recycled waters produced with membrane filtration (N=3), compared to
those of Class A waters where membrane filtration was not employed (N=7), are noteworthy despite
low sample numbers. Endotoxin concentrations in samples collected post membrane filtration (mean =
41 EU/mL) were at least as low as levels obtained for drinking water samples (mean = 66 EU/mL). In
contrast, endotoxin levels in Class A waters without membrane treatment were on average at least an
order of magnitude higher (mean = 2,031 EU/mL) than for drinking waters or Class A waters produced
with membrane filtration. This difference in endotoxin concentration between Class A waters needs
further verification but supports the observation that endotoxins seem to be efficiently removed in
processes that are used to reduce particulate matter in drinking water treatment (Rapala et al., 2002).
Based on recent theoretical assessment of the potential for adverse impacts to human health
(Anderson et al. 2007), Class A waters produced without membrane treatment may contain sufficient
endotoxin to induce inflammatory symptoms such as respiratory problems, chills and fever if used as
humidifier feed water.
The observed difference in endotoxin levels between Class A recycled waters produced with and
without membrane filtration also leads to the hypothesis that endotoxin monitoring of finished recycled
waters produced using membrane technology may provide a means of assessing membrane
treatment performance. Results using the LAL assay can be available in a 2 hour time frame,
providing the laboratory has relevant equipment and expertise. Membranes used in water and
wastewater treatment are classified into micro-filters (MF); ultra-filters (UF) and nano-filters. MF
membranes are generally considered to have a pore size range of 0.1 - 0.2 µm (nominally 0.1 µm).
For UF, pore sizes generally range from 0.01 - 0.05 µm (nominally 0.01µm) or less, with the lower cutoff for a UF membrane at approximately 0.005 µm (US EPA, 2005). Given the size range of individual
monomeric lipopolysaccharide molecules from 0.002 to 0.02 µm and of aggregations in vesicles
ranging from 0.1 to 1.0 µm (Williams, 2001 cited in Anderson et al., 2002), there is potential for the
LAL assay to be used as a measure of membrane treatment efficacy.
67
3.5.3 Relationship between other water quality indicators and endotoxin
concentration
A number of investigators have sought to investigate the use of endotoxin as a surrogate for bacterial
indicators (Evans et al. 1978; Sykora et al. 1980; Haas et al. 1983). Some investigators have found a
high degree of correlation between plate counts and endotoxin (Evans et al. 1978) whereas others
have demonstrated only low correlations (Jorgensen et al. 1976; Haas et al. 1983). Others in
investigating the utility of the LAL procedure as a surrogate for indicator organisms in drinking water
came to the conclusion that as the level of association between classical bacteriological indicators
employed in water microbiology is low, it is not practical to use the LAL assay as a surrogate or rapid
measurement of these classical parameters (Haas et al. 1983).
In this study E. coli results were only available for 66 of the 94 samples analysed. Linear regression
analysis of results showed a weak correlation between endotoxin concentration and E. coli counts (R2
= 0.1803). This result is not surprising given that total endotoxin, which is inclusive of both bound and
free endotoxin present in the water sample, was being measured. Waters subjected to disinfection but
not filtration, as is the case with some recycled waters and drinking waters, will not contain viable
bacteria but will potentially still contain a large amount of endotoxin in association with bacteria
rendered non-viable by disinfection.
3.5.4 Health risk assessment: aerosolised endotoxin
As there are no exposure limits for endotoxin in drinking water, this study focused on the concentration
of endotoxin in recycled water compared with the concentration of endotoxin in drinking waters. Such
a comparison allows the potential risk associated with aerosolised endotoxin arising from the use of
recycled water in the urban and domestic context to be benchmarked against the existing risk
associated with aerosolised endotoxin arising from drinking water use. Where endotoxin levels in
recycled water are equal to, or no greater than, those in drinking water it is possible to conclude that
the risk associated with aerosolised endotoxin in the domestic and urban situation is no greater for
recycled water than for drinking water. Results of the survey indicate that this statement can clearly be
made for recycled water that has received membrane treatment.
Where endotoxin levels in recycled water are greater than those in existing drinking water supplies it is
not possible to conclude that there is no greater risk from aerosolised endotoxin associated with the
use of recycled water for domestic and urban purposes. The increased mean levels of endotoxin in
Class A (2,031 EU/mL) and Class C (9,392 EU/mL) recycled waters where membrane filtration was
not employed, compared with existing drinking water supplies (66 EU/mL), indicate a potential
increase in human health risk from inhalation of endotoxin when these waters are employed for
aerosol-generating water uses. However, this does not mean that the increase in risk is necessarily of
health significance as the risk might still be at an acceptable level.
Determination of safe endotoxin levels in drinking water requires that research is performed to quantify
the aerosolised endotoxin densities arising from water-using activities. Once this work is performed it
will be possible to relate health based endotoxin exposure limits for occupational inhalation exposures
to endotoxin concentrations in the water. This research should be supplemented with additional
endotoxin monitoring of recycled waters.
3.6 Conclusions
This study is an initial investigation into levels of endotoxin in a variety of Australian water types,
including recycled waters.
The performance of this study allowed a comparison to be made between endotoxin levels in existing
Australian drinking water supplies and in recycled waters produced using a variety of water treatment
processes. In addition, it allowed conclusions to be made regarding methodological aspects of sample
preservation and analysis of water samples for endotoxin. This is important if future surveys are to be
performed as sampling, preservation and analysis protocols are all critical in obtaining accurate
quantitation estimates.
68
Results of the study also allowed suggestions to be made relating to endotoxin monitoring of water
supplies and the potential of the use of the LAL assay as a means to assess membrane treatment
efficacy. Potential future research priorities arising from the study were also able to be formulated.
The following conclusions are derived from the Australian endotoxin survey and related preliminary
studies:
•
The addition of sodium thiosulphate to sample containers does not significantly impact on the
measured endotoxin levels in water samples
•
Freezing of water samples at -80°C in pyrogen-free containers for 4 weeks or longer, then
thawing, may lead to considerable endotoxin loss
•
Until further investigations are performed relating to extended storage of water samples, the
best way to preserve water samples is to store them at 4°C for short periods prior to endotoxin
analysis
•
Endotoxin concentration in recycled water may be reduced to levels equal to, or less than,
those in drinking water but for some recycled waters, where membrane filtration is not
practiced, higher endotoxin concentrations may persist in the finished product
•
Survey results indicate that those water treatment processes that give rise to particulate and
biomass removal, rather than oxidative disinfection processes give rise to greatest reduction in
endotoxin levels
•
Survey results showed that wastewater processes, where membrane filtration is employed
may result in the production of finished water with endotoxin concentrations similar to or lower
than those in existing drinking water supplies. This leads to the conclusion that such waters do
not give rise to an increased likelihood of adverse respiratory effects from endotoxin exposure
compared with the use of existing drinking water supplies
•
The increased mean levels of endotoxin in Class A (2,031 EU/mL) and Class C (9,392EU/mL)
recycled waters where membrane filtration was not employed, compared with existing drinking
water supplies (mean 66 EU/mL), indicate a potential increase in human health risk from
inhalation of endotoxin when these waters are employed for aerosol-generating water uses
•
Assay for endotoxin in water immediately post ultra-filtration using the LAL provides a potential
means to assess the integrity and performance of the membrane filter and a possible
substitute for existing methods of membrane filter evaluation.
3.7 Future research directions
Research is required to quantify the aerosolised endotoxin densities arising from water-using
activities so that the inhalation health risk associated with the substitution of drinking water with
recycled water for domestic and urban uses can be elucidated.
This research should be supplemented with additional endotoxin monitoring of water throughout
the recycled water production process so that the fate of endotoxins is better understood and
judgments can be made as to whether or not LAL test results can be interpreted for monitoring
treatment efficacy.
Further validation studies of sampling, sample storage and analytical procedures for endotoxin
testing of waters is required if endotoxin monitoring is to be included as part of existing or future
water quality monitoring programs and for obtaining accurate quantitation estimates of endotoxin
exposure in water for health effects determinations.
69
4 ACKNOWLEDGEMENTS
The authors wish to extend special thanks to Naomi Cooke for her technical assistance and
excellence in the conduct of the endotoxin component of this work. Thanks are also extended to Tom
Jeavons, Department of Health Science, Monash University for his instruction and provision of
equipment employed for the endotoxin assay and to Michelle Aspros at the Monash University
Frankston laboratory for her assistance.
Special thanks are extended to all Water Authority personnel that provided samples for the endotoxin
survey. Sampling and organisation of samples for the survey often required special arrangements to
be made and the cooperation and good will of many people is gratefully acknowledged. Dr Michael
Storey is thanked for provision of samples which often required him to overcome not insignificant
logistical obstacles.
Thanks are also extended to CSIRO personnel responsible for construction of experimental rigs and
for sourcing equipment and materials. CSIRO personnel, Dr Melita Keywood, Stuart Smith, Dr John
Gras and J Ward are acknowledged for their contribution to the aerosol experimental component.
70
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74
APPENDICES
Appendix 1: Major sources of biases that occur in droplet and aerosol
measurement
Aerosol
measurement
error
Original
aerosol
Description of error
•
•
Sampling
efficiency
•
•
Various portions of particles in the size range 0.001 – 100 µm may be non
detectable with a given measurement technique.
Particles 0.4-0.7 µm are generally not detectable by optical means, however by
using a condensation particle counter the particles grow to a size where they
can be detected by optical means.
As the particle enters the sampling inlet of the aerosol measuring device the
ratio of ambient air velocity to sampling velocity, the air turbulence as well as
the size shape and orientation of the inlet may affect the sampling efficiency of
the inlet.
PDPA makes a single point diagnosis and thus accurate mapping of an entire
spray can be a tedious and time consuming process (Widmann 2001).
Internal losses
•
Particle losses may occur in the channels used to transport the aerosol from
the inlet to the sensor. Particle losses may occur due to electrostatic attraction,
impaction or gravitational settling and further reduce the aerosol concentration,
generally in the upper size range. For devices measuring sub-micron particles,
diffusion may also contribute significantly to the losses. Thus, it is important to
make this connection region out of conductive material to reduce electrostatic
losses and to minimise the length of this region to reduce losses due to other
forces.
Sensor
response
•
Bias may occur with optical particle sizing instruments that depend on having
only one particle at a time in the detector view volume. If more than one
particle ‘coincide’ in the view volume the sensor only registers one particle,
possibly of a larger size (Willeke and Liu 1976 cited in Baron and Heitbrink
(2001)).
The spray outside the measurement volume can impact on measurements.
Laser beam attenuation may occur and may lead to over-counting. In short ,
Doppler burst signals are degraded and erroneously interpreted as multiple
bursts (Widmann 2001).
•
Data
processing
•
•
•
If too few particles have been sampled the displayed particle size distribution
may not reflect the true size distribution because of statistical considerations.
Because the particle volume depends on the cube of the particle size a few
large particles outweigh many small particles. Thus the presentation of particle
by count for most naturally occurring aerosol size distributions focuses on a
smaller size range than the size distribution weighted by volume or by mass.
The number of particles in the relevant size range therefore statistically limits
the accuracy of the recorded aerosol concentration indicating that a sufficient
number of particles must be collected in the size range of interest.
PDPA provides only single point measurements. Spatial variation in the flow
can only be determined by making a large number of individual point
measurements and this method can only give time mean droplet size
distributions (Glover et al. 1995).
75
Appendix 2: Summary results of research studies
Finding
(Keating and McKone 1993) cited
in (Mercer 1999) and (Wilkes
1999)
Mass median
diameter
Range: 6.3-7.7 µm (hot water)
Range: 2.5-3.1µm (cold water)
Volume mean
diameter
Range: 7.1-16.3 µm
Aerosol
concentrations
Range: 300-1200/cm3
300-14,000 µg/m3 (hot water)
Range: 20 to 100 µg/m3 (cold
water)
Aerosol mass
Respirable mass
Descriptive
(Zhou et al. 2007)
19% of the mass is in the 0 to 2.5µm
range and 81% of the mass is in the
2.5-10 µm range
• Though droplet size of the
standard nozzles was significantly
larger (1000-1500 µm) than that of
the water saving nozzle (300 µm)
there was no significant difference
in the size distribution of aerosols
produced
76
• Size was independent of flow
rate
• The particle size distribution
did not vary over the 10
minute showering time for
cold water
• The particle size
concentration in the shower
was variable for the hot
water especially within the
first 5 minute of showering
• For hot water total deposition
in the extrathoracic region
was 50% (mouth breathing)
and 86% (nose breathing) of
total deposition which ranged
from 11 to 14 mg
• Alveolar deposition was 610% for oral and 0.9% for
nasal breathing
Appendix 3: Details of individual aerosol measuring instruments and
limitations and uncertainties associated with each
Aerodynamic Particle Sizer (APS)
The APS (TSI Model 3020) is a particle time of flight spectrometer. In this instrument, particles in the
sample air are confined to the centreline of an accelerating flow by sheath air, and pass through two
laser beams, scattering light. This scattered light is collected by an elliptical mirror that focuses it onto
a solid-state photo detector. By electronically timing between the peaks of the pulses, the velocity can
be calculated for each particle. Velocity information is stored in 1024 time-of-flight bins. The APS
converts each time-of-flight measurement to an aerodynamic diameter using a polystyrene latex
sphere calibration. This particle size information is binned into 52 logarithmically-spaced channels.
Scanning Mobility Particle Size (SMPS)
The SMPS (TSI Model 3076) determines aerosol size distributions using the property of particle
electrical mobility. Particles are first introduced into a charger where they interact with bipolar ions,
resulting in a known charge distribution. They then enter the cylindrical column of the analyser, in
which a high voltage rod is housed. Depending on the flow rate and the voltage, the particles are
classified into different sizes. The classified particles then enter a condensation particle counter (TSI
Model 3010) in which they encounter an area saturated in butanol vapour. This enables the particle to
grow to a size that can be detected with a laser.
Phase Doppler Particle Analyser (PDPA)
The Phase Doppler Particle Analyser (PDPA) equipment consists of an Aerometrics Real-Time Signal
Analyser using an argon-ion laser. The laser beam is split into two beams with a small angle between
them; these are focused such that they intersect at the desired interrogation (or sampling) point. At the
intersection point they generate an interference pattern. The back-scattered light from the interference
pattern for any droplets passing through the interrogation point is measured at the transmitter unit and
the velocity component normal to the bisector of the acute angle between the beams is interpreted.
The forward scatter of the interference pattern is measured by the receiver unit and is interpreted into
droplet size. Size measurement is possible because a smaller droplet scatters light through a larger
angle. All measurements were made over a 5-minute time period, sampling at 1.25 MHz. The
3
interrogation point volume is approximately 0.25 mm .
Limitations and uncertainties associated with aerosol measurement
Each sizing method used has its own issues and limitations. The PDPA has a lower sizing capability of
500 nm, uses a passive sampling technique, and suffers from lens fogging in conditions with high
humidity and low airflow.
The APS and SMPS use active sampling, drawing air through a sampling tube, including a dryer and
thence to the measuring devices. The reported sizes of the nuclei are humidity dependent and require
adjustment back to the droplet size at ambient humidity. In addition, drawing particles through sample
tubing can result in loss of particles by impaction of large particles or by diffusion of small particles.
Impaction can occur as particles enter the sample inlet (affecting sampling efficiency) and as particles
encounter obstacles such as bends, tube joins and tube narrowing. To reduce particle loss by
impaction, the number of bends and tube reductions was limited. The efficiency of the inlet (1/4” tubing
at 5 LPM 40 cm long) can be calculated to be 90% for 5 µm particles and 98% for 1µm particles. Loss
of particles in the 3/8“ 90 degree bend at 5 LPM before entering the APS can be calculated to be less
than 5% for 5 µm particles. Loss of particles by diffusion to the sample tubing has the most significant
effect on the very small particles being sampled by the SMPS. For example, for 100 nm particles at
0.3 L/min in 1/4” tube (2 m long) loss of particles due to diffusion is < 3%, for 15 nm particles the loss
is 20%.
The APS diameter measurement is dependent on flow rate which was measured to be within 1.1% of
the ideal flow rate. The accuracy of the diameter was determined by measuring the diameter of a
standard particle mix of known particle diameter comprised of polystyrene latex spheres and was
77
found to be within 5% of the diameter of the standard. The minimum concentration of particles
detectable (mdl) was 0.001 particles/cm3 for any one size bin. This requires counting statistics of 10
particles per bin counted over a 15 minute period at 5 L/min.
The uncertainty on the particle diameter for the SMPS is dependent on the uncertainty of the flow rate
(1%) and the uncertainty of the voltage applied to the instrument (1%). Thus, the uncertainty of the
diameter by SMPS measurements is less than 5%. Measurement of polystyrene latex spheres was
found to be within 3%. The minimum concentration of particles detectable was 0.03 particles/cm3 for
any one size bin. This requires counting statistics of 15 particles per bin counted over a 15 minute
period at 0.3 L/min.
PDPA diameter measurement depends on the forward scattering of the interference fringe pattern
from the intersection point of the laser beams. A droplet passing through the intersection point results
in the interference pattern sweeping across the receiver aperture at the Doppler difference frequency
which is proportional to the drop velocity. The spatial frequency is inversely proportional to the drop
diameter. The minimum concentration of droplets detectable was 0.1 droplets/cm3 for any one size
bin. This requires counting statistics of 10 droplets per bin counted over a 15 minute period, assuming
a velocity of 0.002 m s-1 and sample area of 0.47 mm2.
78
Appendix 4: Victorian classes of reclaimed (recycled) water
Historically in Australia the degree to which recycled water is treated has often been defined in
Classes; Class A being the ‘best’ or most highly treated. The classification system differs from state to
state, and has other complications, as water in some recycling schemes is notably better than typical
Class A (GHD 2007). Victorian Class A to Class D classifications of recycled water (EPA Victoria
2003) are shown in Table 1 below.
Table 1 Classes of reclaimed water and corresponding standards for biological treatment (EPA
Victoria 2003)
Class
Water quality objectives
Treatment processes
Range of uses- uses
include all lower class
uses
A
•
•
•
•
•
<10 E. coli orgs / 100 mL
Turbidity <2 NTU
<10/5 mg/L BOD/SS
pH 6-9
1 mg/L chlorine residual
(or equivalent
disinfection)
Tertiary and pathogen
reduction with sufficient log10
reductions to achieve:
• <10 E. coli per 100 mL
• < 1 helminth per L
• < 1 protozoa per 50 L
• < 1 virus per 50 L
•
•
•
•
<100 E. coli orgs/100 mL
pH 6-9
<20/30 mg/L BOD/SS
Secondary and pathogen
reduction (including Helminth
reduction for cattle grazing)
•
•
<1000 E. coli
orgs/100 mL
<20/30 mg/L BOD/SS
pH 6-9
Secondary and pathogen
reduction (including Helminth
reduction for cattle grazing)
•
B
C
•
•
•
•
•
•
•
•
D
•
•
•
<100 E. coli orgs/100 mL
pH 6-9
<20/30 mg/L BOD/SS
Secondary
79
•
Urban non-potable uses
with uncontrolled public
access
Human food crops
consumed raw
Industrial open systems
with worker exposure
potential
Agricultural (e.g. dairy
cattle grazing)
Industrial (e.g.
washdown water)
Urban non-potable uses
with controlled public
access
Human food crops
cooked/processed
Grazing/fodder for
livestock
Industrial systems with
no potential worker
exposure
Non-food crops including
instant turf, woodlots,
flowers
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• Australian Water Quality Centre
• Australian Water Services Pty Ltd
• Brisbane City Council
• Centre for Appropriate
Technology Inc
• City West Water Limited
• CSIRO
• Curtin University of Technology
• Department of Human Services
Victoria
• Griffith University
• Melbourne Water Corporation
• Monash University
• Orica Australia Pty Ltd
• Power and Water Corporation
• Queensland Health Pathology &
Scientific Services
• RMIT University
• South Australian
Water Corporation
• South East Water Ltd
• Sydney Catchment Authority
• Sydney Water Corporation
• The University of Adelaide
• The University of
New South Wales
• The University of Queensland
• United Water International Pty Ltd
• University of South Australia
• University of Technology, Sydney
• Water Corporation
• Water Services Association
of Australia
• Yarra Valley Water Ltd
Research Report
42

CRCWQT A Series of Exposure Experiments

Transcription

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