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Protozoan Method
Development Workshop
EXECUTIVE SUMMARY
October 20-22, 1997
Arlington, VA
As part of the EPA's ongoing effort to improve the methodology for Cryptosporidium
and Giardia, the Agency held a public meeting on October 20-22,
1997, in Arlington, VA, to discuss the latest research developments. The
purpose of the three-day workshop was to exchange information related
to method development and to discuss possible criteria for method performance,
within regulatory context, so that the EPA can better assess the state
of method development for Cryptosporidium and address the needs
associated with developing a method within the next two years.
Experts presented research findings that have been recently completed
or are underway in the following areas: oocysts/cysts concentration, purification,
quantification, and viability and speciation determination. In addition,
EPA gave a status report on the development and validation of Method 1622,
a new method currently being validated to improve measurement performance
for Cryptosporidium and Giardia.
Background
Cryptosporidium oocysts and Giardia cysts are ubiquitous
in the environment. The standard method for detecting these protozoa in
water samples is the indirect fluorescent antibody (IFA) procedure as
specified by the Information Collection Rule (ICR). The ICR method has
been heavily scrutinized by scientists which led them to conclude that
the method has low capture and recovery efficiencies; the results are
widely variable both within and among laboratories; it is difficult to
perform and requires a skilled microscopist; and it can determine neither
viability nor speciation of oocysts and cysts.
EPA is considering requiring systems to monitor their source water for
Giardia and Cryptosporidium to determine appropriate
levels of treatment as a future regulatory option. To establish the level
of treatment, it is necessary to be able to measure reliably the occurrence
of the organism in the source water. An improved analytical method that
meets certain acceptability criteria (e.g., acceptable recovery efficiencies,
precision, and accuracy) is a precondition for implementation of the proportional
treatment approach or the watershed-based approach (described below).
Summary
As part of the workshop, EPA described three rule options that are currently
being considered for future regulations. These include: 1) fixed treatment
approach (i.e., all systems would be required to provide
at least the same level of treatment); 2) proportional treatment approach
(i.e., level of treatment required to achieve a desired risk level would
be based on the density of Giardia and Cryptosporidium
in source water during the reasonably worst case occurrence period); and
3) watershed-based approach (i.e., systems would be required to monitor
the source water and provide a level of treatment based on a combination
of factors that indicates the level of vulnerability of the source water
to pathogen contamination). The proportional treatment approach and watershed-based
approach require source water monitoring. Therefore, an accurate and precise
analytical method is essential to these two options.
EPA also described the approach that is being considered for defining
method performance criteria. This approach is based on analysis of source
waters with low pathogen occurrence levels (typical of systems with protected
watersheds), assumed probability distributions of protozoan occurrence,
and the assumption that there would be possible ranges of acceptable misclassification
rates. Under the EPA's approach, systems could minimize the misclassification
rate if they voluntarily increased their sampling frequency above the
minimum required. Misclassification might lead to an overestimation or
an underestimation of the treatment removal presumed to be necessary to
comply with a regulation. An overestimation would impose excess compliance
cost, while an underestimation would lead to less treatment than intended
and, therefore, a lower compliance cost, higher microbial risk, and possibly
lower disinfection by-product levels.
EPA's derivation of the method performance criteria is as follows: (1)
for a 15% or less chance of misclassifying the 90th percentile by 0.25
logs, 75% or greater recovery is needed if sampling includes 18 or 24
samples (sample volume of 25 liters or 10 liters, respectively) -or- 50%
or greater recovery is acceptable with respective sample volumes of 50
liters or 25 liters; (2) for a 5% or less chance of misclassifying the
90th percentile by 0.5 logs, 25% or greater recovery is needed if sampling
includes 18 or 24 samples (sample volume of 10 liters). The desired method
performance criteria will probably be at least as good as (2) but depending
upon significant factors (i.e., national distribution of level of treatment
in place, national distribution of site-specific probability distributions
of oocyst concentrations, and other water quality factors, basis of source
water concentration levels: total oocyst count vs. viable oocyst count
vs. infectious oocyst count, and the defined acceptable risk level, 10-4
or 10-3 level) the desired method performance criteria may
approach (1).
The following include some of the method components presented by workshop
experts: hollow fiber filtration, blood cell separator, flow cytometry,
immunomagnetic separation, cell culture techniques, fluorochrome viability
techniques, molecular probes and signature lipids. All of these have varying
degrees of promise depending on site-specific water matrix factors, sensitivity
desired for analysis, and cost considerations. Of the components presented,
the Gelman capsule filter, flow cytometry, and use of fluorescent in
situ hybridization (FISH) probes to determine speciation appeared
most promising (yielding higher recovery efficiencies, low detection limits
and reasonable cost per sample) as for being available by 1999. Under
a proportional treatment technique or a watershed-based technique approach,
where source water monitoring for protozoa would be required, EPA might
allow systems to use a variety of methods that met the EPA's specified
minimum performance criteria. Depending upon the source water characteristics,
different method components may be more appropriate than others for meeting
minimum method performance criteria.
After the presentations, the workgroup discussed the feasibility of incorporating
the method components into an analytical method for Cryptosporidium.
Critical issues raised concerning the methods presented, as well as the
EPA's method performance criteria approach, were: 1) the need for subjecting
the various method components and complete methods to different water
matrices; 2) developing several methods for use with different water matrices
and defining performance criteria for each type of water matrix (level
of organic and inorganic matter, type and level of potentially interfering
microbes, etc.); 3) defining threshold detection levels for the various
method components; 4) need to develop criteria for establishing efficacy
of any antibodies used in the method; 5) need for a reliable, readily
available source of oocysts for research and to establish method performance;
6) identifying age and condition of oocysts used during method development
research; and 7) need for the EPA to explain how it intends to use viable
versus nonviable cysts/oocysts for regulatory purposes.
One workshop expert gave a presentation that demonstrated that to assure
that the development of new methods proceeds as quickly and reliably as
possible, it is essential that scientists document materials (i.e., quality
of oocysts) and methods (i.e., enumeration techniques) when performing
studies with Cryptosporidium oocysts. Such documentation of information
would allow the study to be repeated by others and allow investigators
to properly compare the accuracy and precision of the method with other
analytical methods.
The workgroup agreed that future information exchange meetings would
be beneficial. It was suggested that the workgroup reconvene to discuss
research at annual conferences such as the American Society for Microbiology
(ASM) and the American Water Works Association's Water Quality Technology
Conference (WQTC).
Next steps
There will be a meeting on Tuesday, December 16, 1997 at RESOLVE, 1255
23rd Street, Suite 275, Washington, D.C., to summarize the findings and
other information discussed during the workshop for stakeholders. EPA
will present its programmatic and regulatory needs, describe the method
performance criteria approach presented at the workshop, present available
and near-term Cryptosporidium analytical methods, and discuss
possible methods that might be appropriate for future regulations. The
proceedings from this workshop will be available by March 1998.
For further information about this workshop or future Cryptosporidium
method development meetings, contact Crystal Rodgers at (202) 260-0676,
401 M Street, SW (MC 4607), Washington, D.C. 20460, or by e-mail at rodgers.crystal@epamail.epa.gov.
To register for the December 16 stakeholder meeting, please contact Eddie
Scher of RESOLVE at (202) 965-6203 or Escher@RESOLV.org.
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