Drinking Water Services by Analytical Services, Inc.

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	•	Drinking Water Services
	•	Drinking water capabilities

	•	LT2 Testing & Compliance
• LT2 Schedule 4 Public Water Suppliers • EPA Method 1623 • Budget Planning for LT2 (Please Call)
	•	Emerging Pathogens
	•	Giardia and Cryptosporidium Analysis
	•	Legionella
	•	Microscopic Particulate Analysis
	•	Viral Analysis
	•	Water Treatment Plant (WTP) Optimization Techniques

USEPA Method 1623

Introduction

ASI is an EPA-approved laboratory for LT2 compliance samples, for both Cryptosporidium and E. coli enumeration.

As such, we are authorized by the EPA to enter LT2 data into the Data Collection and Tracking System (DCTS) for both Cryptosporidium and E. coli.

ASI has provided EPA Method 1623 analyses for more than 200 clients since 1999 and has been providing LT2-compliant data since July 2001. ASI is currently processing nearly 300 LT2 samples per month and has successfully assisted nearly eighty clients to date in the submission of grandfathered LT2 data packages to EPA.

ASI is certified by the State of Vermont for Drinking Water Microbiology, Total Coliforms and E. coli, by ColilertT (SM 9223B). ASI is an "approved" laboratory under the LT2 Laboratory QA Program for the quantitative analysis of E. coli in source waters by Colilert/QuantiTrayT (SM9223B).

Please contact our Senior Client Services representative, Lynse Stafford (lstafford@analyticalservices.com or 800.723.4432x18), with questions or for additional information.

Our Technical Advisory concerning EPA Method 1623 is included below and may be downloaded by using this link: Download the complete technical document in pdf format

Method 1623 - General

Cryptosporidium parvum and Giardia lamblia are well-known waterborne pathogens that have caused disease outbreaks around the globe. The 1993 outbreak of cryptosporidiosis in Milwaukee and subsequent outbreaks have stimulated research regarding the occurrence and behavior of these pathogens in natural waters.¹ Substantial research efforts have been expended to identify factors related to pathogen levels in water supplies and to identify problematic aspects of traditional water treatment and handling.^1,2,3 Another research focus has been the development of improved sample collection methods and laboratory techniques for the recovery and detection of Cryptosporidium and Giardia.⁴

This technical document summarizes the development of USEPA Method 1623 and presents options for the simultaneous detection of Cryptosporidium and Giardia (Method 1622 detects only Cryptosporidium).

Methods 1622/3 were developed to address some of the limitations of the Information Collection Rule (ICR) Protozoa Method that have been identified by various researchers.^2,5,6 For improved detection and enumeration of Cryptosporidium and Giardia, Method 1623 incorporates filtration, immunomagnetic separation (IMS) and immunofluorescence assay (FA) microscopy.^6,7 Organisms are confirmed using 4,6-diamidino-2-phenylindole (DAPI) vital dye staining and differential interference contrast (DIC) microscopy.

Analytical Services, Inc. (ASI) participated in the EPA's inter-laboratory method validation studies for Method 1622 (Cryptosporidium) in 1998 and Method 1623 (Cryptosporidium and Giardia) in 1999. ASI was also 1 of 6 laboratories nationally to participate in the EPA's ICR Supplemental Survey (ICRSS). ASI is an EPA approved laboratory for analysis of Cryptosporidium (and E. coli) samples for Long Term 2 Enhanced Surface Water Treatment Rule (LT2) compliance.

Sample Collection

Source Water - The EPA developed and validated Method 1623 for source water for Public Water Suppliers (PWSs) whose sources are surface water or groundwater under the direct influence of surface water (GWUDI).

Samples for analysis by Method 1623 can be collected as bulk ten-liter (10L) "grab" samples or field filtered (10 - 50L samples). Several types of filters have been approved by the EPA, including the Filta-Max T and Filta-Max xpress filters (IDEXX Corp., Westbrook, ME), and the EnvirochekT and Envirochek HV filters (Pall Gelman Laboratory, Ann Arbor, MI). Larger sample volumes (50L) may yield more representative samples and field filtration does reduce shipping costs.

To simplify field filtration of 10L samples, ASI has developed EasyVol, a simple, volumetric system that eliminates the use of a flow totalizer, thus negating totalizer calibration and adjustment issues and improving sample volume accuracy measurement.

In areas with turbid waters, it may not always be possible to collect 10L using a single filter. For LT2 compliance, the sample volume analyzed must be at least 10L sample, or as much volume as two filters can accommodate before clogging, or 2 mL pf packed pellet volume, whichever comes first.

Finished Water - Although there are no current or pending regulations for finished water monitoring in the United States (and therefore no EPA approved procedures), Method 1623 is routinely applied to finished water. In the United Kingdom (UK), the Drinking Water Inspectorate (DWI) has approved Method 1623 for mandatory, daily 1000L finished water samples.⁸

Sampling units and supplies for both bulk and field filtering of source and finished water, instructions and support are available from ASI.

Analytical Method

Bulk ("grab") samples are concentrated in the laboratory following the filter manufacturer's instructions. When samples are filtered (concentrated) in the field, laboratory procedures begin with filter elution.

Elution of captured organisms from the filter media is improved with Method 1623 compared to previous Cryptosporidium methods. For the Envirochek and Envirochek HV filters, a wrist-action shaker is used to physically agitate the capsule after partial filling with elution fluid. This action dislodges the particulates from the surface of the membrane filter, allowing them to backflushed out of the filter capsule by decanting the elution solution, which is subsequently concentrated by centrifugation. Filta-Max and Filta-Max xpress filters are also backflushed to remove Cryptosporidium, Giardia and other particulates, however the IDEXX system uses pressurized backflushing. The Filta-Max xpress system is a rapid, automated filter elution system that dispenses calibrated volumes of elution solution under controlled pressure to capture the elution solution and associated particulates in a centrifuge bottle. The eluant is then concentrated by centrifugation.

After elution and concentration, the concentrate is then purifiedby IMS, which involves adding magnetic beads labeled with Cryptosporidium and Giardia specific antibodies to the sample concentrate and allowing the antibody-antigen reactions to bind the beads to the (oo)cysts. The sample is then magnetized, separating the (oo)cyst-magnetic bead complex from the sample debris, which is then discarded. The beads and (oo)cysts are then dissociated, the beads removed. The purified sample is then applied to a well slide, dried and stained with immunofluorescent antibody (IFA) stain and vital dyes. Examination is by fluorescence microscopy with confirmation by DAPI staining and DIC microscopy.

Method Approval and Recovery Efficiency

The EPA validates analytical methods when they have a regulatory mandate to do so. Method 1623 was developed and validated specifically for use in the Information Collection Rule Supplemental Survey (ICR-SS), and was conducted to augment the data generated from the ICR monitoring period regarding the occurrence of protozoa in source waters. Method 1623 has recently been revised and is included in the LT2 Rule by reference. The current is Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA; December 2005.⁷

Method 1623 is a performance based method and, as such, allows users to modify the method as improved technologies become available, as long as validation trials are conducted and specific performance criteria are achieved.⁷

Matrix Spike (MS) Samples

In addition to analyst requirements and process controls, Method 1623 requires preparation and analysis of a matrix spike (MS) sample in conjunction with 1 in 20 samples from each source.⁷

A MS sample is collected in parallel with the corresponding monitoring sample and submitted to the laboratory where it is spiked with known quantities of protozoa to determine the analytical recovery efficiency. MS samples are an important quality assurance / quality control (QA/QC) aspect of Method 1623, and allow an assessment of whether the water from the site contains any substances that interfere with the analysis. This is particularly important in providing confidence in negative analytical results.

MS samples are site-specific and are distinct from laboratory QA/QC, which includes positive and negative staining controls and weekly Ongoing Precision and Recovery (OPR) samples.

Ideally, matrix spike (MS) samples are exact duplicates of the corresponding field samples; samples should be collected in parallel using a "Y" plumbing arrangement, although sequential sampling is acceptable. ASI recommends collection and submission of two 10L bulk water samples to ensure that the Field Sample and Matrix Spike Samples are treated identically. However, for LT2 compliance, the EPA allows the field sample to be field filtered and the MS sample to be submitted as a bulk sample. In addition, if large volumes samples (up to 50L) are collected, the EPA allows the MS sample to be partially field filtered (the last 10L must be submitted as a bulk sample for laboratory spiking). For example, a utility can collect a 50L field filtered sample with an MS sample which is 40L field filtered plus 10L bulk water). It should be noted that the volume of the Matrix Spike sample analyzed is required to be within 10% of the volume analyzed for the associated field sample.

In the lab, an MS sample is seeded with known amounts of Giardia and Cryptosporidium (using flow cytometry counted (oo)cysts) and then analyzed. The recovered number of Giardia and Cryptosporidium are then compared to the seeded quantities, and the recovery percentage is calculated. It should be noted that while Method 1623 includes "acceptance criteria" for recovery of protozoa from MS samples, there is no regulatory requirement in LT2 to meet these recoveries. The EPA recognizes that some water matrices contain substances that will interfere with Method 1623, and have stated that MS recoveries have no effect on data acceptability for LT2 compliance.

ColorSeed^TM

Although an improvement over the preceding method(s), the Matrix Spike remains less than optimal because it is only done periodically. Intermittent MS samples only determine the recovery efficiency in the given matrix at that time, and fail to reveal variation in recovery efficiency caused by different matrices or temporal variability in water quality.¹⁰ Variability can be pronounced with surface water samples and more so with other matrices such as wastewater. Submitting a MS sample with every sample would enhance confidence in the data, but may be cost prohibitive.

Internal positive controls called ColorSeed^TM, (BioTechnology Frontiers, Pty Ltd., Sydney, Australia) are prestained Cryptosporidium and Giardia which are added to each sample (rather than to duplicate samples as in the MS approach) thus allowing an assessment of recovery efficiency for all samples vs. a 1 in 20 frequency as provided for by Method 1623. ColorSeed Giardia cysts and Cryptosporidium oocysts are labeled with a red dye such that they can be distinguished from indigenous organisms. ColorSeed (oo)cysts are gamma irradiated, flow cytometer enumerated and packaged in aliquots for single sample use. A vial of ColorSeed organisms is added to a water sample and the sample is otherwise processed in accordance with Method 1623. ColorSeed (oo)cysts behave in the same way as naturally occurring organisms in tests performed thus far.^{10, 11} Therefore, the percent recovery of labeled (oo)cysts should be an accurate estimate of the recovery efficiency of indigenous protozoa. IPCs minimize the potential for false negative results and allow managers to make more accurate estimates of actual protozoa occurrence rates and concentrations.

Note: EPA Method 1623 has been validated only for use on raw (untreated) surface water samples. Adapting the method to finished, ground or wastewater samples is a modified use of the method and is therefore not "validated". Also, please note that ColorSeed is currently not approved for LT2 compliance samples. The use of ColorSeed with samples of any matrix is considered a modification to Method 1623.