The need for improved Cryptosporidium sampling and analytical methods was recognized just as the United States Environmental Protection Agency (USEPA) Information Collection Rule (ICR) utility monitoring program was being developed (Clancy et al, 1994). In response, the USEPA set about to devise a better means of enumerating protozoa in source water. The result was USEPA Method 1622: Cryptosporidium in Water by Filtration/IMS/FA (USEPA, 1998). The original method used a 10 L sample volume, concentration of the particulate fraction on capsule filters, and immunomagnetic separation of the oocysts from other debris for microscopic examination (Clancy et al, 1999). A follow-up, Method 1623: Giardia and Cryptosporidium in Water by Filtration/IMS/FA, expanded the procedure to allow analysis of Giardia along with Cryptosporidium (USEPA, 1999). The Envirochek capsule, employing a 1 µm polyethersulfone pleated membrane, was a key component in the successful development of these methods by allowing complete capture of cysts and oocysts from the water AND a simple and efficient elution system for recovering the captured target organisms from the filter for detection. Methods 1622 and 1623 have been revised slightly since the development to improve efficiency and the latest versions are found at USEPA 2001a, b.

The USEPA conducted the Supplemental Surveys to ICR using Methods 1622 and 1623, and developed acceptance criteria for both methods (USEPA, 2001c; 2001d). The acceptance criteria for Methods 1622 and 1623 are based on the results from 293 Cryptosporidium ongoing precision and recovery (OPR) samples and 186 Giardia OPR samples analyzed by six laboratories during the Information Collection Rule Supplemental Surveys (USEPA, 2001e). The USEPA has recently proposed Methods 1622 and 1623 in the Guidelines Establishing Test Procedures for the Analysis of Pollutants; Analytical Methods for Biological Pollutants in Ambient Water (Federal Register 66:169:45811-45829), known as the Ambient Bio Rule. The USEPA plans to use Methods 1622 and 1623 in the upcoming Long Term 2 Enhanced Surface Water Treatment Rule to develop occurrence data on Cryptosporidium levels in waters used as sources for drinking water intakes. Laboratories around the world have implemented methods 1622 and 1623 successfully.

The Envirochek capsule was adopted by the USEPA for Methods 1622 and 1623 for use with 10 L sample volumes. When larger volumes were sampled, recoveries were noted to be lower (data not shown). To address the issue of high sample volume, Pall Life Sciences developed a second product, the Envirochek HV capsule. The Envirochek HV capsule is identical to the standard Envirochek capsule except that the pleated filter in the capsule is a polyester track-etch membrane material in place of polyethersulfone. Studies were undertaken to develop performance data for use of the Envirochek HV capsule for 50 L source water samples and for 1000 L finished drinking water samples. The Envirochek HV capsule has been validated for use in Method 1622 for recovery of Cryptosporidium from 50 L source water samples. The high volume finished drinking water method was developed and tested recently and will undergo USEPA validation trials in late 2002. This document presents performance data on the Envirochek HV capsule developed in independent testing laboratories.

The Envirochek HV capsule was evaluated for Tier 2 validation for use in Method 1622 in place of the standard for collecting samples volumes up to 50 L. The USEPA office of Groundwater and Drinking Water has determined that the Envirochek HV meets the criteria for methods 1622 and 1623 for the determination of Cryptosporidium. A collaborative trial using the Performance Based Measurements System or PBMS criteria developed by the USEPA (USEPA, 1996) was designed and conducted as described in the EPA Guide to Method Flexibility and Approval of EPA Water Methods (USEPA, 1997). These criteria are used in PBMS studies to determine method equivalency from the data developed by EPA in the Supplemental Surveys (USEPA, 2001e). Three laboratories received blind spike vials of live Cryptosporidium oocysts prepared by flow cytometry. Each laboratory prepared and analyzed eight samples. Five samples were reagent water—four seeded with Cryptosporidium oocysts and a fifth seeded with a blank; three samples were 50 L source water, two seeded with Cryptosporidium oocysts and the third seeded with a blank vial containing no oocysts. Laboratories did not know which vials contained oocysts nor at what level until the results were reported to the study manager. The protocol used to spike the samples was that used in the USEPA 1622 validation studies and described in Method 1622. Contents of the vials were added directly to the 50 L volume as it was being stirred, and stirring continued throughout the filtration.

The elution of the Envirochek HV capsule is similar to that of the original standard Envirochek capsule method, with the exception that following the first 5 minute shake and decant, the capsule is rotated 120°, as opposed to 90°.

After the second shake, the capsule is not decanted, but rotated an additional 120° to the 240° position. It is then given a third 5 minute shake, and the eluant is then decanted to a 250 mL sample tube. This protocol has been adopted for the standard Envirochek capsule in the most recent revision of Methods 1622 and 1623. Each sample was concentrated by centrifugation (1500 x g; 15 min.) and the supernatant aspirated to above the pellet. The pellet was vortexed and transferred to a 50 mL conical tube. The 250 mL sample tube was rinsed with phosphate buffered saline with Tween (PBST). The rinse was transferred to the conical tube containing the sample. The sample was diluted to 50 mL with PBST and concentrated by centrifugation (1500 x g; 15 min.). The supernatant was aspirated to 10 mL. Pellet material equivalent to 1.0 mL or less was vortexed and transferred to a Leighton tube containing IMS buffers. Where packed pellet volumes exceeded 1 mL, the entire pellet was analyzed. Cryptosporidium oocysts were recovered from the interfering debris using IMS and recovered oocysts were stained and enumerated using epifluorescence microscopy according to Method 1622. The validation data are presented in Table 1, and show that the Envirochek HV capsule with 50 L source water samples achieved equivalent performance to the standard Envirochek capsule with 10 L (USEPA, 2001c, d).

Table 1: IPR and MS/MSD Validation Data for Cryptosporidium Recovery Using Method 1622 and the Envirochek HV Capsule with 50 L Source Water Samples

Sample Description	Lab	Sample Turbidity (ntu)	Packed Pellet Volume (mL)	Spike Dose (#)	No. Oocysts Recovered	Background Adjusted Count	Percent Recovery	Mean % Recovery	RSD or RPD
Reagent Blank	1	< 0.1	< 0.1	0	0	NA
IPR1	1	< 0.1	< 0.1	95.9	43	43	44.9
IPR2	1	< 0.1	< 0.1	95.9	48	48	50.1
IPR3	1	< 0.1	< 0.1	95.9	43	43	44.9
IPR4	1	< 0.1	< 0.1	95.9	38	38	39.6	44.9	9.5%
Reagent Blank	2	0.05	< 0.05	0	0	NA
IPR1	2	0.05	< 0.05	95.9	78	78	81.4
IPR2	2	0.05	< 0.05	95.9	69	69	72.0
IPR3	2	0.05	< 0.05	95.9	53	53	55.3
IPR4	2	0.05	< 0.05	95.9	59	59	61.6	67.6	17.0
Reagent Blank	3	0.03	0.01	0	0	NA
IPR1	3	0.03	0.01	95.9	45	45	47.0
IPR2	3	0.03	0.01	95.9	53	53	55.3
IPR3	3	0.03	0.01	95.9	68	68	70.9
IPR4	3	0.03	0.01	95.9	66	66	68.9	60.5	18.8
IPR Acceptable Range								24-100	< 55%

Sample Description	Lab	Sample Turbidity (ntu)	Packed Pellet Volume (mL)	Spike Dose (#)	No. Oocysts Recovered	Background Adjusted Count	Percent Recovery	Mean % Recovery	RSD or RPD
Matrix Blank	1	10.6	3.0	0	0	NA
MS1	1	9.4	3.0	95.9	28	28	29.2
MS2	1	10.1	4.0	95.9	28	28	29.2	29.2	0.0%
Matrix Blank	2	2.1	0.5	0	0	NA
MS1	2	2.1	0.5	95.9	62	62	64.7
MS2	2	2.1	0.5	95.9	76	76	79.3	72.0	20.3
Matrix Blank	3	1.8	0.45	0	0	NA
MS1	3	1.8	0.45	95.9	57	57	59.5
MS2	3	1.8	0.45	95.9	51	51	53.2	56.3	11.1
Matrix Acceptable   Range								13-111	< 61%

IPR = Initial Precision and Recovery
MS/MSD = Matrix Spike and Matrix Spike Duplicate
RSD = Relative Standard Deviation
RPD = Relative Percent Difference

As part of the Tier 2 validation for Cryptosporidium, a Tier 1 validation was performed for Giardia according to the PBMS criteria for Tier 1 validation of Method 1623: Giardia and Cryptosporidium in Water by Filtration/IMS/FA. A Tier 1 validation is done with reagent water (IPR) samples only. The protocol was identical to that described for spiking and recovery in the Tier 2 validation for Method 1622 for Cryptosporidium, except that Giardia cysts were used. The data are shown in Table 2, and indicate that the Envirochek HV capsule can be used by the Tier 1 laboratories for Giardia cyst recovery as described in Method 1623. Laboratories wishing to use the Envirochek HV capsule for Methods 1622 or 1623 with 50 L source water samples must develop their own IPR and OPR data demonstrating that they can achieve acceptable data using the modification before adopting it. Once this is demonstrated, QC for ongoing precision and recovery follows.

Table 2: IPR Tier 1 Validation Data for Giardia Recovery Using Method 1623 and the Envirochek HV Capsule

Sample Description	Lab	Sample Turbidity (ntu)	Packed Pellet Volume (mL)	Spike Dose (#)	No. Oocysts Recovered	Background Adjusted Count	Percent Recovery	Mean % Recovery	RSD or RPD
Reagent Blank	1	< 0.1	< 0.1	0	0	NA
IPR1	1	< 0.1	< 0.1	101.0	27	27	26.7
IPR2	1	< 0.1	< 0.1	101.0	27	27	26.7
IPR3	1	< 0.1	< 0.1	101.0	27	27	26.7
IPR4	1	< 0.1	< 0.1	101.0	29	29	28.7	27.2	3.6%
Reagent Blank	2	0.05	< 0.05	0	0	NA
IPR1	2	0.05	< 0.05	101.0	71	71	70.3
IPR2	2	0.05	< 0.05	101.0	72	72	71.3
IPR3	2	0.05	< 0.05	101.0	67	67	66.3
IPR4	2	0.05	< 0.05	101.0	51	51	50.5	64.6	17.8
Reagent Blank	3	0.03	0.01	0	0	NA
IPR1	3	0.03	0.01	95.9	45	45	47.0
IPR2	3	0.03	0.01	95.9	53	53	55.3
IPR3	3	0.03	0.01	95.9	68	68	70.9
IPR4	3	0.03	0.01	95.9	66	66	68.9	60.5	18.8
IPR Acceptable Range								24-100	< 49%

IPR = Initial Precision and Recovery
RSD = Relative Standard Deviation
RPD = Relative Percent Difference

Background
Adapting the Envirochek HV capsule for high volume finished water samples proved to be more of a challenge and required an alteration in the buffer composition to achieve high and reproducible recoveries. While capture on the filters was complete, elution of the target organisms proved difficult and recoveries were low. When 1000 L samples were spiked with 100-500 cysts and oocysts, recoveries were often in the single digits, in stark contrast to the performance of the standard Envirochek capsule or Envirochek HV capsule in source water matrices. It was hypothesized that chemistry of finished drinking water, either from the treatment process itself, or from the distribution system, was interfering with the elution and/or separation processes. Similar isolated findings were reported by laboratories in the UK and Australia. It was also recognized that when using the standard elution procedures for these filters following filtration of a high volume (500-1000 L) of finished water, residual material remained on the filter surfaces, and that extraordinary means would be necessary to complete the elution. The answer was the inclusion of sodium hexametaphosphate in the elution buffer. Sodium hexametaphosphate has been used for many years to reduce scaling (Fireman and Reitemeier, 1944), and is the main ingredient used in household products to aid cleansing in hard water conditions. In drinking water applications, it is used as a scale inhibitor in membrane pre-treatment (Schock, 1990; Kasper, 1993) and in lime softening processes.

Research Effort
Experiments were designed to examine the effect of sodium hexametaphosphate-enhanced elution on the recovery of oocysts spiked into high volume finished water (0.1 NTU) samples. Each of the filter capsules would be spiked with ~100 oocysts at the beginning of the run, so that a worse-case scenario could be evaluated. Flow rates through each filter were set at 1 L/min; and runs were set up to be 16.6 h, or 1000 minutes; resulting in 1000 L samples. Flow decay during one series caused total volumes to be 700 L per filter (sampling was stopped when the pressure drop reached 30 psid, well before reaching the maximum published rating of 60 psid). In addition to the tap water tests, a series of capsules would be spiked and applied to a filtered water matrix to evaluate initial precision and recovery (IPR) of the method. While recoveries from these would be compared to previous data (also spiked at the beginning of sampling), control tests were run which were spiked similarly but underwent the standard elution procedure without the sodium hexametaphosphate step.

Improved Elution Using Sodium Hexametaphosphate
The method used for sodium hexametaphosphate elution was performed as described in McCuin et al, 2001. The capsules were filled with a 5% weight by volume in a deionized (DI) water solution of sodium hexametaphosphate, and placed on the shaker at the standard speed for 5 minutes. The solution was allowed to drain through the filter, so that any materials dissolved or disaggregated could exit through the filter pores, but any oocysts or other intact particles greater than 1 µm would remain in the capsule. This action was followed by a DI rinse, which was poured into the inlet of the capsule and allowed to drain through the filter membrane, continuing the purge of dissolved or sub-micron material. These preliminary steps were followed by the standard Envirochek HV capsule eluting procedure in its entirety, as described above. The samples were centrifuged and the resulting pellets analyzed by IMS as described in Method 1622.

The Envirochek HV has been approved by Drinking Water Inspectorate in the United Kingdom, to be used for the regulatory analysis for Cryptosporidium.

Sodium hexametaphosphate was shown to significantly improve performance of the Envirochek HV elution process in high volume finished drinking water samples. Table 3 shows recoveries using the standard Envirochek HV elution process with Laureth-12 as described in Method 1622. Recoveries in filtered tap water, used to create water for IPR testing, showed higher recoveries (36%) than did tap water taken from a point in the distribution system (4%). Recoveries were consistently lower than with Method 1622 for source water samples.

Table 3: Recoveries of Cryptosporidium Oocysts from Envirochek HV Capsules Using the Method 1622 Standard Envirochek HV Capsule Elution Process

Sample Volume (L)	Matrix	Number of Tests	Percent Recovery (Average)	Percent Recovery (Range)
1000	Filtered tap water (IPR matrix)	13	36	16-61
1000	Tap water	6	4	1.8-7.4

IPR = Initial Precision and Recovery
C. parvum spike doses ranged from 89 to 104 oocysts

Table 4 shows recoveries with the improved elution step employing sodium hexametaphosphate. All recoveries fall within the acceptable range for Method 1622, although the sample matrix is finished drinking water and the volume is significantly increased.

Table 4: Enhanced Recoveries of Cryptosporidium Oocysts Using Sodium Hexametaphosphate

Sample Volume (L)	Matrix	Number of Tests	Percent Recovery (Average)	Percent Recovery (Range)
700*	Tap water	3	37.7	35-41
1000	Tap water	3	45.6	37-53
1000	Tap water	6	51.3	35-68
1000	Tap water	6	69.2	59-78

IPR = Initial Precision and Recovery
C. parvum spike doses ranged from 89 to 104 oocysts
*Sampling was stopped at 30 psid, prior to the published maximum rating of 60 psid.

During the elution steps, the tap water samples that were eluted using the sodium hexametaphosphate procedure were visibly different from the standard Laureth-12 elution samples. The sodium hexametaphosphate solution that was drained out of the capsule was discolored by dissolved material. The resulting pellet volume of the standard samples were approximately 200 µl, but contained only a trace (<10 µL) when eluted with sodium hexametaphosphate. These observations indicate that significant material was dissolved or reduced to sub-micron particles and eliminated from the elute, freeing the oocysts for subsequent separation by IMS. This method variation was successfully validated using the PBMS Tier 1 validation criteria as shown in Table 5.

Table 5: IPR Tier 1 Validation Data for Cryptosporidium Recovery Using Method 1622 and the Envirochek HV Capsule for 1000 L Finished Drinking Water Samples Using the Sodium Hexametaphosphate Elution

Sample Description	Lab	Sample Turbidity (ntu)	Spike Dose (#)	No. Oocysts Recovered	Background Adjusted Count	Percent Recovery	Mean % Recovery	RSD or RPD
Reagent Blank	1	< 0.1	0	0	NA
IPR2	1	< 0.1	99.3	68	68	68.5
IPR2	1	< 0.1	99.3	58	58	58.4
IPR3	1	< 0.1	99.3	60	60	60.4
IPR4	1	< 0.1	99.3	61	61	61.4	62.2	7.0%
IPR Acceptable Range							13-143	< 67%

IPR = Initial Precision and Recovery
RSD = Relative Standard Deviation
RPD = Relative Percent Difference

The Envirochek HV capsule was developed in response to the need for an improved capsule filter that would permit filtration and recovery of Giardia cysts and Cryptosporidium oocysts from high volume source and finished water samples. The Envirochek HV capsule achieves high and reproducible recoveries of cysts and oocysts from 50 L source water samples and has been validated using the PBMS criteria demonstrating that it meets the performance criteria for Method 1623. The Envirochek HV capsule can also be used for sampling high volumes (1000 L) of finished drinking water, but the elution procedure requires a pre-treatment step using sodium hexametaphosphate to permit maximum recovery of oocysts from the sample matrix. The high volume finished drinking water method can now be used to develop meaningful data on finished waters, both for IFA analysis as described in Method 1622 or for capture and recovery of oocysts for infectivity testing by any of the cell culture/polymerase chain reaction (CC/PCR) methods currently coming into use (Rochelle et al, 1997; DiGiovanni et al, 1999).

Envirochek capsules on Pall Life Sciences Laboratory Shaker.

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2. Clancy, J.L., Z. Bukhari, R.M. McCuin, Z. Matheson, and C.R. Fricker. 1999. USEPA Method 1622. J. American Water Works Assoc., 91:9 60-68.
3. DiGiovanni, G.D., F.H. Hashemi, N.J. Shaw, F.A. Abrams, M.W. LeChevallier, and M. Abbaszadegan. 1999. Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Cell Culture-PCR. J. Appl. Environ. Microbiol. 65 (8):3427-3432.
4. McCuin, R.M., T.M. Hargy, J.E. Amburgey, and J.L. Clancy. 2001. Improving Methods for Isolation of Cryptosporidium Oocysts and Giardia Cysts from Source and Finished Waters. Proc. AWWA WQTC. Nashville, TN, Nov. 9-14, 2001.
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8. Schock, M.R. 1990. Internal Corrosion and Deposition Control. Ch. 17 in Water Quality and Treatment, A Handbook of Community Water Supplies. F. Pontius, (ed.) Am. Water Works Assn.
9. USEPA. 1996. Guidelines and Format for Methods to be Proposed at 40 CFR Part 136 or Part 141. EPA-821-B-96-003. USEPA Office of Water Engineering & Analysis Division (4303), Washington, DC.
10. USEPA. 1997. 40 CFR Parts 136 and 141. EPA Guide to Method Flexibility and Approval of EPA Water Methods, EPA Proposed Rule. Fed. Reg. 62:14975.
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12. USEPA. 1999. Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA. United States Environmental Protection Agency. Office of Water, Washington, DC. EPA 821-R-99-006.
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Research was conducted by Clancy Environmental Consultants, Inc., St. Albans, VT. Laboratories participating in the collaborative trials were Metropolitan Water District of Southern California, La Verne, CA and the New York City Department of Environmental Protection, Shokan, NY.