Office of Ground Water and Drinking Water

January 20, 2012

MEMORANDUM

SUBJECT: Basis for Expedited Approval of EPA Method 1623.1

FROM: Carrie Miller

Standards and Risk Management Division, Technical Support Center

TO: The Record

A U.S. Environmental Protection Agency (EPA) method 1623.1 was developed for monitoring 

Cryptosporidium oocysts under the Long Term 2 Enhanced Surface Water Treatment Rule (LT2SWTR). 

This regulation establishes or extends treatment technique requirements in lieu of maximum 

contaminant levels for Cryptosporidium. Public water systems must conduct an initial and a second 

round of source water monitoring for each plant that treats a surface water or ground water under 

direct surface water influence. To implement these requirements, EPA must assess Cryptosporidium

occurrence in raw surface waters used as source waters for drinking water treatment plants. EPA 

Method 1623.1 was developed to support this assessment. EPA has approved one method for 

Cryptosporidium through the notice-and-comment rulemaking process. This memo provides the 

requisite data to support approval of EPA method 1623.1 as an optional alternate method for meeting 

the monitoring requirements of EPA. 

Method 1623.1 is for the detection of Cryptosporidium (CAS Registry number 137259-50-8) in water by 

concentration, immunomagnetic separation (IMS), and immunofluorescence assay microscopy. 

Cryptosporidium is further characterized using 4',6-diamidino-2-phenylindole staining and differential 

interference contrast (DIC) microscopy.

Method 1623.1 relies on fluorescent and DIC microscopy for detection; thus, the performance 

characteristics of method 1623.1 were compared to the characteristics of EPA Method 1623 which is 

also a microscopy-based method that is approved in the regulations at 40 CFR 141.704(a) for 

Cryptosporidium analysis in source water. The substantial change from Method 1623 is the addition of 

sodium hexametaphosphate for use with the Envirochek HV filter. Method 1623.1 also incorporates a 

rinse to remove excess debris during immunomagnetic separation of the oocysts and cysts.

The method performance data presented in this memo includes two EPA studies which showed 

improved accuracy and precision for detecting the concentration of Cryptosporidium oocysts in water 

when NaHMP was added: 1) a single laboratory side-by-side analysis that compared samples from nine 

public water system sources processed by 1623, and by 1623.1, and showed an average improvement of 

18 percentage points (p = 0.0001); and 2) a multi-laboratory side-by-side analysis that resulted in an 

average Cryptosporidium recovery improvement with the NaHMP, compared to no addition, of 15 

percentage points for the three source waters tested (p=0.0197). 
 

Figure 1. Results of single laboratory side-by-side analysis. Cryptosporidium matrix spike recoveries 

from 9 sources comparing methods 1623 and 1623.1 (n=4). Error bars are ± one standard error of the 

mean.

Figure 2. Results of side-by-side analysis in 4 laboratories. Cryptosporidium matrix spike recoveries 

from 3 sources comparing analyses with, and without, dispersant addition (n=12). Error bars are ± 

one standard error of the mean. 

An additional study successfully validated Method 1623.1 in fourteen laboratories and demonstrated a 

mean Cryptosporidium recovery from source water of 61.2% with an average within laboratory RSD of 

12.7%. A summary of the precision and recovery for Method 1623.1 was compared to the previous 

validation study for Method 1623 (Table 1). The Cryptosporidium reagent water and source water mean 

percent recoveries are ≥20 percentage points higher than the previous validation study. The mean RSD 

has decreased for Cryptosporidium in both matrices with Method 1623.1. 

Table 1.Comparison of Study Results to Previous Validation

Cryptosporidium

Matrix Statistic Method 1623 Method 1623.1

Reagent Water Mean % Recovery 40 60

Mean RSD 24 16

Source Water Mean % Recovery 38 61

Mean RPD 36 13

These data from the 1623.1 validation were used to develop quality control criteria for laboratory 

performance. For each criterion, the distribution of recovery was estimated by using random effects 

ANOVA. The recovery limits were estimated as the 5th percentile of the predictive distribution for each 

criterion. The lower limit acceptable for recovery of Cryptosporidium detected in reagent and source 

water increased by 22 and 19 percentage points, respectively, over Method 1623 criteria. Laboratories 

performing Method 1623.1 should have better detection rates and follow more stringent quality control 

criteria than those laboratories following Method 1623.