Appendix A.4: Effects of Holding Time, Storage, and the Preservation of 

Samples on Sample Integrity for the Detection of Fecal Indicator Bacteria by 

Quantitative Polymerase Chain Reaction

COMPLETION NOTICE

Study the effects of sampling holding time, sample storage, and preservation on sample 

integrity for future use (P16)

Summary of the Study 

This study investigated how qPCR measurements were affected by holding fresh water samples 

for 24 and 48 hours. This study also investigated how qPCR measurements were affected by 

storing frozen samples for incremental time periods of 6, 12 and 24 months. Finally, this study 

evaluated the feasibility of using freezer-archived NEEAR epidemiologic study samples for 

determining health relationships using qPCR measurements from new and/or improved fecal 

indicators with the health data from the NEEAR epidemiologic studies. The integrity of frozen 

archived samples was investigated by measuring the fecal indicators (Enterococci and 

Bacteroidales) in freezer-archived water samples (using the same methods that were used in the 

NEEAR studies). To determine if the archived samples had degraded, the fecal indicator 

measurement results from the archived NEEAR samples were compared to the fecal indicator 

measurement results from the samples analyzed during the NEEAR studies using statistical 

analyses. 

Summary of Findings 

The summary of results from this study are: 

• The 24-48 hour holding time results for fresh/refrigerated samples were limited, conflicting, 

and difficult to interpret. 

• The 6, 12 and 24 month frozen samples significantly changed over the 24 months, but not 

consistently across indicators and not progressively over time. 

• Reanalyzed archived frozen filters from prior NEEAR studies were degraded. 

This study has been completed as of December 15, 2010.
Effects of Holding Time, Storage, and 

the Preservation of Samples on 

Sample Integrity for the Detection of 

Fecal Indicator Bacteria by 

Quantitative Polymerase Chain 

Reaction

R E S E A R C H A N D D E V E L O P M E N T

EPA/600/R-10/150

December 2010

EPA/600/R-10/150

December 2010

Effects of Holding Time, Storage, and the 

Preservation of Samples on Sample Integrity 

for the Detection of Fecal Indicator Bacteria by 

Quantitative Polymerase Chain Reaction 

(qPCR)-based Assays

Larry Wymer1, Kevin Oshima1, Jack Paar III2, Mark Doolittle3, Jennifer Lavender4, 

Manju Varma1 and Rich Haugland1

1 U. S. Environmental Protection Agency

National Exposure Research Laboratory

Cincinnati, OH 45268

2 U. S. Environmental Protection Agency

New England Regional Laboratory

North Chelmsford, MA, 01853

3 TechLaw, Inc.

Environmental Services Assistance Team (ESAT)

U.S. EPA Region 1

Lowell, MA 01853

4 Student Services Contractor to the U. S. Environmental Protection Agency

National Exposure Research Laboratory

Cincinnati, OH 45268

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, OH 45268
i

Disclaimer

Although this work was reviewed by U.S. EPA and approved for publication, it may not 

necessarily reflect official Agency policy. Mention of trade names or commercial products does 

not constitute endorsement or recommendation for use.
ii

Executive Summary

The purpose of this project was to answer questions related to storage of samples to be analyzed 

by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. 

The report is divided into two parts. The first part describes studies that were performed to 

determine if filters that are used to collect fecal indicator bacteria can be stored frozen and 

analyzed at a later date. These studies were primarily directed at a specific, targeted question: 

can qPCR results from freezer archived samples be used to establish valid relationships between 

fecal indicator densities and health effects data collected from previous epidemiological studies.

The second part describes studies that were performed to determine if refrigerated water samples 

can be held for 24 to 48 hours prior to analysis by qPCR. These studies addressed a question that 

may be relevant to the implementation of the qPCR method for water quality monitoring at 

remote locations where immediate analysis of the samples is not possible.

Part 1.

Archived Sample Stability Study and Long-Term Holding Time Study: Evaluation of 

effects of freezer-storage on the preservation of filter samples for qPCR analysis

The Beach Environmental Assessment and Coastal Health (BEACH) Act of 2000 directed the 

U.S. EPA to conduct studies concerning pathogen indicators in coastal recreation waters. The 

results of these studies are to be used by U.S. EPA to publish new or revised water quality 

criteria for the purpose of protecting human health in coastal recreational waters. U.S. EPA has 

conducted a number of studies pursuant to the BEACH Act including studies: 

• To develop new rapid methods for measuring water quality 

• To establish the relationship between water quality and health using the rapid methods 

• To develop a system for monitoring water quality 

• To provide guidance to states on the application of the new methods 

The U.S. EPA has conducted the National Epidemiological and Environmental Assessment 

Research (NEEAR) Water Study at four beaches on the Great Lakes and three on the Atlantic 

and Gulf Coasts. Water quality at each of the beaches was impacted by point sources that 
iii

received combined treated sewage discharges from communities with populations of at least 

15,000.

The NEEAR studies contained a health data collection component as described in Wade et al. 

(2006, 2008, 2010). The objective of the health portion of the studies was to quantify the 

symptomatological observations of illnesses in the swimmer vs. non-swimmer groups. The 

second component of the studies was to collect exposure data based on fecal indicator bacteria 

(FIB) densities in the water as determined by rapid methods such as quantitative polymerase 

chain reaction (qPCR) for Enterococcus. Data from the two components of the studies were 

compared to assess the relationships between risk of swimming-related illness and exposure 

(Wade et al. 2006, 2008, 2010). 

As part of the NEEAR study design, additional replicate aliquots of each water sample were 

filtered and the filters were archived by freezing at -40 °C. The ultimate objective of the 

archived NEEAR sample stability study was to establish if the results of several more recently 

developed qPCR methods (e.g., for alternative indicators such as Escherichia coli and 

Bacteroidales) or method modifications might be suitable for generating meaningful health 

relationship assessments. If the archived sample results did not show a change with respect to 

the original sample results using the original qPCR methods, this would lend credibility to using 

the results of the newer qPCR methods employed in the analyses of the archived samples for also 

assessing health relationships using the health data collected from the original NEEAR studies. 

To address the question of archived sample stability, the analysis results from each of the qPCR 

methods that were performed from 2003–2007 on the original NEEAR study samples 

(Enterococcus for both fresh water and marine beach samples and Bacteroidales for marine 

beach samples only) were compared with corresponding results obtained two to six years later in 

2009 from the archived samples.

To further evaluate the effects of filter freezer storage on qPCR analysis results, a supplemental 

long-term holding time study was also conducted by the U.S. EPA, Region 1 Research 

Laboratory in North Chelmsford, MA. In this study, replicate filters from 29 fresh water and 23

marine water samples from diverse non-recreational beach locations in the Boston, MA area 
iv

were held in freezer storage for varying lengths of time from 1 day up to 2 years prior to 

analysis. In addition to qPCR analysis for Enterococcus, this study also sought to provide data 

for some of the newer qPCR-based methods that were not included in the original NEEAR 

studies (specifically Bacteroidales in fresh water and E. coli in both fresh and marine waters). A 

demonstration that the filter analysis results in this study did not show a change over time, would 

lend further credibility to using the analysis results of the archived NEEAR study samples by 

these newer qPCR methods to assess health relationships.

Results from the NEEAR archived sample stability study showed, however, that substantial and 

statistically significant degradation of samples had occurred. The highly significant changes and 

low or absent correlation between archived and original sample analysis results indicate that the 

archived filters cannot be used in a credible manner to establish health relationships involving

Enterococcus qPCR or, by extension, any other indicator, pathogen, or method. Any health 

relationships based on data derived from the archived samples is not useful because they do not 

reflect data that would have been obtained from the original samples in actual beach monitoring 

circumstances. 

The results from the long-term holding time study, though of reduced importance due to the 

findings from the archived sample stability study, showed statistically significant decreases in 

qPCR estimates of Bacteroidales and E. coli after 2 years of freezer storage. These results 

complement the findings from the archived NEEAR study sample analyses for Enterococcus in 

indicating that archived sample analysis results for the two alternative indicator bacteria groups 

(Bacteroidales and E. coli) cannot be used in a credible manner to establish health relationships.

It is noted that the holding temperature used in this study was -20 °C.

Part 2.

Short-Term Holding Time Study: Evaluation of holding refrigerated surface 

water samples for up to 48 h for qPCR analysis

The short-term holding time study was conducted to determine whether holding refrigerated 

surface water samples for 24 and 48 hours affects qPCR-determined density estimates of 

Enterococcus, Escherichia coli, and Bacteroidales bacteria as compared to results from samples 
v

processed within the currently accepted holding time limit of six hours. Additional subsamples 

of the same water samples, collected in the Boston, Massachusetts area for the U.S. EPA Region 

1 long term holding study, were held with refrigeration for 24 and 48 hours prior to the collection 

of target organisms by filtration and qPCR analysis. A supplemental study, motivated by 

ambiguous results from the main study of Boston area water samples, was also conducted using 

an Ohio River water sample. 

Results from the short-term holding time study of Boston area water samples showed small but 

in some cases statistically significant changes in qPCR-estimated densities of the three different 

fecal indicator organism groups in subsamples of the water samples that were held for 24 and 48 

hours prior to filtration compared to corresponding subsamples that were filtered within six 

hours. However, these changes were not consistent for the 24 and 48 hour holding times nor 

were they consistent between indicators. In comparisons with subsamples of the Ohio River 

water sample held for just one hour prior to filtration, statistically significant declines in density 

estimates of one of the indicator groups were observed in subsamples held for 24 hours and of all 

three indicator groups in subsamples held for 48 hours. Overall, the results of these studies were 

inconclusive and, as a result, can neither support nor refute recommendations to hold 

refrigerated water samples for 24 hours or longer prior to filtration and analysis. 
vi

Table of Contents

Disclaimer........................................................................................................................................ i

Executive Summary........................................................................................................................ ii

Table of Contents........................................................................................................................... vi

 Part 1

Archived Sample Stability Study and Long-Term Holding Time Study: Evaluation of effects 

of freezer-storage on the preservation of filter samples for qPCR analysis ................................ 1-1

Introduction............................................................................................................................. 1-1

Material and Methods ............................................................................................................. 1-2

Materials and methods for archived sample stability study............................................. 1-2

Water samples........................................................................................................... 1-2

Sample collection and distribution. .......................................................................... 1-3

Sample archiving. ..................................................................................................... 1-4

Sample analyses........................................................................................................ 1-4

Materials and methods for long-term holding time study................................................ 1-5

Water samples........................................................................................................... 1-5

Sample collection and archiving............................................................................... 1-6

Sample analyses........................................................................................................ 1-7

Computational methods..................................................................................... 1-7

Statistical analyses............................................................................................. 1-9

Quality control/ Quality assurance........................................................................................ 1-10

Laboratory...................................................................................................................... 1-10

Cultures.......................................................................................................................... 1-10

Water samples................................................................................................................ 1-11

Instruments..................................................................................................................... 1-11

Microscopes and general laboratory equipment. .................................................... 1-11

Real-time sequence detection instruments.............................................................. 1-11

Data ................................................................................................................................ 1-11

qPCR data. .............................................................................................................. 1-11

Quality controls, QC acceptance criteria and responses................................................ 1-12

CFU enumeration.................................................................................................... 1-12

qPCR analyses. ....................................................................................................... 1-12

QC failures and responses. ..................................................................................... 1-12

Results................................................................................................................................... 1-14

Archived sample stability study..................................................................................... 1-14

Long-term holding time study ....................................................................................... 1-19

Discussion ............................................................................................................................. 1-20

Conclusions........................................................................................................................... 1-25

References............................................................................................................................. 1-26

 Part 2

Short-Term Holding Study: Evaluation of holding refrigerated water samples for up to 48 

hours for qPCR analysis............................................................................................................... 2-1

Introduction............................................................................................................................. 2-1

Material and Methods ............................................................................................................. 2-2
vii

Water samples.................................................................................................................. 2-2

Sample treatments............................................................................................................ 2-2

Sample analyses............................................................................................................... 2-5

Computational methods................................................................................................... 2-5

Statistical analyses........................................................................................................... 2-6

Ohio River water holding time study............................................................................... 2-7

Quality control/ Quality assurance.......................................................................................... 2-7

Results..................................................................................................................................... 2-7

Discussion ............................................................................................................................. 2-11

Conclusions........................................................................................................................... 2-15

References............................................................................................................................. 2-16

Tables

Table 1-1. Master standard curve variables for qPCR assays..................................................... 1-8

Table 1-2. QC acceptance criteria for current U.S. EPA Enterococcus and Bacteroidales

qPCR methods ......................................................................................................... 1-12

Table 1-3. NEEAR study archived sample characteristics....................................................... 1-13

Table 1-4. NEEAR study mean log10(qPCR calibrator cell equivalents) based on qPCR 

calibrator cell equivalents ÷ 2 for non-detects......................................................... 1-15

Table 1-5. Maximum likelihood estimates of difference between archived and original 

samples..................................................................................................................... 1-17

Table 1-6. Long-term holding time study samples mean log10(qPCR calibrator cell equivalents) 

and change from initial results (24 hr) for samples held for two years ................... 1-20

Table 2-1. Boston, MA area sample locations and descriptions................................................. 2-3

Table 2-2. Description of sample treatments.............................................................................. 2-4

Table 2-3. Primers and probes for qPCR assays......................................................................... 2-6

Table 2-4. Holding time effects: qPCR recovery comparisons between refrigerated water 

(RW) and the recovery standards (RS)...................................................................... 2-9

Table 2-5. Evaluation of difference in holding time effects between fresh and marine waters2-10

Table 2-6. Ohio River holding time effects: qPCR recovery comparisons .............................. 2-11

Table 2-7. Holding time effects: qPCR recovery comparisons between refrigerated water 

(RW) held for 24 or 48 hours and respective recovery standards (RS)................... 2-12
viii

Figures

Figure 1-1. Probability plot of residuals from the MCMC model for differences in observed 

log10 qPCR analysis between archived and original samples............................... 1-16

Figure 1-2. Scatter plots of archived sample stability study samples, Enterococcus qPCR 

archived (“EPA”) vs. initial (“EMSL”) value........................................................ 1-18

Figure 1-3. Scatter plots of archived sample stability study samples, Bacteroidales qPCR 

archived (“EPA”) vs. original (“EMSL”) values................................................... 1-19

Figure 2-1. Fresh water box and whisker plot1 of differences in log10(calibrator cell 

equivalents per 100 ml): refrigerated water – recovery standards........................... 2-8

Figure 2-2. Marine water box and whisker plot1 of differences in log10(calibrator cell 

equivalents per 100 ml): refrigerated water – recovery standards........................... 2-8
ix
1-1

Part 1. Archived Sample Stability Study and Long-Term Holding Time 

Study: Evaluation of effects of freezer-storage on the preservation of 

filter samples for qPCR analysis

Introduction

The Beach Environmental Assessment and Coastal Health (BEACH) Act of 2000 directed the 

U.S. EPA to conduct studies concerning pathogen indicators in coastal recreation waters. The 

results of these studies are to be used by the U.S. EPA to publish new or revised water quality 

criteria for the purpose of protecting human health in coastal recreational waters. The U.S. EPA 

has conducted a number of studies pursuant to the BEACH Act including studies: 

• To develop new rapid methods for measuring water quality 

• To establish the relationship between water quality and health using the rapid methods 

• To develop a system for monitoring water quality 

• To provide guidance to states on the application of the new methods

The U.S. EPA has conducted the National Epidemiological and Environmental Assessment 

Research (NEEAR) Water Study at four beaches on the Great Lakes and three on the Atlantic 

and Gulf Coasts. Water quality at each of the beaches was impacted by point sources that 

received combined treated sewage discharges from communities with populations of at least 

15,000.

The NEEAR studies contained a health data collection component as described in Wade et al. 

(2006, 2008, 2010). The objective of the health portion of the studies was to quantify the 

symptomatological observations in the swimmer vs. non-swimmer groups. The second 

component of the studies was to collect water quality data on fecal indicator bacteria (FIB) using 

rapid methods such as quantitative polymerase chain reaction (qPCR) for Enterococcus and 

Bacteroidales. 
1-2

As part of the NEEAR study design, additional replicate aliquots of each water sample were 

filtered and the filters were archived by freezing. The key objective of the archived NEEAR 

sample stability study was to establish if the results of several more recently developed qPCR 

methods (e.g., for alternative indicators such as E. coli and Bacteroidales) or using various 

method modifications might be suitable for generating meaningful health relationship 

assessments. If the archived sample results did not show a change with respect to the original 

sample results using the original qPCR methods, this would lend credibility to using the results 

of the newer qPCR methods that were employed in the analyses of the archived samples for also 

assessing health relationships. To address the question of archived sample stability, the analysis 

results from each of the qPCR methods that were performed from 2003–2007 on the original

NEEAR study samples (Enterococcus for both fresh water and marine beach samples and 

Bacteroidales for marine beach samples only) were compared with corresponding results 

obtained two to six years later in 2009 from the archived samples.

To further evaluate the effects of filter freezer storage on qPCR analysis results, a supplemental 

long-term holding time study was also conducted by the U.S. EPA, Region 1 Research 

Laboratory in North Chelmsford, MA. In this study, replicate filters from 29 fresh water and 23

marine water samples from diverse non-recreational beach locations in the Boston, MA area 

were held in freezer storage for varying lengths of time from 24 hours up to2 years prior to 

analysis. In addition to qPCR analysis for Enterococcus, this study also sought to provide data 

for newer qPCR-based methods that were not included in the original NEEAR studies

(specifically Bacteroidales in fresh water and E. coli in both fresh and marine waters). A 

demonstration that the filter analysis results in this study did not show a change over time, would 

lend further credibility to using the analysis results of the archived NEEAR study samples by 

these newer qPCR methods to assess health relationships.

Material and Methods

Materials and methods for archived sample stability study

Water samples. Water samples for the NEEAR study were collected over a four year period 

from 2003 to 2007. Sampling sites were West Beach at the Indiana Dunes National Lakeshore in 
1-3

Porter, Indiana on Lake Michigan in 2003; Huntington Beach in Bay Village, Ohio on Lake Erie 

in 2003; Silver Beach, near St. Joseph, Michigan, and Washington Park Beach in Michigan City, 

Indiana on Lake Michigan in 2004; Edgewater Beach in Biloxi, MS on the Gulf of Mexico in 

2005; Fairhope Municipal Beach in Fairhope, AL on the Gulf of Mexico in 2007; and Goddard 

State Memorial Park Beach in West Warwick, RI on Long Island Sound in 2007.

Sampling designs were similar at each of the sites. Sampling visits occurred on Saturdays, 

Sundays and holidays over time periods varying from approximately 10 to 12 weeks from either 

May through August or June through September. Sampling occurred three times daily, at 8 AM, 

11 AM, and 3 PM in waist-level (1 m deep) and shin-level water (0.3 m deep) locations along 

three transects perpendicular to the shoreline. The sampling design at Huntington Beach 

included three additional shin-level locations.

Sample collection and distribution. One liter water samples were collected at each location by 

standard methods as recommended in Section 9060 of Standard Methods for the Examination of 

Water and Wastewater, American Public Health Association (1998). Following collection, all 

samples were placed in coolers and maintained on ice during transport to a local laboratory and 

at 1–4 °C during the time interval before they were processed. Processing of all samples by 

filtration was performed within six hours of collection.

Duplicate 50 or 100 ml volumes of each water sample (either 50 or 100 ml was used consistently 

for all samples within a beach) were filtered through 47-mm, 0.4-μm pore size polycarbonate 

filters (catalog #K04CP04700, Osmonics Inc., Minnetonka, MN) and the sides of the funnels 

were rinsed twice with 20 ml of sterile, phosphate buffered saline. The filters were transferred to 

a Petri dish with the sample side facing up. Using sterile forceps, each filter was folded into a 

cylinder with the sample side facing inward, and then inserted into a 2 ml semiconical screw-cap 

microcentrifuge tube (extraction tube; catalog #506-636, PGC Scientific, Gaithersburg, MD) 

containing 0.3 g of acid-washed glass beads (catalog #G-1277, Sigma, St. Louis, MO). The 

filters were held at -20 °C for no more than three days until shipment to the analytical 

laboratories on dry ice. One filter from each water sample was shipped to EMSL Analytical, 
1-4

Inc., Cinnaminson, NJ for analysis within seven days while the duplicate was shipped to the U.S. 

EPA’s National Exposure Research Laboratory (NERL), Cincinnati, OH for archiving.

Sample archiving. Filters received by the U.S. EPA NERL laboratory were immediately 

transferred to a -40 °C, 25 cubic ft. capacity, 208V upright freezer (model A25-40T, So-Low 

Environmental Equipment Co., Cincinnati, OH) where they were stored continuously (except as 

noted below) until analysis in 2009. The freezer temperature was continuously monitored by a 

centralized monitoring system within the Cincinnati facility. On several occasions samples were 

briefly removed from the freezer for reorganization and/or while defrosting of the freezer and on 

one occasion they were temporarily transferred to other freezers due to a facility power outage. 

Possible temperature changes of the filters during these activities were not determined.

Sample analyses. Original filters sent to the EMSL lab and archived filters stored at the NERL 

laboratory were extracted to recover total DNA and the DNA extracts were subjected to qPCR 

analysis by the basic procedures described in Haugland et al. (2005). Briefly, cells were 

suspended from the filters and lysed in a bead mill for 60 seconds at maximum speed and the 

debris was removed by centrifugation. For all samples analyzed after 2004, including the 

archived samples, the published DNA extraction procedure was modified slightly by increasing 

the total volume of extraction buffer, containing 0.2 μg ml-1 salmon DNA in AE buffer (Qiagen,

Valencia, CA), from 0.3 ml to 0.6 ml and decreasing the dilution of extracts prior to analysis 

from 10-fold to 5-fold. Calibrator samples (three to six replicates), consisting of clean 

polycarbonate filters amended with known cell quantities of Enterococcus faecalis (ATCC# 

29212) and/or Bacteroides thetaiotaomicron (ATCC # 29741), and negative control samples 

(three to six replicates), consisting of clean filters only, were extracted in the same manner with 

each batch of test samples. Cells used by EMSL in the calibrator samples originated from 

laboratory grown cultures and were enumerated as previously described (Haugland et al. 2005,

Siefring et al. 2008). Cells used by U.S. EPA NERL were enumerated by flow cytometry and 

were acquired in the form of commercially available, lyophilized pellets (BioballsTM, BTF, 

Sydney Australia). QPCR analyses were performed using previously described primer and 

TaqManTM hybridization probe assays for Enterococcus and Oncorhynchus keta (salmon) DNA 

target sequences (Haugland et al. 2005) on all samples (Great Lakes and marine) and a 
1-5

previously described primer and TaqManTM hybridization probe assay for total Bacteroidales

target sequences (Siefring et al. 2008) were performed on marine samples only. Primer and 

probe characteristics of each of these assays are listed in part II of this report. QPCR 

amplification of water sample and calibrator sample DNA extracts, and negative control samples, 

was performed by using 5 μL of equally diluted extracts in a total reaction volume of 25 μL. 

Reagent mixes were prepared by combining 12.5 μL of TaqMan® Universal Master Mix 

(Applied Biosystems, Foster City, CA), 2.5 μL of 2 mg/ml bovine serum albumin, 1 μM of each 

primer, and 80 nM of probe for each reaction. Amplification occurred with an initial start at 50

°C for 2 min followed by 95 °C for 10 min, then forty PCR cycles of 95 °C for 15 s and 60 °C for 

1 min. All analyses of the fresh samples were performed by EMSL Analytical Inc., Cherry Hill, 

NJ in a Cepheid SmartCycler® II(Cepheid, Sunnyvale, CA). All analyses of the archived 

samples were performed in the U.S. EPA NERL laboratory in either a Cepheid SmartCycler® II 

(freshwater samples) or in an Applied Biosystems StepOnePlus® (marine samples). Results 

obtained on these two instruments have been previously compared in simultaneous analyses of

DNA extracts from multiple, replicate filter retentates of 12 marine and 12 freshwater samples 

from diverse locations. No significant difference (p > .05) in log10 target sequence copy 

recovery estimates, based on three-way ANOVA with fixed factors: instrument, matrix, 

instrument*matrix; and random factors: sample (nested in matrix) and inst*sample (nested in 

matrix), nor in precision among these estimates (p > .05), based on the one-way ANOVA of 

Levene's Test for Homogeneity of Variance was observed in the results from the two instruments 

(unpublished data). For this reason results from the present study treated results from both 

instruments as equivalent or interchangeable.

Materials and methods for long-term holding time study

Water samples. Water samples analyzed in this study were collected from 29 freshwater sites 

and 23 marine water sites in the Boston, MA area. Different subsamples of the same water 

samples used in this study were used for the short term holding study, presented in part II of this 

report. Sampling locations and considerations in the selection of these locations are described in 

part II of this report. Although 25 samples from both marine and freshwater sites were planned 

for, some sites thought to be saltwater were later reclassified after measurements revealed low 

levels of salinity. 
1-6

Sample collection and archiving. Either three or four water samples were simultaneously 

collected in 1 liter bottles at each site by standard methods as recommended in Section 9060 of

Standard Methods for the Examination of Water and Wastewater, American Public Health 

Association (1998). Following collection, all samples were placed in coolers and maintained on 

ice during transport to the U.S. EPA Region 1 laboratory and at 1–4 °C during the time interval 

before they were processed.

Upon arrival to the laboratory, and within six hours of collection, the contents of the individual 1 

liter collection bottles for each sampling site were combined in a carboy and mixed. A total of 

eight 50 ml, or for some samples 100 ml, volume aliquots of each composite water sample were 

filtered through 47-mm, 0.4-μm pore size polycarbonate filters (catalog #K04CP04700, 

Osmonics Inc., Minnetonka, MN) and the sides of the funnels were rinsed twice with 20 ml of 

sterile, phosphate buffered saline. The filters were then transferred to a Petri dish with the 

sample side facing up. Using sterile forceps, each filter was folded in half three times to form an 

umbrella and then inserted into a 2 ml semiconical screw-cap extraction tubes, containing 0.3 g 

of siliconized ceramic beads (Roche MagNA Lyser Green BeadsTM). All filter samples were 

flash frozen by placing the tubes in a cooling block, pre-chilled to -20 °C for 1 hour, and then 

held in a -20 °C freezer until they were extracted and analyzed. Duplicate filter samples from 

each site were extracted and analyzed after being held for 1 day, and 6, 12, and 24 months.

Multiple replicate calibrator sample filters were prepared just prior to the study by placing 

aliquots of a single mixed cell suspension containing pre-determined cell quantities of 

laboratory-grown, representative target strains of each of the assays (E. faecalis, B. 

thetaiotaomicron and E. coli) on clean filters. These calibrator filter samples were held at -20 °C 

and replicate filters were extracted and analyzed in parallel with each batch of test samples at 

each time point in the holding study. Since the calibrator sample filters were held in freezer 

storage for the same lengths of time as the water sample filters, the holding time effects 

presented in this report are specific to the water samples and do not take into account any 

potential holding time effects on the cultured calibrator cells. However, target sequence 

recoveries from the calibrator samples were also assessed independently at each time point based 
1-7

on comparisons of raw CT values and after calibration of the CT results with standard curve 

data.

Sample analyses. Filter samples were extracted to recover total DNA and the DNA extracts 

were subjected to qPCR analysis as described for the NEEAR study samples. Duplicate analyses 

were performed on each filter sample extract. In addition, analyses for E. coli 23S rRNA gene 

target sequences were performed in the same manner except using Gene Expression PCR Master 

Mix (Applied Biosystems) and an unpublished TaqMan® probe and primer set (Primer and 

probe characteristics of this assay are listed in part II of this report). All of the qPCR analyses 

were conducted by the U.S. EPA Region 1 laboratory in a Cepheid SmartCycler® II.

Computational methods 

Analysis data consisted of paired observations of filters from each sample: (1) a qPCR 

measurement soon after the sample had been collected and (2) a qPCR measurement after 

archival and storage over a six month to two year period (long-term holding time study) or a two 

to six year period (archived sample stability study). Statistical analysis was performed on log 

transformed calibrator cell equivalents (CCE) estimated by qPCR using the “∆∆CT” approach as 

previously described (Haugland et al. 2005) and as used in the NEEAR study analysis of 

swimmers’ risk-exposure relationship (Wade et al. 2006, 2008, 2010). 

The ∆∆CT computational approach, which is derived from the comparative cycle threshold (CT) 

method (Applied Biosystems 1997), employs an arithmetic formula to determine the ratio of 

target sequence quantities in DNA extracts from test sample filters relative to those in similarlyprepared DNA extracts from calibrator sample filters containing a known quantity of target 

organism cells based on the difference in CT values obtained from qPCR analyses of these 

samples. Similar comparisons of CT values from qPCR assays for an exogenous target sequence 

from salmon sperm DNA, added in equal quantities to both the test and calibrator sample filters 

before DNA extraction, were used both as a reference to normalize results for differences in the 

amount of total DNA recovered from each sample (e.g., caused by test sample effects on DNA 

recovery) and as a sample processing control (SPC) to signal potentially non-quantifiable test 

sample results caused by PCR inhibition or low DNA recoveries (Haugland et al. 2005). The 

calculation can be expressed by the following equations:
1-8

∆∆CT = ∆CT,target - ∆CT,reference (1)

CCE = NCalibrator⋅A-∆∆CT (2)

in which ∆CT,target represents CT,sample - Mean CT,calibrator for the target sequence (e.g., enterococci) 

and ∆CT,reference represents the corresponding difference for the salmon sperm reference sequence.

Ncalibrator is the known number of cells in the calibrator sample and A is the amplification factor 

for the assay. Ideally, A=2 but typically it is in the range 1.9 – 2.0 with values less than 2 

resulting from less than 100% replication of the target sequence at each cycle. In practice, A is 

either assumed to be 2 or is calculated based on the slope of a standard curve (Applied 

Biosystems 1997). The calculation can be expressed by the following equation:

A = 10^(1/-stand curve slope) (3)

For both the archived sample stability and the long-term holding time studies, slope values were 

obtained from standard curves generated by each of the laboratories from pooled results of 

repeated qPCR analyses of serially diluted DNA standards nominally containing target 

sequences in a range from 10 to 4 x 104 copies per analysis. Table 1-1 shows the Y-intercept, 

slope and amplification factors calculated by each of the laboratories from their respective 

master standard curves from each of the qPCR assays.

Table 1-1. Master standard curve variables for qPCR assays

Laboratory (instrument) Indicator qPCR Assay Y-intercept Slope A

EMSL (Smart Cycler) Enterococcus 38.32 -3.42 1.96

Bacteroidales 38.61 -3.34 1.99

NERL (Smart Cycler) Enterococcus 38.16 -3.62 1.89

NERL (StepOnePlus)) Enterococcus 38.94 -3.50 1.95

Bacteroidales 39.19 -3.62 1.89

U.S. EPA Region 1 (Smart Cycler) Enterococcus 37.01 -3.24 2.05

Bacteroidales 37.41 -3.22 2.05

E. coli 39.62 -3.45 1.95
1-9

Statistical analyses 

For both the archived sample stability and the long-term holding time studies, the analyses were 

conceptually the same, although differences in the designs of the respective studies necessitated 

slightly different treatment of the data. In the long-term holding time study, duplicate filters 

were analyzed for each sample and the extract from each filter was analyzed by qPCR in 

duplicate reactions at each time point. All of the results were well above the detection limit, this 

having been a criterion of sample selection in the first place. Therefore, for each water type, a 

nesting sampling scheme was present (duplicate analysis nested within filter and filter nested 

within sample). A mixed model was used to account for these as nested random effects. Data 

from the Frozen Storage study were analyzed using the MIXED procedure in SAS v. 9 for 

Windows (SAS Institute, Cary, NC 2009). Explicitly, the model used was:

∆∆CT = W + T + WT + Sample(W) + S*T(W) +Filter(W*T*S) + ε(0,σ2) (4)

in which W represents water type (fresh, marine), T represents time (0, 6 m, 12 m, 24 m), S 

uniquely identifies an individual random sample within each water type, and F is a uniquely 

identified filter (filter 1 or 2) within each sample. Within filter, there is random variability 

represented by ε(0,σ2

), which is assumed normal with mean zero and variance σ2 of the duplicate 

runs performed on each filter.

In the archived sample stability study, a single analysis of a single filter of each sample was 

performed shortly after collection and again for an archived filter two to six years later. The 

samples were not collected with the intention that they would necessarily all contain sufficiently 

high densities of the target fecal indicator organisms to allow their detection and, in fact, many 

samples were non-detects (up to 20% depending on beach). Data from the archived sample 

stability study could thus be analyzed for differences (archived result minus original result), but 

it was necessary to consider the non-detects as censored results. If the censored result was the 

original analysis, the difference was right censored, that is representing a lower bound on the 

actual difference since the original result could be lower than the stated value. Similarly, if the 

censored result was the archived analysis, the difference was left-censored, that is, likely to be 

smaller than the stated value. Sufficient statistics comprised the set of samples for which one or 
1-10

both results were quantifiable so that samples for which both analyses yielded non-quantifiable 

results were ignored.

Maximum likelihood estimation (“Tobit analysis”) was used for the archived sample stability 

study analysis where the difference in log (base 10) between the qPCR results before and after 

archival were censored as above. Monte Carlo Markov Chain (MCMC) estimation using 

WinBUGS v. 1.4 (Lunn et al. 2000, Ntzoufras 2009) with diffuse priors was employed for this 

purpose because of the software’s ability to capture estimates of individual differences. These 

estimates were useful for evaluating the reasonableness of the normality model used and for 

further evaluating paired observations, particularly in terms of prediction of archived results 

based on the original results.

Other data analyses and summaries were performed in R v. 2.8 (2008) and Excel 2003 for 

Windows. An alpha level of 0.05 is used to determine statistical significance of differences or 

effects.

Quality control/ Quality assurance

Laboratory 

General guidance for overall laboratory quality assurance for environmental PCR analyses have 

been developed (U.S. EPA 2004) and was followed in this project. 

Cultures 

Culture collection strains of representative target species E. faecalis, B. thetaiotaomicron, and E

.coli were maintained as cell suspension freezer stocks. Fresh cultures of these strains were

prepared as described in the qPCR methods (U.S. EPA 2010a, 2010b, Chern et al. 2010) and 

routinely checked for evidence of contamination by microscopic examination of cell morphology 

and by examination of colony morphology on the appropriate agar plates. Freezer stocks were

maintained at -80 °C. For NERL Cincinnati analyses, lyophilized BioballTM cell preparations 

were used to prepare positive control (calibrator) samples rather than lab-grown cell 

preparations. 
1-11

Water samples 

Collection and storage of water samples were conducted following general quality control 

guidelines described in sections 9060 A and B of Standard Methods for the Examination of 

Water and Wastewater (1998) and specific guidelines for the qPCR methods (U.S. EPA 2010a,

2010b). 

Instruments

Microscopes and general laboratory equipment. General guidelines described in section 9030 

of Standard Methods for the Examination of Water and Wastewater (1998) were followed for the 

maintenance of microscopes and general laboratory equipment.

Real-time sequence detection instruments. Calibrations of instruments were performed on-site 

or off-site as specified by the instrument manufacturers. Potential instrument performance 

problems were identified from positive control QC sample results. Service contracts for the 

instruments were maintained which included telephone troubleshooting support and on-site or 

off-site instrument service by a factory trained technician when required.

Data

qPCR data. Raw fluorescence growth curve data and other diagnostic information generated by 

the sequence detectors (run files) were archived on compact disks or flash drives that were 

specifically designated for this purpose. Run files were labeled with the date of the analysis and 

the analyst’s initials. CT results tables from each instrument run were exported and saved as 

Excel files. Data from analyses performed by the sequence detectors consisted of CT values 

from the different assays for positive control (calibrator), negative control, and test samples. The 

CT data were copied into Excel template worksheets that were custom designed for performing 

comparative cycle threshold analysis and simple descriptive statistics tests (e.g., means and 

standard deviations of replicate samples).
1-12

 1

Quality controls, QC acceptance criteria and responses

CFU enumeration. Quality controls for Enterococcus, B. thetaiotaomicron and E. coli cultured 

cell enumeration were performed as described in the qPCR methods (U.S. EPA 2010a, 2010b, 

Chern et al. 2010). Certificates of analyses indicating mean and standard deviations of cell 

quantities per BioballTM for each lot were either provided directly by the manufacturer or are 

available online at: http://www.btfbio.com/cofa.php?nav=BioBall.

qPCR analyses. Quality controls and QC acceptance criteria used in the Enterococcus and 

Bacteroidales qPCR methods have been reported (U.S. EPA 2010a, 2010b). The same quality 

controls and QC acceptance criteria were employed in qPCR analyses for E. coli. These QC 

acceptance criteria are identified in Table 1-2.

QC failures and responses. Frequencies of samples that failed the QC acceptance criterion for 

the salmon DNA SPC in the archived sample stability study are indicated in Table 1-3. These 

samples were excluded from further analyses. All other QC acceptance criteria for sample 

analyses by qPCR were consistently met.

Table 1-2. QC acceptance criteria for current U.S. EPA Enterococcus and Bacteroidales qPCR methods

Control Method QC acceptance criteria

Negative controls qPCR assay for target 

sequences in no 

template controls

At least 66% of analyses yield no logarithmic amplification traces. 

All analyses yield CT values >35.

qPCR assay for target 

sequences in method 

blanks

At least 66% of analyses yield no logarithmic amplification traces. 

All analyses yield CT values >35.

Positive controls qPCR assay for SPC 

sequences

Test sample analyses are within 3 CT units of mean value from daily 

analyses of at least 3 equally diluted calibrator samples.

qPCR assay for target 

sequences in 

calibrator samples

Baseline mean CT value and standard deviation are determined from 

analyses of a minimum of 9 calibrator samples. Mean CT values from 

daily analyses of calibrator samples from same source are within 3 

standard deviations of the laboratory’s baseline mean CT value.

Standard Curves Baseline slope values are determined from linear regression analysis 

of CT values from a minimum of three separate analyses (performed 

in duplicate) of DNA standards. Slope values determined from 

subsequent analyses are within 95% confidence range of baseline 

values. 1 Unless otherwise specified, same QC acceptance criteria are applied to methods for alternative indicators.
1-13

Table 1-3. NEEAR study archived sample characteristics

West 

Beach

Huntington 

Beach

Silver 

Beach

Washington 

Park Beach

Edgewater 

Beach

Fairhope 

Beach

Goddard 

Beach

Years stored 6 6 5 5 4 2 2

Enterococcus qPCR-CCE

Total Samples 294 420 423 421 396 438 426

- Failed QC1 11 5 16 19 29 36 8

- Both times < DL2 8 10 18 7 9 49 35

Samples Used 275 405 389 395 358 353 383

# samples

- Original < DL 5 7 38 23 38 74 24

- Archived < DL 102 111 105 113 46 85 109

- Neither < DL 168 287 246 259 274 194 250

% < DL before or 

after archival3 38.9 29.1 36.8 34.4 23.5 45.0 34.7

Bacteroidales qPCR-CCE

Total Samples -

4 - - - 396 438 426

- Failed QC - - - - 16 5 12

- Both times < DL - - - - 26 36 5

Samples Used - - - - 354 397 409

# samples - - - -

- Original < DL - - - - 14 36 29

- Archived < DL - - - - 33 14 20

- Never < DL - - - - 307 347 360

% < DL before or 

after archival - - - - 13.3 12.6 12.0

1 Sample Processing Control out of range of ±3 cycle thresholds from mean. 

2 Detection limit (DL) of 40 cycles reached without a positive signal in analyses for both the initial and archive 

sample. 

3 Among samples used. 

4 Bacteroidales qPCR was not performed at the 4 freshwater beaches.
1-14

Results

Archived sample stability study

Table 1-3 summarizes sample characteristics from the NEEAR original and archived data 

analyses. A total of 2818 samples were collected at the four fresh water and three marine water 

beach sites. An average of 4% and 5% of these samples did not meet quality control standards or 

gave assay values below the detection limit for both the original and archived samples, analyzed 

by EMSL Analytical Inc. and U.S. EPA, respectively. These samples were excluded from 

further analyses. Quality control failure and unusable data rates were similar for both fresh and 

marine samples (Table 1-3). 

Of particular note is the relative number of sample data that were below the detection limit 

(“censored”) in the original analysis vs. when they were reanalyzed after a period of archival.

For the Enterococcus assay, 24 to 45% of the samples from the different beaches yielded at least 

one qPCR result that was below the limit of detection. Among the Great Lakes samples, as well 

as Goddard Beach, non-detects were much more frequent after archival than before. This is as 

expected if there is a decline in the qPCR signal as a result of archival. However, at the other 

two marine beaches, non-detects were about as likely to occur after as they were before being 

archived. The imbalance in results below the limit of detection in original vs. archived samples 

was one more impetus to incorporate the non-detects in the analysis. Otherwise, results would 

be biased by discarding more samples that had declined than those that had increased.

Non-detect results for Bacteroidales were considerably less prevalent because of the larger 

number of these organisms, generally amounting to about an order-of-magnitude. Assays for 

Bacteroidales were performed at only the three marine beaches, where non-detect results 

amounted to only about 13% of all usable samples. While the percentage of non-detects may be 

small in comparison to those for Enterococcus, a 13% non-detect rate is still substantial. Note 

that samples with non-detects both before and after archival do not inform the estimation of an 

archival effect. We may infer that where this occurred, there was increased likelihood that that 

particular sample was devoid of the target DNA for the respective qPCR assay.
1-15

Using one-half the detection limit for non-detect results, Table 1-4 shows that when qPCR-CCE 

are calculated for the fresh and archived samples at each beach, the mean values for archived 

samples are consistently lower than those of fresh samples, although this decline was relatively 

small at Edgewater and Fairhope compared to the other beaches. These mean differences by 

beach, as well as a pooled precision parameter based on substituting one-half of the detection 

limit for censored data, were used as initial values in the MCMC estimation procedure.

Table 1-4. NEEAR study mean log10(qPCR calibrator cell equivalents) based on qPCR calibrator cell 

equivalents ÷ 2 for non-detects

West Beach

Huntington 

Beach

Silver 

Beach

Washington 

Park Beach

Edgewater 

Beach

Fairhope 

Beach

Goddard 

Beach

Enterococcus qPCR-CCE

Original 2.201 2.244 1.696 1.746 2.062 1.847 2.239

Archived 0.831 1.246 1.098 0.965 1.935 1.670 1.451

Change -1.370 -0.998 -0.598 -0.781 -0.128 -0.178 -0.788

Bacteroidales qPCR-CCE

Original -

1 - - - 2.999 3.050 3.120

Archived - - - - 2.494 2.862 2.807

Change - - - - -0.505 -0.187 -0.313

1 Bacteroidales qPCR was not performed at the 4 freshwater beaches.

A probability plot of the residuals from the MCMC estimation procedure, that is, log10(∆∆CT, 

archived) – log10(∆∆CT, fresh) less it’s respective MCMC mean, is given in Figure 1-1. The plot 

justifies using the Normal model for these data. 
1-16

Residual Value

-2 -1 0 1 2

Cumulative Frequency (%)

0.1

1

10

30

50

70

90

99

99.9

Figure 1-1. Probability plot of residuals from the MCMC model for differences 

in observed log10 qPCR analysis between archived and original samples

Final analytical results from MCMC estimation that explicitly accounted for values below their 

respective limits of detection (Table 1-5) indicated strong, statistically significant declines in 

qPCR-CCE yields from the archived NEEAR samples from most beaches. Samples from all but 

Edgewater Beach in Biloxi and Fairhope Beach exhibited highly significant declines with respect 

to Enterococcus recoveries. With respect to Bacteroidales qPCR-CCE, Edgewater and Goddard 

Beach samples exhibited highly significant changes. The analysis was performed on logarithmtransformed qPCR-CCE. To put these numbers in perspective, Table 1-5 also shows the 

equivalent percent declines that corresponded to the differences in geometric means (the 

antilogarithms of the mean log differences). In many cases, these declines imply that only 10% 

or less of the original DNA remained in the archived samples.

Pearson product moment correlations based on the MCMC estimates are given in the last column 

of Table 1-5. For enterococci, the correlations were particularly low where there was any 

correlation whatsoever. A common measure of predictability in regression analysis is R2

, which 

is equal to the square of the Pearson correlation. Multiplied by 100, an R2 indicates the percent 
1-17

Table 1-5. Maximum likelihood estimates of difference between archived and original samples

Log10 

change P-value1

Equivalent % 

change

Pearson 

correlation

Enterococcus qPCR-CCE

West Beach -1.65 < 0.001 -98% 0.36

Huntington Beach -1.19 < 0.001 -94% 0.39

Silver Beach -0.70 < 0.001 -81% -0.01

Washington Park Beach -0.94 < 0.001 -89% -0.04

Edgewater Beach -0.14 0.195 -28% 0.11

Fairhope Beach -0.21 0.113 -38% -0.04

Goddard Beach -1.00 < 0.001 -90% 0.11

Bacteroidales qPCR-CCE

Edgewater Beach -0.51 < 0.001 -69% 0.52

Fairhope Beach -0.06 0.255 -12% 0.46

Goddard Beach -0.28 < 0.001 -47% 0.48

1 P-values in bold-face indicate statistically significant differences.

of variation explained by the independent variable. In this study, the independent variable would 

be regarded as the initial qPCR-CCE result, and knowing this value was seen to account for only 

10% or less in the variation among archived sample qPCR results. Scatter plots of archived vs. 

original log10(qPCR-CCE) results for Enterococcus are shown by beach in Figure 1-2.

Bacteroidales qPCR-CCE results from the archived samples show much better correlation with 

their respective initial analyses compared to the Enterococcus analyses. These correlations still 

are not substantial, however, and amount to R2 values on the order of 0.25, thus explaining only 

25% of the variation among archived sample analyses for Bacteroidales. We show the scatter of 

archived vs. original log10 qPCR-CCE results for Bacteroidales in Figure 1-3.
1-18

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

West Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Huntington Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Silver Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Washington Park Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Edgewater Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Fairhope Beach

log10(EMSL qPCR-CE)

0 1 2 3 4 5

log10(EPA qPCR-CE)

0

1

2

3

4

5

Goddard Beach

Figure 1-2. Scatter plots of archived sample stability study samples, Enterococcus qPCR archived 

(“EPA”) vs. initial (“EMSL”) value
1-19

log10(EMSL qPCR-CE)

1 2 3 4 5

log10(EPA qPCR-CE)

1

2

3

4

5

Edgewater Beach

 log10(EMSL qPCR-CE)

1 2 3 4 5

log10(EPA qPCR-CE)

1

2

3

4

5

Fairhope Beach

 log10(EMSL qPCR-CE)

1 2 3 4 5

log10(EPA qPCR-CE)

1

2

3

4

5

Goddard Beach

Figure 1-3. Scatter plots of archived sample stability study samples, Bacteroidales qPCR archived 

(“EPA”) vs. original (“EMSL”) values

Long-term holding time study 

Some significant changes in qPCR-CCE results for the long-term holding time study samples 

between their initial analysis and their reanalysis two years later were observed (Table 1-6).

Marine water Enterococcus assay results actually showed a statistically significant increase of 

0.16 logs (44%) over this period (p=0.002). Mean log qPCR-CCE in this case rose between the 

initial analysis and the reanalysis at six months and stayed more or less constant at that level at 

the one and two year marks. Meanwhile, Enterococcus qPCR-CCE results from freshwater 

samples exhibited a small “marginally significant” (p=0.063) decline. Among the other assays, 

both Bacteroidales (p<0.001) and E. coli (p=0.022) re-analyses indicated significant declines in 

the PCR target in freshwater samples after two years amounting to 40% and 21% of their initial 

values (0.22 and 0.10 logs), respectively. The decrease in Bacteroidales signal was already 

evident at six months, but E. coli declined incrementally. Another decrease in qPCR results over 

the two year period was for Bacteroidales from marine water samples where the change was 
1-20

Table 1-6. Long-term holding time study samples mean log10(qPCR calibrator cell equivalents) and change

from initial results (24 hr) for samples held for two years

 Mean log10(qPCR-CCE) 2 yr net 

change1 P-value2

Equivalent 

% change

Pearson 

24 h 6 mo 1 yr 2 yr correlation

Enterococcus qPCR-CCE

Freshwater3 3.82 3.93 3.77 3.73 -0.08 0.063 -17 0.94

Marine4 3.34 3.61 3.45 3.50 0.16 0.002 44 0.92

Bacteroidales qPCR-CCE

Freshwater 6.49 6.23 6.32 6.27 -0.22 < 0.001 -40 0.96

Marine 6.10 6.30 5.98 6.00 -0.10 0.056 -20 0.96

E. coli qPCR-CCE

Freshwater 4.65 4.64 4.58 4.55 -0.10 0.022 -21 0.95

Marine 3.99 4.02 3.98 4.01 0.02 0.658 5 0.95

1 Difference from 24 h to 2 yr mean log10(qPCR-CCE). 

2 P-values in bold-face indicate statistically significant differences. 

3 N=29. 

4 N=23.

only marginally significant (p=0.056). Target sequence recoveries from the calibrator samples 

showed no decreases over this time period for any of the indicator organisms. 

Pearson correlations between initial and two-year log10(qPCR-CCE) values were all above 0.9, 

being in all but one case about 0.95 (Table 1-6). Correspondingly, R2 values were all 0.84 or 

higher. This fact is reflected in the plots of two-year vs. initial log10(qPCR-CCE) values in 

Figure 1-3.

Discussion

The primary purpose of the present analysis is to evaluate the feasibility of using the archived 

NEEAR study samples as surrogates for fresh samples in determining relationships between 

results of new or revised methods for potential indicator bacteria of fecal pollution and 

swimmers’ health risks. New methods that were examined in the analyses of the archived 
1-21

samples included qPCR assays for E. coli and total Bacteroidales (the former assay being of 

interest due to the widespread acceptance of E. coli as an indicator of fecal pollution in fresh 

waters and the latter assay being of interest due to its use in the original analyses of marine 

samples only). Modified methods included purification and concentration of DNA in the filter 

extracts as well as the incorporation of additional and/or improved positive controls for the 

detection of PCR inhibition. For the archived data to be used in this manner, the most desirable 

outcome would be for there to be no change between the original analyses and the archived 

sample analyses two to six years later. In addition there should be a relatively high correlation 

between the two sets of results (the latter implying a low variance of the difference). Lacking 

this, at least a high correlation between the two sets of results might allow some adjustment to be 

made to the archived data so that they would reliably reflect the fresh sample data. Devising an 

adjustment factor would necessarily involve non-provable, critical assumptions, such as that 

other qPCR assays would follow the predictive model established by the results that were 

available. Results from analyses of the archived samples by the new or revised methods may at 

least be more readily accepted if one or both of the conditions described above are indicated by 

the available data.

The analysis of the previous section shows, however, that suitability of the archived samples as 

surrogates for original samples is contraindicated by comparisons between archived and original 

sample results with respect to the available data, i.e., qPCR results for Enterococcus and marine 

Bacteroidales. Not only are large, highly significant declines in qPCR-CCE recoveries 

observed, but the differences are largely unpredictable from sample to sample as evidenced by 

the low correlation between results of archived and original sample analyses. This precludes the 

possibility of using any sort of “adjustment factor” to rectify this change. 

A short-coming of the archived sample data from the archived sample stability study, with 

respect to the interpretation of their change, is that the initial analysis of original samples and 

analysis of archived samples two to six years later were performed by different laboratories.

Thus, the effects of change over time may be confounded with effects of different analysts and 

equipment. However, even for the long-term holding time study data, time effects are 

confounded with any potential changes that may have occurred within the single laboratory that 
1-22

performed all of the qPCR analyses. An experiment designed to properly capture all of the 

between lab and between time variation would have to involve several labs performing both the 

original and archived sample qPCR analysis. The cost of doing so would be prohibitive and 

even then, one would have to assume that there were no systematic changes in labs and/or qPCR 

quality over time. By way of a rough comparison of the changes observed among the archived 

samples over time and the magnitude of changes that may be expected from lab-to-lab variation, 

some preliminary data on qPCR inter-laboratory variance are available (U.S. EPA Office of 

Water 2008, unpublished data; Ad Hoc multi-laboratory study 2010, unpublished data). These 

data indicate that a two standard deviation (i.e., 95% confidence level) difference on the order of 

about 0.5–0.7 logs might be expected between two labs analyzing the identical sample. Most of 

the significant changes observed in the archived samples are outside this range. Therefore, it 

does not seem likely that inter-laboratory differences themselves could have accounted for all of 

the observed changes.

Given the negative results for the archived sample stability study data, results from long-term 

holding time study have a greatly reduced relevance. The value of the long-term holding time 

information would have been in supporting the inference that the equivalency or predictability of 

the NEEAR archived sample data could be extrapolated to other qPCR targets (most notably E. 

coli and fresh water Bacteroidales), but neither equivalency nor predictability were observed for 

any of the NEEAR study results that could be directly compared. Even if one or both of these 

conditions had been met, there would have been additional challenges in making such 

extrapolations. Results from the long-term holding time study, particularly the high correlations 

between held and initial sample qPCR-CCE values, indicate that archived samples may be 

capable of serving as surrogates for fresh samples under some circumstances, at least with a 

statistical correction. However, the predictive relationship observed in the long-term holding 

time study would need to have been extrapolated from two years out to as long as six years in 

order to be applicable to the archived sample stability study samples. The length of time that 

samples are held would logically seem to be an important factor, as is borne out by the archived 

data, except for enterococci results at Goddard Beach. The Goddard Beach data showed a 90% 

2-year decline in mean log10 (qPCR-CCE) that was more in line with the five and six year 

declines seen for the fresh water beaches (Table 1-5). However, the long-term holding time
1-23

study data themselves, which were designed to track change over time, were not consistent 

among the different indicator and water matrix combinations with respect to showing progressive 

declines, if any at all, over time, nor were they consistent with the changes seen in the archived 

sample stability study Fairhope, and particularly Goddard marine beach, samples that were held 

for the same total amount of time of two years.

The finding of a lack of stability in the archived samples in this study does not preclude the 

possibility that more favorable results might have been achieved using alternative storage 

conditions. The conditions used for storing the archived NEEAR study samples were adopted 

primarily for practical reasons, such as the availability of freezer space in the NERL Cincinnati 

laboratory, rather than on the basis of what might have been the optimal storage conditions.

Because of our intention of being able to link the results of the two studies, the storage 

conditions used in the long-term holding study were also dictated in large part by the storage 

conditions used for the archived NEEAR study samples. 

It was difficult to interpret results in the literature for their relevance to the specific questions 

posed in this study. Particularly important studies to consider would be those involving similar 

types of cells, similar influences of organic, chemical and other constituents in water samples, 

low copy numbers of the target of interest, and similarities in the conditions and duration of 

storage. However, no studies meeting all of these conditions were identified. 

Recovery of DNA standards after storage under various conditions has been reported. Storage 

conditions that appeared to give the greatest recoveries included storage at -20 °C in buffer or 

glycerol or lyophilized at +4 or -20 °C (Podivinsky et al. 2009, Roder et al. 2010). A number of 

studies have characterized DNA recovery from cells that were in high abundance and not from 

environmental samples (Cannas et al. 2009, King et al. 2009, Rohland and Hofreiter 2007, Smith 

and Morin 2005, Wallenius et al. 2010, Wong et al. 2008). Some of these studies indicate better 

recovery at -80 °C compared to -20 °C. A number of additives have been studied however there 

is generally mixed results with some reporting that the recovery was just as efficient with no 

additives (Smith and Morin 2005). Recovery of DNA from urine samples stored for up to 28 

days indicated a log or more loss of signal from storage at temperatures ranging for 4 °C to -80
1-24

°C (Cannas et al. 2009). Another study indicated more efficient recovery at -20 °C over -80 °C 

for DNA samples analyzed by RFLP from stomach tissue (Molbak et al. 2006). Recovery of 

DNA from ancient and forensic samples has also been studied (King et al. 2009, Rohland and 

Hofreiter 2007, Wallenius et al. 2010, Wong et al. 2008). However, the relevance of these 

studies is also questionable since the recovery efficiency requirements were generally not as 

great and the duration of storage was not comparable. Efficient recovery of viral DNA by qPCR 

was observed from clinical samples after storage of 16 months at -20 °C (Jerome et al. 2002).

Recovery and qPCR amplification of DNA from stored bacterial cells have been reported, 

however, there were still significant differences in the types of cells, samples and storage 

conditions examined in these studies as compared to those examined in the present study. 

Efficient recoveries have been reported from Bacillus anthraces spores stored at 4 °C for 182 

days (Alemeida et al. 2007). However spores may be more stable to environmental influences 

and the duration of storage was much shorter. Studies on the recovery of bacterial DNA from 

stored soil samples reported efficient recoveries in samples stored at -20 or -80 °C with (phenol–

chloroform–isoamyl alcohol) or without additives for storage for 14–30 days (Lauber et al. 2010,

Rissenen et al. 2010). 

As a final point in the comparison of original and archived sample results from the NEEAR 

study, we note that Enterococcus and Bacteroides target organism cells used for the preparation 

of calibrator sample filters by the two laboratories came from different sources. Cells used by 

EMSL originated from laboratory grown cultures and were enumerated as previously described 

(Haugland et al. 2005, Siefring et al. 2008). Cells used by U.S. EPA NERL were enumerated by 

flow cytometry and were acquired in the form of commercially available, lyophilized pellets 

(BioballsTM, BTF, Sydney Australia). To evaluate the comparability of target organism CCE 

estimates in the test samples using calibrator samples prepared from these two cell sources, 

target sequence recoveries from calibrator sample DNA extracts prepared by each of the 

laboratories were examined. Quantitative estimates of target sequence recoveries per calibrator 

cell were obtained by interpolating qPCR CT values from analyses of the calibrator extracts on 

master standard curves generated by each of the laboratories from pooled results of repeated 

qPCR analyses of DNA standards nominally containing from 101 to 4x104 target sequence copies 
1-25

per analysis. Results from these analyses indicated that the mean target sequence recoveries 

from the laboratory grown Enterococcus cells used by EMSL were approximately two-fold 

higher than those from the BioballTM Enterococcus cells used by U.S. EPA NERL. This 

apparent difference may have been related to uncertainty in the applicability of the EMSL 

standard curves. Uncertainty in the EMSL recovery estimates was associated with the fact that 

known concentration DNA standards were only analyzed by this lab during one year of the 

study. In other years only slope (amplification efficiency) values were determined from serial 

dilutions of the calibrator sample extracts. The slope values from this laboratory were not 

considered to be different from year to year as indicated by overlapping 95% confidence ranges. 

If real, the observed difference in target sequence recovery estimates from the two sources of 

cells would result in a systematic bias towards relatively high CCE estimates for the archived 

U.S. EPA NERL test samples compared to those obtained for the original EMSL samples 

implying that, if anything, the actual difference between initial and archived data were even 

greater than the differences in enterococci results of Table 1-5. In contrast, the mean target 

sequence recoveries from the laboratory grown Bacteriodes calibrator cells used by EMSL were 

nearly identical to those from the BioballTM cells used by U.S. EPA NERL as determined from 

the master standard curves generated by the respective laboratories, a condition that does not 

lead to change in Table 1-5 for Bacteroidales.

Conclusions

The archived sample stability study results from Great Lakes and marine beaches indicate that 

significant degradation of samples had occurred. The highly significant changes and low or 

absent correlation between archived and original sample analyses indicate that the archived 

filters cannot be used in a credible manner to establish health relationships involving

Enterococcus qPCR or, by extension, any other indicator, pathogen, or method. Any health 

relationship based on data derived from the archived samples is not useful because these do not 

reflect data that would have been obtained from fresh samples in actual beach monitoring 

circumstances. 

The results from the long-term holding time study, though minor in importance compared to the 

analysis of the archived sample stability study samples, showed small but significant differences 
1-26

in the 24-month holding time study for Bacteroidales and E. coli that further complement the 

findings of the archived sample stability study samples that differences are observed between the 

original and archived samples. 

References

Almeida, J. L., B. Harper, and K. D. Cole. 2008. Bacillus anthracis spore suspensions: 

determination of stability and comparison of enumeration techniques. J. Appl. Microbiol. 

104:1442-1448. 

American Public Health Association (APHA). 1998. Standard Methods for Examination of 

Water and Wastewater. 20th ed. Washington, D.C.

Applied Biosystems. 1997. User Bulletin #2. ABI PRISM 7700 Sequence Detection System. 

http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cm

s_040980.pdf.

Cannas, A., G. Kalunga, C. Green, L. Calvo, P. Katemangwe, K. Reither, M. D. Perkins, L. 

Maboko, M. Hoelscher, E. A. Talbot, P. Mwaba, A. I. Zumla, E. Girardi, and J. F. Huggett.

2009. Implications of Storing Urinary DNA from Different Populations for Molecular Analyses. 

PLoS ONE 4(9): e6985.

Chern, E. C., S. C. Siefring, J. Paar, M. Doolittle, and R.A. Haugland. 2010. Comparison of 

quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes.

Manuscript Submitted.

Haugland R. A., S. C. Siefring, L. J. Wymer, K. P. Brenner, and A. P. Dufour. 2005. Comparison 

of Enterococcus measurements in freshwater at two recreational beaches by quantitative 

polymerase chain reaction and membrane filter culture analysis. Water Res. 39(4):559-568.
1-27

Jerome, K. R., M-L. Huang, A. Wald, S. Selke, and L. Corey. 2002. Quantitative stability of 

DNA after extended storage of clinical specimens as determined by real-time PCR. J. Clin. 

Microbiol. 40:2609-2611.

Lauber C. L., N. Zhou, J. I. Gordon, R. Knight, and N. Fierer. 2010. Effect of storage conditions 

on the assessment of bacterial community structure in soil and human-associated samples. FEMS 

Microbiol. Lett. 307:80-86.

Lunn, D. J., A. Thomas, N. Best, and D. Spiegelhalter. 2000. WinBUGS -- a Bayesian modelling 

framework: concepts, structure, and extensibility. Stat. Comput. 10:325-337.

Ntzoufras I. 2009. Bayesian Modeling Using WinBUGS, John Wiley & Sons, Hoboken, NJ.

Molbak, L., H. M. Sommer, K. Johnsen, M. Boye, M. Jahansen, K. Moller, and T. D. Leser.

2006. Freezing at -80 oC distorts the DNA composition of bacterial communities in intestinal

samples. Curr. Issues Intestinal Microbiol. 7(1):29-34.

Podivinsky, E., J. L. Love, L. van der Colff, and L. Samuel. 2009. Effect of storage regime on 

the stability of DNA used as calibration standard for real-time polymerase chain reaction. Anal. 

Biochem. 394:132-134.

R Foundation for Statistical Computing. 2008. R: A Language and Environment for Statistical

Computing, Vienna, Austria.

Röder B., K. Frühwirth, C. Vogl, M. Wagner, and P. Rossmanith. 2010. Impact of long-term 

storage on the stability of standard DNA for nucleic acid based methods. J. Clin. Microbiol. 

48:4260-4262.

Siefring S. C., M. Varma, E. Atikovic, L. J. Wymer, R. A. Haugland. 2008. Improved real-time 

PCR assays for the detection of fecal indicator bacteria in surface waters with different 

instrument and reagent systems. J. Water Health 6:225-237.
1-28

Smith S., and S. A. Morin. 2005. Optimal storage conditions for highly dilute DNA samples: A 

role for trehalose as a preserving agent. J. Forensic Sci. 50(5):1101-1108.

Wade T. J., R. L. Calderon, E. Sams, M. Beach, K. P. Brenner, A. H. Williams, and A. P. 

Dufour. 2006. Rapidly measured indicators of recreational water quality are predictive of 

swimming-associated gastrointestinal illness. Environ Health Persp. 114(1):24-28.

Wade T. J., R. L. Calderon, K. P. Brenner, E. Sams, M. Beach, R. Haugland, L. Wymer, and A. 

P. Dufour. 2008. High sensitivity of children to swimming-associated gastrointestinal illness: 

Results using a rapid assay of recreational water quality. Epidemiology. 19(3):375-383.

Wade T. J., E. Sams, K. P. Brenner, R. Haugland, E. Chern, M. Beach, L. Wymer, C. C. Rankin, 

D. Love, Q. Li, R. Noble, and A. P. Dufour. 2010. Rapidly measured indicators of recreational 

water quality and swimming-associated illness at marine beaches: A prospective cohort study. 

Environ Health, in press.

Wallenius K., H. Rita, S. Simpanen, A. Mikkonen, and R. M. Niemi. 2010. Sample storage for 

soil enzyme activity and bacterial community profiles. J Microbiol. Meth. 81:48–55.

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Analyses on Environmental Samples. Office of Science and Technology, Washington, D. C. 

EPA- 815-B-04-001.

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Reaction (qPCR) Assay. Available at: http://www.epa.gov/waterscience/methods/method/

biological/rapid1.pdf.

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Chain Reaction (qPCR) Assay. Available at: http://www.epa.gov/waterscience/methods/

method/biological/ methodb2010.pdf.
2-1

Part 2. Short-Term Holding Study: Evaluation of holding refrigerated water 

samples for up to 48 hours for qPCR analysis.

Introduction

This study was conducted to determine whether holding surface water samples under 

refrigeration for 24 and 48 hours affects qPCR-estimated recoveries of DNA target sequences 

from Enterococcus, Escherichia coli, and Bacteroidales bacteria as compared to samples held for 

no more than the currently accepted time limit of six hours. While the qPCR method is normally 

intended to provide rapid determinations of fecal indicator bacteria densities in surface or 

recreational waters, which is contrary to the holding of water samples, instances could arise 

where temporary holding of water samples would be beneficial or necessary. Such instances 

might include when water samples are collected at remote locations where it is not possible to 

transport them to an analytical laboratory immediately after collection. Another example might 

be where it is desired to transport samples to several different laboratories for round-robin 

testing. The current recommended limit on water sample holding time is six hours for 

microbiological culture methods (U.S. EPA 2000). This time limit imposes constraints on the 

applicability of the culture methods for reasons such as those mentioned above. Previous studies 

have suggested that the DNA target sequences of the qPCR method may be more stable than 

cultivability of the respective target organisms (Duprey et al. 1997, Walters et al. 2009, Wery et 

al. 2008). Thus water samples might be held longer than the 6 hour microbiological culture 

method time limit without significant effects on quantitative density estimates of these organisms

as determined by qPCR. The objective of the current study was to provide additional data to 

support this hypothesis.

Sample size (the number of different fresh water and marine water samples) used in this study 

was based on results from a U.S. EPA study in which filter retentates of replicate water samples 

with greater than 100 enterococci cells were distributed to multiple laboratories (U.S. EPA 

Office of Water 2008, unpublished data). A design of approximately 25 samples per water 

matrix (fresh or marine water), with 2 subsamples (filters) per sample was estimated as the 
2-2

required number to enable detection of a decline in target recovery by qPCR of 36% at the 0.05 

critical level (alpha=0.05).

Material and Methods

Water samples

Water samples from 29 freshwater and 23 marine locations in the Boston, MA area were 

collected from July through October, 2008 (Table 2-1). Selection of freshwater and marine 

sampling sites was based on their ambient levels of fecal indicator bacteria (FIB) and their 

proximity to the laboratory that conducted the study. All locations had to be close enough to the 

laboratory to allow collection, transport to the laboratory and filtration of the samples within the 

currently recommended time limit of six hours for microbiological culture methods. A further 

consideration in the selection of the sampling sites was that historic data gathered by the 

laboratory and various collaborators indicated that the water samples could reasonably be 

expected to contain more than the estimated 95% confidence detection limit of the qPCR method 

of approximately 100 target organism cells per sample (unpublished data). Actual samples 

giving lower mean results than 100 enterococci in initial 24 hour analyses were excluded from 

the study to eliminate the possibility of having subsequent results below the detection limit of the 

qPCR methods.

Sample treatments 

In most studies of this nature, samples would be analyzed as soon as possible (within six hours) 

to establish the initial concentration against which samples that have been held for 24 or 48 hours 

would be compared. Complete analysis of the samples within six hours was not possible in the 

present study because of the amount of time necessary to collect samples from diverse locations 

during the day and transport them to the laboratory as well as the amount of time necessary to 

filter multiple aliquots of each water sample to support both this study and the parallel long-term 

holding time study. Instead, within six hours of collection aliquots of each sample was filtered in 

the lab and the filters were stored in a freezer at -20 °C to serve as recovery standards as 

described below. 
2-3

Table 2-1. Boston, MA area sample locations and descriptions

ID Date Collected Source

Freshwater samples

1–5 08/05/08 Charles River, Stony Brook Tributary

6–8 08/11/08 Charles River, Boston Water & Sewer Outfalls

9–11 08/13/08 Charles River, Muddy River Outfalls

22, 24–26 08/25/08 Winns Brook

30 08/27/08 Newtown

36 09/03/08 Salem Sound

37 09/08/08 Ell Pond, Melrose

38 09/08/08 Mill Brook, Melrose

39, 40 09/08/08 Lower Mystic Lake, Arlington

41 09/08/08 Spy Pond, Arlington

52, 53 09/22/08 York Beach, ME, River Rd, Sewage Spiked

58 09/25/08 Concord, NH WWTF, pre-UV

59 09/25/08 Concord, NH WWTF, post-UV

61 09/25/08 Outfall at Wollaston Beach, Sewage Spiked

62 09/25/08 Furnace Brook, Sewage Spiked

63 10/08/08 Lowell WWTF (chlorinated)

Marine samples

14–15 08/18/08 Mystic River, Marine Side of Dam

17–21 08/20/08 Mill Creek, Chelsea

27, 28 08/27/08 E. Boston

29 08/27/08 Revere

32–36 09/03/08 Salem Sound

43, 44 09/15/08 Kings Beach, Lynn

45 09/15/08 Fisheries Beach, Lynn

54–57 09/22/08 York Beach, ME, Clark Rd

60 09/25/08 Wollaston Beach, Sewage Spiked

The different treatments to which subsamples of each water sample were subjected are described 

below and in Table 2-2.
2-4

• Recovery Standard: Multiple 50 or 100 ml aliquots of each water sample were filtered 

within six hours of collection. Replicate filters from each water sample were held at -20

°C until 24 and 48 hours after collection and then extracted for DNA and analyzed by

qPCR. 

• Refrigerated Water: The remainder of each of the water samples was refrigerated and 

then equivalent aliquot volumes to those used to prepare each of the respective recovery 

standard filters were filtered at 24 and 48 hours after collection. After filtration, the 

filters were immediately flash frozen in a -20 °C cooling block for 1 hour and then 

thawed, extracted for DNA and analyzed by qPCR. A freeze/thaw cycle was important in 

order to be consistent with the also frozen and thawed recovery standard filters.

Table 2-2. Description of sample treatments

Treatment Treatment description Extraction and analysis

Recovery Standard Water samples filtered within six hours of 

collection, filters immediately stored at –20 °C 

until extraction and analysis

24 and 48 hours after sample collection

Refrigerated Water Refrigerated water samples filtered 24 and 48 

hours after sample collection, filters 

immediately frozen and thawed and then 

extracted and analyzed

24 and 48 hours after sample collection

The purpose of the recovery standard filters was to establish standards representing the initial 

densities of indicator organisms that could be recovered by the qPCR method when the water 

samples were filtered within six hours of collection. This study design assumed that results from 

samples held for 6 hour or less prior to filtration could be considered to be representative of 

freshly collected samples. This assumption was based on previous holding time studies that have 

shown no significant losses of culturable bacteria counts over this time period (The Public Health 

Laboratory Service Water Sub-Committee 1953). The recovery standard filters were frozen and 

stored instead of processing and analyzing the water samples on the same day that they were 

collected (e.g., within six hours) in part because of the logistical and time challenges associated 

with collecting, transporting and filtering multiple water samples, as indicated above. A second 
2-5

important consideration in the decision to use frozen recovery standard filters was that this 

procedure enabled side-by-side extraction and analysis of these filters under the same conditions 

as the refrigerated water samples at each holding time. Freezing the recovery standard filters 

assumed that the DNA targets of the qPCR method would be preserved in this manner and thus 

allowed these filters to be used as a basis for comparing target organism density estimates in the 

original samples with those in the samples that had been refrigerated. By using this approach, 

run to run variation (batch effects) was eliminated from the analysis.

Each water sample was subjected to both of the treatments (recovery standard and refrigerated 

water) described above and each treatment was analyzed at 24 and 48 hours. Each of the 4 

combinations of treatment and holding time (recovery standard and refrigerated water at 24 and 

48 hours) was performed on duplicate filters and the DNA extracts from each filter were 

analyzed via qPCR in duplicate, giving a total of 8 subsamples and 16 qPCR analyses for each 

sample. 

Sample analyses

Sample analyses were performed as described for the long-term holding time study in part I of 

this report. QPCR assays for Enterococcus (Entero1), general Bacteroidales (GenBac3), E. coli

(EC23S857), and spiked salmon sperm as sample processing controls (Sketa2) were performed 

on each subsample. Primer and probe characteristics of each of these assays are listed below in 

Table 2-3.

Computational methods

Quantitative estimates of target organism calibrator cell equivalents (CCE) in the test samples 

were obtained as described in part I of this report. Multiple replicate calibrator sample filters

were prepared from a single mixed suspension containing pre-determined cell quantities of 

representative target strains of each of the assays (E. faecalis, B. thetaiotaomicron and E. coli) 

just prior to the study. It is noted that while quantitative estimates of target organism densities in 

the samples were calculated by the comparative cycle threshold method and are reported as CCE 

as described above, the role of the calibrator sample CT measurements had no influence on the 

comparisons between recovery standard and refrigerated water samples in this study. Common 
2-6

Table 2-3. Primers and probes for qPCR assays

Assay 

Name

Target Species 

and (Gene) 

Sequences (5’ to 3’)* GenBank Reference

(Base Positions)

Entero1 Enterococcus

(23S rRNA)

F: AGAAATTCCAAACGAACTTG

R: CAGTGCTCTACCTCCATCATT

P: TGGTTCTCTCCGAAATAGCTTTAGGGCTA

AJ295306 (818-837)

AJ295306 (889-909)

AJ295306 (846-874)

GenBac3 Bacteroidales

(16S rRNA)

F: GGGGTTCTGAGAGGAAGGT

R: CcGTCATCCTTCACGCTACT†

P: CAATATTCCTCACTGCTGCCTCCCGTA

M58763 (299-318)

M58763 (409-428)

M58763 (354-380)

EC23S857 E. coli

(23S rRNA)

F: GGTAGAGCACTGTTTtGGCA†

R: TGTCTCCCGTGATAACtTTCTC†

P: TCATCCCGACTTACCAACCCG

DQ682619 (857-876)

DQ682619 (923-944)

DQ682619 (883-903)

Sketa2 Oncorhynchus 

keta (rRNA 

ITS region 2)

F: GGTTTCCGCAGCTGGG

R: CCGAGCCGTCCTGGTCTA

P: AGTCGCAGGCGGCCACCGT

AF170538 (23-38)

AF170538 (82-99)

AF170538 (41-59)

* F = Forward primer, R = reverse primer, P = probe

† Lower case denotes deliberately mismatched base

calibrator sample CT measurements were used in the calculations for the two sets of samples in 

all instances.

Statistical analyses

Statistical analysis was performed using a linear mixed model on logarithm (base 10) of the 

number of CCE as calculated by ∆∆CT. Samples were treated as random. Filters, which were 

performed in duplicate for each sample, were treated as another random factor. Holding time (24 

and 48 hours), water type (freshwater and marine water), and treatment (recovery standard, 

refrigerated water) comprise fixed, controllable, effects. Of particular interest are comparisons 

between recovery standard and refrigerated water at each holding time as the estimator of the 

respective holding time effect.
2-7

Ohio River water holding time study

Given that it was not possible to complete analyses of samples described above within six hours, 

a supplemental study was conducted for the purpose of obtaining data from a more traditional 

design in which a sample was analyzed immediately after holding times of 1, 24 and 48 hours 

without freezing. In February, 2009, a sample of Ohio River water was collected in Cincinnati.

The sample was refrigerated and subsequently 12 aliquots were filtered each at 1 hour, 24 hours 

and 48 hours. All filters were extracted immediately and the DNA extracts from each filter were 

immediately analyzed in duplicate. Identical assays, methods and calculations to those described 

above were performed and used in evaluating the results. This portion of the study also provided 

data on variability among replicate aliquots from the same sample that were used in evaluating 

the importance of any variability introduced by sample holding time, but does not account for 

run to run or “batch” variation in the analysis. Rather than comparison with a parallel recovery 

standard as was necessary in the case of the Boston area samples, 24 and 48 hour holding time 

results from the Ohio River water sample were compared back to the one hour recovery values.

Quality control/ Quality assurance

Quality control and quality assurance measures described in Part I of this report were also 

applied in this project. All QC acceptance criteria were consistently met.

Results

The samples described in Table 2-1 were analyzed by the three qPCR assays and the results are 

given in Figures 2-1 and 2-2 and Table 2-4. Figures 2-1 and 2-2 show box and whisker plots of 

the difference in log10(qPCR calibrator cell equivalents per 100 ml) between refrigerated water 

and their respective recovery standards at 24 and 48 hours for freshwater and marine samples, 

respectively. The box indicates the interquartile range, wherein 50% of the samples lie, and the 

horizontal line indicates the median value for the respective difference. “Whiskers” indicate the 

range of the 90th percentile. Data lying outside the 90th percentile range are individually plotted. 
2-8

Ent 24 h Ent 48 h Bac 24 h Bac 48 h E coli 24 h E coli 48 h

Log10(Cell Equivalents) difference RW-RS

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1 Box shows 50% range, “whiskers” show 90% range, individual points shown 

outside this range. Median value is indicated within each box.

Figure 2-1. Fresh water box and whisker plot1 of differences in log10(calibrator cell 

equivalents per 100 ml): refrigerated water – recovery standards

Ent 24 h Ent 48 h Bac 24 h Bac 48 h E coli 24 h E coli 48 h

Log10(Cell Equivalents) difference RW-RS

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1 Box shows 50% range, “whiskers” show 90% range, individual points shown 

outside this range. Median value is indicated within each box.

Figure 2-2. Marine water box and whisker plot1 of differences in log10(calibrator cell 

equivalents per 100 ml): refrigerated water – recovery standards
2-9

Table 2-4 shows that there are small but statistically significant mean differences in enterococci 

and Bacteroidales assay results for 24 hr refrigerated fresh water samples compared to the 

corresponding recovery standards. No significant differences were found for 24 hr refrigerated 

marine water samples or for 48 hour refrigerated fresh or marine water samples with their 

corresponding recovery standards. 

Table 2-4. Holding time effects: qPCR recovery comparisons between refrigerated water (RW) 

and the recovery standards (RS)

Fresh/ Marine

Holding 

time (h)

Mean log Calibrator Cell 

Equivalents

RS P-value 1 RW2 Difference

Entero1

Combined 24 3.58 3.52 -0.06 0.042

48 3.51 3.53 0.02 0.457

F 24 3.82 3.74 -0.08 0.028

48 3.75 3.71 -0.03 0.356

M 24 3.34 3.31 -0.03 0.460

48 3.28 3.35 0.07 0.132

GenBac3

Combined 24 6.29 6.23 -0.06 0.023

48 6.23 6.26 0.02 0.380

F 24 6.49 6.38 -0.11 0.001

48 6.41 6.41 0.00 0.892

M 24 6.10 6.09 -0.01 0.887

48 6.06 6.10 0.04 0.289

EC23S857

Combined 24 4.32 4.31 -0.01 0.577

48 4.32 4.36 0.03 0.159

F 24 4.65 4.62 -0.03 0.276

48 4.60 4.65 0.05 0.111

M 24 3.98 3.99 0.01 0.823

48 4.04 4.06 0.02 0.641

1 Recovery Standards: filtrates taken within six hours of sample collection and 

representing the initial densities of indicator organisms that could be recovered 

by the qPCR method. 

2 Refrigerated Water: water held for 24 or 48 hours prior to filtration and 

analysis by qPCR.
2-10

These data showed a lack of significant “interaction effects” between water type 

(freshwater/marine water) and treatment (recovery standard / refrigerated water) regardless of 

organism or holding time as shown in Table 2-5. This suggests that the effect of holding a 

sample on qPCR results may be the same regardless of whether it is a freshwater or marine water 

sample. Comparing data combined from both water types continued to show small but 

statistically significantly lower recoveries in refrigerated water samples held for 24 hours for the 

Entero1 and GenBac3 assays (Table 2-4). After 48 hours holding time, on the other hand, none 

of refrigerated water samples, using combined freshwater and marine results, were seen to be 

significantly different from the frozen and presumably fixed, recovery standard samples. As 

Table 2-4 shows, Entero1 and GenBac3 recoveries among recovery standards were slightly 

lower at 48 hours compared to the 24 hour analyses, while refrigerated water samples remained 

fairly constant.

Table 2-5. Evaluation of difference in holding time effects between fresh and 

marine waters

 "Interaction" P-value1

24 hours 48 hours

Entero1 0.53 0.08

GenBac3 0.06 0.65

EC23S857 0.57 0.25

1 Test of difference between fresh and marine waters with 

respect to holding time effect on recovery.

Table 2-6 shows the data from the Ohio River sample. These data show holding time effects at 

48 hours for all assays and at 24 hours for the GenBac3 assay (Table 2-6).
2-11

Table 2-6. Ohio River holding time effects: qPCR recovery comparisons

Holding 

time (h)

Mean log 

CCE

Difference from 

1 hour P-value

Entero1

1 2.98 -

24 2.79 -0.19 0.073

48 2.74 -0.24 0.029

GenBac3

1 4.59 -

24 4.32 -0.27 0.024

48 4.26 -0.33 0.007

EC23S857

1 3.12 -

24 2.87 -0.25 0.080

48 2.69 -0.43 0.004

Discussion

The results from the samples described in Table 2-1 indicated small but statistically significant 

lower recoveries of qPCR targets from two of the three target organism groups among 

refrigerated water samples held for 24 hours based on comparison with their respective recovery 

standards. Ninety five percent confidence intervals for these differences between the held 

refrigerated water and their recovery standards among both freshwater and marine water samples 

combined are shown in Table 2-7. These are further interpreted in terms of equivalent 

percentage differences simply by taking the antilogarithms of the differences in log10(cell 

equivalents per 100 ml). This is the corresponding percentage change in average (geometric

mean) recovery by qPCR over 24 or 48 hours of holding time.
2-12

Table 2-7. Holding time effects: qPCR recovery comparisons between refrigerated water (RW) 

held for 24 or 48 hours and respective recovery standards (RS)

 24 hours 48 hours

RW-RS 95% CI RW-RS 95% CI

Difference in log10(CCE)

Entero1 -0.06 (-0.11, -0.00) 0.02 (-0.03, +0.07)

GenBac3 -0.06 (-0.11, -0.01) 0.02 (-0.03, +0.07)

EC23S857 -0.01 (-0.06, +0.03) 0.03 (-0.01, +0.08)

Difference as a percent of geometric means

Entero1 -12% (-22%, -0%) +5% (-7%, +18%)

GenBac3 -13% (-22%, -2%) +5% (-6%, +18%)

EC23S857 -3% (-13%, +8%) +8% (-3%, +21%)

Relative increase in root mean square error

Entero1 1% (< 5%) 0.2% (< 2%)

GenBac3 3% (< 10%) 0.4% (< 5%)

EC23S857 0% (< 2%) 0.6% (< 4%)

RW-RS: refrigerated water minus recovery standard (log10 difference)

At 24 hours, geometric mean recoveries for both the Entero1 and GenBac3 assays were 12–13% 

lower in the refrigerated water samples than in the recovery standards, with a potential range of 

up to 28% lower. For EC23S857 the difference amounted to 3%, possibly as high as 13%. Near 

parity between refrigerated water and their recovery standards for EC23S857 was indicated by 

the low percent differences and the fact that their respective 95% confidence intervals bracket 

zero.

The use of recovery standards for these samples was an approach to evaluating holding time 

effects on microorganisms in water samples that would not be available for culture based 

methods because samples for culture cannot be preserved through freezing like DNA. By 

controlling predation, chemical reactions, or other factors that might degrade DNA in a water 

sample, the target DNA in organisms deposited on a filter and subsequently frozen were assumed 

to persist without any losses for at least the short holding times involved in this investigation.
2-13

Thus, the recovery standards acted as a control group against which the refrigerated water 

analyses were compared in a side-by-side manner for the 24 and 48 hour time points.

Differences in the qPCR results of the two sets of samples using this side-by-side analysis 

approach was anticipated to minimize any potential influences of method-related “batch” effects, 

i.e., variability between the results of two sets of samples associated with their being extracted 

and analyzed at different times. A potential illustration of this benefit can be seen in 

comparisons of the mean CCE densities estimated in the recovery standards at 24 and 48 hours in 

Table 2-4. Although the overall mean CCE estimates obtained by the E. coli EC23S857 assay 

remained constant, the overall mean CCE estimates obtained by the Entero1 and GenBac3 assays 

differed appreciably between 24 and 48 hours. While not found to be statistically significant, the 

latter differences may be indicative of the aforementioned “batch” effects. A similar difference 

was observed in the 24 and 48 hour refrigerated water results obtained by the E. coli EC23S857 

assay. In this instance an unexpected increase in the 48 hour CCE densities was observed. 

Although the possibility of growth by these indicator organisms in stored water samples can not 

be completely ruled out, we are not aware of any published results demonstrating growth of these 

organisms in refrigerated surface water samples.

Despite the efforts taken to eliminate as many method-related variables as possible that might 

confound the results of this water sample holding time study, the observation of going from a 

significant difference between refrigerated water and recovery standard results at 24 hours to no 

significant difference at 48 hours was unexpected. It is noted that the net change in recovery 

standards between 24 and 48 hours was not significant, even though the differences themselves 

go from being significant at 24 hours to non-significance at 48 hours. As always, lack of 

statistical significance is not convincing evidence for no difference, but only lack of convincing 

evidence for a difference. 

Nevertheless, it is possible that there are variables still unaccounted for in this study. An effect 

associated with the freezing of the recovery standard samples could be such a variable. While an 

attempt was made to control for the potential influence of sample freezing on target DNA 

recovery in the qPCR method by also flash-freezing the refrigerated water filters prior to 

extraction, it could only be assumed that these two freezing methods had the same net effects on 
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DNA recovery. It was also necessary to assume that there were no differential effects of holding 

frozen recovery standard samples for 24 vs. 48 hours. This uncertainty associated with the 

potential effects of sample freezing in this portion of the study was part of the rationale for also 

conducting the supplemental Ohio River water portion of the study where no freezing of the 

samples was involved. It is noted, however, that while the Ohio River water portion eliminated 

freezing effects as a variable, it reintroduced the method-related variables indicated above. 

The experimental design used in the Ohio River sample allowed for the comparison of recoveries 

for 24 and 48 hours based on recoveries at 1 hour which may be a more standard approach for 

comparing recoveries over time. However this analysis approach created the potential to 

introduce greater uncertainties in comparing time point results than with the samples from 

Massachusetts. The uncertainties are associated with extracting and analyzing the different 

holding time samples in different batches. The results from the Massachusetts samples indicate 

that there was run to run or batch effects but they were accounted for in the study design. These 

effects were not considered in the Ohio River sample analysis. The Ohio River results indicate 

that different conclusions may be reached if run to run or batch effects are not a component of 

the analysis.

To put the results of this study in further perspective, any bias as a result of holding a sample 

should be compared to the difference that might be expected among different aliquots from that 

sample. In practice only a single aliquot most likely would be drawn from the sample for 

analysis. A 12% difference in recovery, such as shown in Table 2-7 for the Entero1 and 

Genbac3 assays at 24 hours, might be important if the range of results that could be reasonably 

expected from the “luck of the draw” is, for example, ±10%, but of little importance if the range 

is more like ±50%. Representative data that could be used for determining such differences in 

the analyses of a single aliquot were available from the Ohio River study, where twelve aliquots 

were taken for analysis at each time point. The variances in the indicator density estimates from 

the twelve aliquots that were taken just one hour after collection were used as a basis for 

comparisons with the potential biases that could be attributable to holding time effects. 
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To use the Ohio River data for evaluating a holding-time bias, the concept of mean square error 

(MSE) can be used (Cochran 1963). MSE is the average of the squared deviations of “all 

possible” results from the “true” value of what is being measured (the overall mean log qPCRCE of the water sample at or near the time of collection). If there is no bias (i.e., the sample is 

analyzed immediately), the MSE is obviously the same as the variance of log10(qPCR-CE). If 

there is a bias (e.g., due to holding time degradation) the MSE is equal to this variance plus the 

square of the bias. The square root of the MSE is the root mean square error (RMSE), the same 

as the standard deviation for the unbiased result, and larger than this, influenced by the size of 

the bias, for a biased result. Dividing MSE by variance gives the MSE relative to pure variance, 

and the square root of this (minus 1), the relative increase in RMSE due to bias. Based on the 

observed sample aliquots variances for the respective targets from the 1-hour log10 variances 

pooled over subsamples of Ohio River water, the last section of Table 2-7 shows the relative 

increase in RMSE corresponding to biases represented by the refrigerated water minus respective 

recovery standard log10 differences after 24 and 48 hours of holding time. These range from nil 

up to an 8% increase in RMSE. At the 95% confidence level, the increase in RMSE amounts to 

less than 17%.

This indicates that the potential difference in mean log recovery of enterococci and 

Bacteroidales in held samples may not be an important factor in relation to normal sampling 

variation. The existence of a “statistically significant” difference in recovery (i.e., bias) simply 

means that we are fairly certain that holding affects recovery, not that the bias would be 

considered substantial, and evaluating the magnitude of the estimate bias or potential range of 

this bias in terms of normal sampling variability is one way to evaluate how important the bias 

might be. Note, however, that while sample variability can be compensated for by the collection 

and analysis of additional sample replicates, bias cannot.

Conclusions

There were small, and in some cases statistically significant changes in qPCR-estimated fecal 

indicator densities in subsamples of refrigerated water samples that were held for 24 and 48 

hours prior to filtration compared to recovery standards consisting of additional subsamples of 
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the corresponding water samples that were filtered within six hours. However, these changes 

were neither consistent for the 24- and 48-hour holding times nor were they consistent among 

different indicator groups. In comparisons with subsamples of the Ohio River water sample held 

for just one hour prior to filtration, statistically significant declines in density estimates of one of 

the three indicator groups were observed in subsamples held for 24 hours and of all three 

indicator groups in subsamples held for 48 hours with refrigeration, Taken together, the results of 

the two studies were inconclusive and, as a result, can neither support nor refute

recommendations to hold refrigerated water samples for 24 hours or longer prior to filtration and 

analysis. 

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