United States National Risk Management

Environmental Protection Research Laboratory

Agency Cincinnati, OH 45268

Research and Development EPA/600/SR-97/025 July 1997

Removal of Cryptosporidium and

Giardia through Conventional

Water Treatment and Direct

Filtration

Project Summary

Eva C. Nieminski

Pilot- and full-scale evaluations of

Giardia and Cryptosporidium cyst removals through direct filtration and conventional water treatment were conducted by the Utah Department of Environmental Quality. Cysts were seeded

continuously in a step dose at a 0.5

gpm pilot plant, and in a spike at a 900

gpm full-scale plant; both plants were

operated under conventional treatment

and direct filtration regime. The results

of 20 pilot-scale cyst seeding trials and

8 full-scale trials indicated that source

water quality (turbidity and algal content), as well as treatment effectiveness in removing turbidity, controlled

the removal of seeded Giardia and

Cryptosporidium. Changes in source

water quality influenced removal rates

more than the mode of treatment.

Higher removal rates were consistently

observed for Giardia cysts (3.3-log) than

for Cryptosporidium oocysts (3.0-log).

A high correlation was found between

cyst removal rates and removal of the

respective size particles; poorer correlation existed between cysts and turbidity removal, while no significant correlation was established between the

removals of cysts and heterotrophic

bacteria.

To assure that the best available detection method was used in enumeration of the cysts in raw and treated

water, two versions of the immunofluorescence staining method were evaluated for their efficiencies in detecting

Giardia cysts and Cryptosporidium oocysts seeded at known concentrations

in water: (1) the ASTM method for detection of Giardia cysts and Cryptosporidium oocysts in low-turbidity water and (2) a modified Sauch’s procedure employing sampling by 2.0 μm membrane filters, Percoll/Percoll step gradient

flotation, and immunofluorescence staining on 2.0 μm porosity polycarbonate membrane filters. The second method was selected, since it was characterized by higher

recovery rates in all three types of waters

tested: raw surface water, partially treated

water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water

turbidity regardless of the method used.

Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts

in all types of water sampled.

This Project Summary was developed

by EPA’s National Risk Management

Research Laboratory, Cincinnati, OH,

to announce key findings of the research project that is fully documented

in a separate report of the same title

(see Project Report ordering information at the back).

Introduction

Problem Statement and Study

Objectives

The Surface Water Treatment Rule requires all public water system treating surface water to effectively remove enteric

viruses and Giardia cysts. The removal of

microbial contaminants by filtration is being re-evaluated by the U.S. Environmental Protection Agency in conjunction with
2

the publication of the proposed Interim

Enhanced Surface Water Treatment Rule.

While the removal requirements for Giardia may be increased depending on cyst

concentration in raw water, additional, more

stringent regulations may be developed to

control Cryptosporidium in response to health

concerns surrounding this pathogen. As new

rules are being developed, allowable filtration

credits should be revisited and possibly revised. Also, as the analytical methods for

detection of Giardia and Cryptosporidium cysts

in water are being improved and new methods developed, surrogate water quality parameters should be established to allow for

an accurate, economical, and practical evaluation of cyst removal effectiveness through

treatment.

The project objectives were designed to

address some of the questions associated with the development of the new

regulations. The specific tasks were to

examine the most critical relationships in

removal of Giardia and Cryptosporidium

by comparing the effectiveness of Giardia

and Cryptosporidium removal through conventional treatment with that resulting from

direct filtration, the effectiveness of Giardia removal with Cryptosporidium removal,

and the effectiveness of Giardia and

Cryptosporidium removals with the removals of turbidity, cyst-size particles, and heterotrophic bacteria.

To enable accurate evaluation of Giardia and Cryptosporidium removal efficiency

in water treatment processes, a reliable

method for measuring the concentration

of these pathogens in water must be used.

Therefore, another objective of this

project was to select an analytical method

capable of measuring the concentration of

Giardia cysts and Cryptosporidium oocysts

that would be accurate, reliable, flexible,

and verifiable, and could be applied to

measuring the cyst/oocyst removal performance of water treatment processes.

Procedure

Evaluation of Analytical

Methods

Two methods for finding Giardia cysts

and Cryptosporidium oocysts in water were

compared. Both methods follow flotation

steps and immunofluorescence staining.

These methods were 1) The American

Society for Testing and Materials (ASTM)

method and 2) another immunofluorescence antibody (IFA) method, referred to

as the alternate method, and applied principally by Ongerth and Stibbs. The objective was to evaluate the two IFA methods

using three factors as criteria for comparison. First, the applicability to cyst seeding

experiments in full- or pilot-scale water

treatment plant was evaluated. Second,

the applicability of the methods to cyst

detection in environmental water samples

of varying water quality was assessed.

The third criterion was the economics associated with the two methods. The superior method was then used in the cyst

seeding experiments in the pilot- and in

the full-scale treatment plant.

The ASTM method involves sampling

100 L or more of water through a 1.0 μm

porosity polypropylene yarn cartridge filter, extracting the particulates from the

cartridge filter, and concentrating the extracted particulates by centrifugation. The

concentrated particulates are then processed to selectively concentrate cysts

and oocysts by flotation in 50 mL tubes

on a Percoll/sucrose gradient. The particulates recovered at the interface of the

Percoll/sucrose are stained with fluorescent-tagged antibodies on 25 mm diameter, 0.2 μm pore size cellulose acetate

filters. After mounting on slides, the membrane filters are scanned using a UV

epifluorescent microscope for objects of

the right size, shape, and fluorescence

characteristic as Giardia cysts and Cryptosporidium oocysts. On finding such objects the microscope optics are switched

to phase contrast to look for internal characteristics of the organisms.

The alternate method involves filtration

of the water sample through either a 293

or 142 mm diameter, 2.0 μm pore-size

polycarbonate membrane filter; recovery

of particles from the filter by rinsing and

scraping them from the surface; and concentration of the particulates by centrifugation. The cysts and oocysts are then

selectively concentrated from other particulates by flotation in 15 mL tubes on a

two-step Percoll/Percoll gradient, followed

by IFA staining on 13 mm diameter, 2.0

μm pore-size polycarbonate membrane filters. After mounting on slides, the membrane filters are scanned using an UV

epifluorescent microscope for objects of

the right size, shape, and fluorescence as

Giardia cysts and Cryptosporidium oocysts.

Confirmation of internal structures is not

performed in this method.

Cyst Seeding and Sampling

Procedures

Monitoring of raw and filtered water quality

was conducted throughout the seeding trials.

In addition to monitoring the major water quality parameters, particle counting in four size

ranges (2-4 μm, 4-7 μm, 7-14 μm, and 14-25

μm), was performed during the seeding trials.

Raw water sources were sampled and analyzed for background count of Giardia and

Cryptosporidium, naturally occurring in the

two watersheds.

Inactivated, formalin-fixed Giardia

lamblia cysts and Cryptosporidium parvum

oocysts were used in seeding experiments

in a pilot- and a full-scale treatment plant.

A total of 20 trials were conducted in the

pilot plant, and 8 trials in the full-scale

plant, alternating between conventional

treatment and direct filtration.

The first site for testing was a pilot plant

residing at the 180 MGD Jordan Valley

Water Treatment Plant in Bluffdale, UT.

The pilot plant simulated the actual, conventional treatment plant, treating Provo

River water downstream from Deer Creek

Reservoir. Water flow rate was maintained

at 0.5 gpm. Alum was used as a coagulant, at dosages established through jar

testing. After each seeding trial using the

conventional treatment train, the water was

re-routed through the direct filtration train

for seeding trials the following day. Alternating between the conventional treatment

and direct filtration allowed for a comparison of treatment effectiveness of the water of comparable quality.

The second site was a 900 gpm Huntington Water Treatment Plant, situated

near Price, UT. The plant was operated at

600 gpm for cyst seeding experiments.

Polyaluminum chloride was used as a coagulant. The Huntington Plant was operated by conventional treatment during the

first four seeding trials. After converting

the plant to direct filtration mode, another

four seeding trials were performed.

Detection of Giardia and

Cryptosporidium Cysts

The alternate IFA method for sampling,

processing, and detection of Giardia and

Cryptosporidium cysts was chosen for the

seeding trials, based on results of the method

comparison. Samples were collected by membrane filtration through 2.0 μm porosity, 293

mm diameter polycarbonate membrane filters, processed on Percoll/Percoll step gradient in 15 mL centrifuge tubes, stained on 2.0

μm porosity, 13 mm diameter polycarbonate

membrane filters, and enumerated under an

epifluorescent microscope.

In calculations of cyst removal through

treatment, a direct ratio of the difference

between the cysts seeded and the cysts

detected was calculated for each run in

the pilot plant. Two cyst removal rates

were determined based on two different
3

initial cyst concentrations: concentration

in the seeding solution prior to being

pumped into the raw influent, and concentration in the seeded influent already mixed

with the raw influent. The cyst removal

rates, achieved in the full-scale plant, were

also calculated as relative differences between the influent and effluent concentrations, but the influent concentrations were

adjusted for dilution of cysts in respective

basins.

Three conservative assumptions were

made for data interpretation in calculations of the removal rates, both in the

pilot- and full-scale plant. First, the removals based on the cyst concentrations found

in the seeded influent were used in data

analysis. Otherwise, the higher cyst concentrations found in the seeding solutions

would result in higher removal rates reported. Secondly, cyst removal rates were

calculated only for trials in which cysts

were detected in both influent and effluent

samples. Otherwise, calculations of removals during trials when cysts were not detected in the effluent would be based on

very low detection limits, and therefore

would result in higher removal rates. Finally, no adjustments were made for the

differences in cyst recovery efficiencies in

turbid raw influent samples versus clean

filtered effluent samples. Adjusting for low

recovery rates in influent samples would

also result in higher removal rates being

calculated.

Results and Discussion

Evaluation of Two IFA Methods

for Detection of Giardia and

Cryptosporidium

The effectiveness of cyst recovery from

spiked water samples was impacted primarily by the number of analytical steps

involved in the cyst detection. Both IFA

methods were characterized by low recovery efficiency, when seeded raw water

samples were filtered, then concentrated,

transferred to gradients, stained, and enumerated. Higher recovery rates were observed in detecting Giardia cysts when

the alternate method was employed (Figure 1). In spiked raw water samples, an

average 12% of the seeded Giardia cysts

were detected by the ASTM method, while

the alternate method was characterized

by an average 49% Giardia cysts recovery efficiency. Recovery rates for Cryptosporidium oocysts in spiked raw water averaged 8% detected by the ASTM method

and 9% detected by the alternate IFA

method. The recovery rates in filtered water were 14% and 52% for Giardia and

12% and 12% for Cryptosporidium, using

the ASTM, and the alternate method, respectively. In flocculated water samples,

an average 22% of Giardia cysts were

detected using the ASTM method and 40%

using the alternate method. The recoveries of Cryptosporidium in flocculated water were 7% with the ASTM method and

1% with the alternate method.

Losses were demonstrated to occur due

to incomplete yarn cartridge filtration. In

sampling raw water, 5% Giardia cysts and

6% Cryptosporidium oocysts were captured from the yarn cartridge filtrate by

passing it through a 293 mm diameter 2.0

μm pore size Nuclepore membrane filter.

An average 7% of Giardia and 8% of

Cryptosporidium were recovered from

membrane filters after cartridge filtration.

The sampling step resulted in a high loss of

seeded Giardia cysts and Cryptosporidium

oocysts. When the sampling step was eliminated and cysts were seeded directly onto

flotation gradients, the resulting recovery rates

increased dramatically (Figure 2). An average

53% of Giardia cysts and 27% of Cryptosporidium oocysts was detected from seeded

Percoll/sucrose gradients used in the ASTM

method. The alternate method employing

Percoll/Percoll flotation, yielded recoveries of

82% for seeded Giardia cysts and 69% for

Cryptosporidium oocysts.

The highest cyst recovery rates were

reported when both sampling and flotation

steps were avoided and spiked with Giardia cysts and Cryptosporidium oocysts

samples were stained directly onto the

membrane filters used for IFA assay. The

ASTM method resulted in recoveries of

72% and 56% for Giardia cysts and CryptoFigure 1. Water quality vs. recovery of cysts seeded into water.

60

50

40

30

20

10

0

Giardia Cryptosporidium Giardia Cryptosporidium

S.D.=16

S.D.=18

S.D.=28

S.D.= 6

S.D.= 13

S.D.= 1

S.D.= 13

S.D.= 91

S.D.= 47

S.D.= 4

S.D.= 2

S.D.= 3

ASTM method Alternate method

Water source

Flocculated Raw, 5 NTU Filtered

% Recovery of seeded cysts
4

sporidium oocysts, respectively. The alternate method was characterized by an

average recovery of 86% for Giardia cysts

and 78% for Cryptosporidium oocysts.

Testing of cyst recovery efficiencies from

raw, flocculated, and filtered water indicated that any increase in water turbidity,

whether due to presence of algae or to

that of chemical floc, resulted in a significant decrease in parasite recoveries. The

results also indicate both methods were

more effective in detecting seeded Giardia cysts than Cryptosporidium oocysts.

The results of testing the processing

and detection methods indicated that recoveries of cysts were substantially and

consistently higher with the alternate

method, employing Percoll/Percoll step

gradient combined with IFA on 13 mm

polycarbonate filters, than with the ASTM

method, in all three types of water. Consequently, the alternate method was selected as the method of choice for seeding experiments. Added support for this

choice was provided by a comparison of

the qualitative advantages and disadvantages of the two methods. In summary,

the main advantage of the ASTM method

was its ability to confirm presumptive cysts

and oocysts. The most serious disadvantages of this method were its relatively

high cost and the amount of time required

to complete it. The alternate method, on

the other hand, was found to be less expensive and required less time to complete than the ASTM method. Attractive

features of the membrane filter sampling

method include relatively small sample

volumes, flexibility, and compatibility with

frequent seeded controls. The major limitation of the alternate method was its lack

of a confirmation step.

Experience with detecting Giardia cysts

and Cryptosporidium oocysts in the waters tested during this study and the results generated during this part of the

study, indicate that the alternate method

be recommended to evaluate water treatment processes that use high concentrations of seeded parasites in which algae,

occurring in concentrations much lower

than the seeded parasites, are not of concern. The alternate method can be considered for analysis of environmental

samples, particularly for low-turbidity waters. When high water turbidity requires

higher-volume samples to be collected and

examined, and when cross-reacting algae

should be differentiated from the organisms of interest, the ASTM method should

be used with environmental samples.

The results of this stage of the study

have indicated Giardia cysts and especially, Cryptosporidium oocysts are lost

during the gradient flotation steps of both

methods. Therefore, it is recommended

that the flotation step should be avoided,

whenever possible when processing

treated (filtered) water samples.

A hybrid method, combining the most

efficient steps from the two methods,

should be investigated. Such a hybrid

method should include sampling by membrane filtration only for low-turbidity waters. High-turbidity waters should be

sampled by the ASTM cartridge sampling

method. Since the Percoll/Percoll step gradient in 15 mL tubes is more economical

and had higher cyst recovery than the

Percoll/sucrose gradient, it should be used.

Staining on cellulose acetate membranes,

as opposed to polycarbonate membranes,

allows the demonstration of the internal

morphological characteristics of the organisms. Consequently, staining on cellulose

acetate membranes should be incorporated into a hybrid method. Elvanol mounting medium should not be incorporated

into a hybrid method. As a water-based

medium, it is not compatible with the dehydrated cellulose acetate membrane and

does not allow the membrane to be cleared

so that the cyst’s internal structure can be

visualized by contrast microscopy.

Removal of Giardia and

Cryptosporidium through

Conventional Water Treatment

and Direct Filtration

A general observation about removal of

seeded Giardia and Cryptosporidium was

made that was valid in both pilot- and fullscale plant throughout the entire seeding

studies. Consistent removal rates of Giardia and Cryptosporidium were achieved,

when the treatment plant was producing

water of consistently low turbidity (0.1-0.2

Figure 2. Water quality vs. recovery of cysts seeded into flotation gradients. % Recovery of seeded cysts

100

80

60

40

20

0 Giardia Cryptosporidium Giardia Cryptosporidium

S.D.= 13

ASTM method Alternate method

Water source

Flocculated Raw, 5 NTU Filtered

S.D. = 8

S.D. = 33

S.D. = 14

S.D. = 22

S.D. = 9

S.D. = 32

S.D. = 23

S.D. = 26

S.D. = 21

S.D. = 25

S.D. = 8

S.D. = 8
5

NTU). As soon as the plant’s performance

changed, and resulting filtered water turbidity fluctuated, a high variability in cyst

concentrations was detected in collected

samples.

Removal of Cryptosporidium was compared with removal of Giardia by both

methods of treatment, conventional and

direct filtration, at both the full-scale plant,

and the pilot plant. Trials at the full-scale

plant were impacted by the change in

seasonal water temperature and algal content. Because of the need for a construction to bypass the sedimentation basin,

the first four trials by conventional treatment were conducted from June through

September, while the four trials using direct filtration were conducted in November and December. Greater flexibility of

the pilot plant allowed trials by both treatment methods to be conducted within one

day of each other and enhanced not only

the comparison between removal of

Cryptosporidium with Giardia removal, but

also the comparison between conventional

treatment and direct filtration, as well as

the comparison of cyst removal with removal of other water quality indicators.

Removal of Seeded Giardia and

Cryptosporidium During PilotScale Seeding Trials

Table 1 summarizes cyst removal rates

calculated based on cyst concentration in

seeded influent (after mixing the cysts with

the incoming raw water). These removal

rates, based on seeded influent concentration, were consistently lower than the

removal rates based on cyst concentrations in seeding solution (indicating 99.99%

or 4 log removal for both Giardia and

Cryptosporidium regardless of the treatment mode). Calculations of cyst removals, observed during seeding experiments,

can also be highly impacted by measurements of cyst concentration in filter effluent samples. When cysts were not detected in filter effluent samples, their concentration could be estimated based on

analytical detection limits, determined for

each sample batch. Such estimates lead

to underestimation of cyst concentration

in finished water samples and in turn,

result in overestimation of calculated cyst

removal rates.

Removal of Seeded Giardia and

Cryptosporidium During Full-Scale

Seeding Trials

Table 2 presents a summary of results

and removal rates calculated only from the

trials where cysts were detected both in

influent and effluent in the full-scale plant.

Similar to the pilot-scale experiments, the

removal of Giardia and Cryptosporidium can

be overestimated when calculations are

based on estimated effluent concentrations.

Removal rates based on cyst concentrations detected and enumerated both in influent and effluent can be considered conservative.

Several factors impacted the results of

the full-scale seeding trials, which made

the comparison between conventional

treatment and direct filtration more dependent on uncontrolled variables. Changes

in raw water quality, observed from the

time the plant was in operation by the

conventional mode, compared to raw water quality during operation by the direct

filtration mode, influenced removal rates

more than the mode of treatment. The

water was treated in the conventional plant

during summer, when treatability was more

difficult, while direct filtration was used in

late fall, when the water was easier to

treat. The presence of prolific algal blooms

in samples collected during the first four

trials, and the lack of algal content in

samples from the last four trials, was another variable making the comparison of

removal data problematic.

The results of the pilot-plant experiments

indicate that Giardia cysts were removed

more effectively than were Cryptosporidium oocysts. This observation was valid

regardless of the treatment mode. The

difference between log removals of Giardia and Cryptosporidium ranged from 0.1

Giardia removal Cryptosporidium removal

Percent Log Percent Log

Trial No. Date removal removal removal removal

Conventional treatment

1-C 4/27/93 ND ND 99.65 2.81

2-C 5/11/93 99.16 2.20 98.66 1.94

3-C 5/25/93 ND ND 99.87 2.94

4-C 6/8/93 99.98 3.90 99.95 3.98

5-C 6/22/93 ND ND ND ND

6-C 7/6/93 99.95 3.69 99.88 2.94

7-C 7/20/93 99.95 3.69 99.45 2.64

8-C 8/4/93 ND ND ND ND

9-C 8/17/93 99.91 3.03 99.69 2.84

10-C 8/31/93 99.98 3.90 99.96 3.78

Average log removal 3.40 2.98

Standard deviation 0.67 0.64

Direct Filtration

1-D 4/29/93 ND ND 99.95 3.60

2-D 5/13/93 ND ND ND ND

3-D 5/28/93 99.78 2.90 92.06 1.31

4-D 6/15/93 ND ND 99.96 3.78

5-D 6/23/93 ND ND ND ND

6-D 7/8/93 ND ND ND ND

7-D 7/22/93 99.90 3.00 99.80 2.90

8-D 8/5/93 ND ND ND ND

9-D 8/19/93 ND ND 99.92 3.31

10-D 9/2/93 99.99 4.00 99.84 2.93

Average log removal 3.30 2.97

Standard deviation 0.77 0.89

Table 1. Removal of Giardia and Cryptosporidium Through Conventional Treatment

 and Direct Filtration at Jordan Valley

ND indicates that cysts were not detected in filter effluent.
6

to 1.1 log and averaged 0.3 log, as calculated across all seeding runs. The difference between cysts and oocysts removals were even more pronounced in the

full-scale plant than those observed in the

pilot plant.

Among the seeding trials, where the

cysts were detected both in influent and

effluent, the following average removals

were calculated for the pilot plant runs:

• average removal of Giardia

through conventional treatment:

3.40 log; S.D. = 0.67

• average removal of Cryptosporidium

in conventional treatment:

2.98 log; S.D. = 0.64

• average removal of Giardia through

direct filtration:

3.30 log; S.D. = 0.77

• average removal of Cryptosporidium

through direct filtration:

2.97 log; S.D. = 0.89

Similarly, the following average removals were reported from the full-scale seeding trials:

• average removal of Giardia through

conventional treatment:

3.26 log; S.D. = 0.67

• average removal of Cryptosporidium

in conventional treatment:

2.25 log; S.D. = 0.47

• average removal of Giardia through

direct filtration; 3.87 log

• average removal of Cryptosporidium

through direct filtration:

2.79 log; S.D. = 0.19

Taking into consideration that the reported removal rates of Giardia and

Cryptosporidium were calculated very conservatively, the values presented above

can be interpreted as expected removals,

resulting from a consistent performance

and steady operation of the treatment

plants.

Surrogate Parameters for

Evaluation of Giardia and

Cryptosporidium Removal

Both surface water sources treated in the

treatment plants were characterized by a high

quality water, with low levels of inorganic,

organic, and microbial contamination. Both

sources, however, were tested positive for

both Giardia and Cryptosporidium cysts, confirming previous hypothesis and observations

about these pathogens being ubiquitous in

surface waters.

Both effectiveness and consistency of removal of seeded Giardia and Cryptosporidium cysts depended on the effectiveness

and consistency of the removal of turbidity.

When raw water turbidity was high and it

could not be removed by direct filtration, resulting removals of seeded cysts were low

and inconsistent. On the other hand, if treatment by direct filtration consistently produced

low turbidity effluent, resulting cyst removals

were comparable to those achieved from conventional treatment.

The results of seeded cyst removals,

generated throughout the study regardless of treatment mode, were compared

with the respective results from particle

counting, turbidity measurements, and heterotrophic bacteria counts. Correlation between Giardia and Cryptosporidium cyst

removal and removal of these potential

surrogates are presented in Figures 3

through 5.

The analysis of correlation between cyst

removal and particle removal was performed separately for Giardia cyst and

cyst-size particles, and for Cryptosporidium oocysts and oocyst-size particles

(Figure 3). High correlation was reported

between both sets of data. A correlation

coefficient of 0.82 was calculated (p<0.1)

for the relationship between Giardia cyst

removal and removal of particles ranging

between 7 μm and 11 μm. Similarly, a

correlation coefficient for the relationship

between Cryptosporidium oocyst removal

and removal of 4 μm to 7 μm particles

was 0.79.

The results indicated that particle counting could serve as a reliable indicator of

cysts and oocysts removal. Particle

counters, even though capital intensive,

are cheap to operate and are more sensitive than the assays used in Giardia and

Cryptosporidium analyses.

Much lower correlation was established between removals of Giardia and Cryptosporidium and removal of turbidity (correlation coefficients of 0.64 and 0.55, respectively). As

presented in Figure 4, log removal of turbidity can be used as an indicator of cyst

and oocyst removals, but with lower accuracy than particle counting. The most pronounced differences between removal of

Giardia and Cryptosporidium and the expected removals of turbidity were observed,

when very high cysts removals (4-log) were

reported.

Giardia removal Cryptosporidium removal

Percent Log Percent Log

Trial No. Date removal removal removal removal

Conventional treatment

1-C 6/11/92 99.95 3.7 99.60 2.78

2-C 7/7/92 ND ND 99.05 2.07

3-C 8/5/92 ND ND 97.87 1.89

4-C 10/6/92 99.66 2.82 ND ND

Average log removal 3.26 2.25

Standard deviation 0.67 0.47

Direct Filtration

1-D 11/10/92 99.97 3.87 99.75 2.88

2-D 11/20/92 ND ND 99.82 2.92

3-D 12/8/92 99.97 3.87 99.37 2.57

4-D 12/22/92 ND ND ND ND

Average log removal 3.87 2.79

Standard deviation 0.00 0.19

ND indicates that cysts were not detected in filter effluent.

Table 2. Removal of Giardia and Cryptosporidium Through Conventional Treatment and

Direct Filtration at Huntington
7

Heterotrophic plate count was not shown

to be a surrogate in evaluation of cyst

removals (Figure 5). No correlation was

found between log removal of seeded cyst

and log removal of HPC. Despite the fact

that the filters in both pilot- and full-scale

plants were backwashed with chlorinated

water, a growth of heterotrophic bacteria

was reported in the filters during the seeding experiment and plant run.

Plant performance evaluation using particle counting and turbidity measurement

can be an effective tool in evaluating expected removals of Giardia and Cryptosporidium. The search for a biological surrogate for Giardia and Cryptosporidium

should continue and result in identification

of a parameter that defines both occurrence and removal of Giardia and Cryptosporidium. The results of the study coincide with previously reported relationships

between cysts and particulates. Effective

removal of Giardia and Cryptosporidium

from the water would, however, require

treatment plants to consistently produce

very low turbidity (0.1-0.2 NTU)—much

lower than the levels currently required.

Conclusions and

Recommendations

Analytical Procedures for

Detection of Cysts in Water

Based on the results generated during

the evaluation of the effectiveness of the

IFA methods in enumeration of Giardia

cysts and Cryptosporidium oocysts, it was

concluded that the alternate IFA method

was more suitable for meeting the project

objectives. This method employed samFigure 3. Relationship between removal of cysts and particles.

Figure 4. Relationship between removal of cysts and turbidity.

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

0 1 2 3 4

Log removal of 7-14 m particles u

Log removal of Giardia

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

0 2 4 6

Log removal of 4-7 m particles u

y = 0.8856x + 0.4647

R = 0.7935 2 y = 0.9246x + 0.7221

R = 0.8247 2

Log removal of Cryptosporidium

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

0 1 2 3

Log removal of turbidity

Log removal of Giardia

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

0 1 2 3

Log removal of turbidity

y = 0.9631x + 1.1000

R = 0.5623 2 y = 1.0093x + 1.6697

R = 0.6429 2

Log removal of Cryptosporidium
8

pling through a 2.0 μm polycarbonate

membrane filter, centrifugation in 15 mL

tubes, flotation on a two-step Percoll/

Percoll gradient, IFA staining on 13 mm

diameter, 2.0 μm pore-size polycarbonate

membrane filters, and enumeration under

an UV epifluorescent microscope. The following conclusions were formed:

• The membrane filtration for low turbidity samples outperformed the

polypropylene yarn sampling method

in terms of recovery efficiency of

seeded organisms.

• The Percoll/Percoll step gradient had

better recoveries of seeded organisms then the Percoll/sucrose gradient. Since 15 mL tubes were used in

place of 50 mL tubes, the procedure

of the step gradient flotation was more

economical.

• The alternate method has proven

more effective in recovering seeded

cysts, and therefore, was considered

more suitable in parasite seeding experiments, where evaluation of water

treatment process efficiencies was

conducted using high concentrations

of seeded cysts.

The ASTM method, employing staining

on cellulose acetate membranes, had the

advantage since the gradients could be

cleared and the internal structure of the

organisms could be visualized under the

phase- or differential-interference contrast

microscopy. The ASTM method, due to its

ability to confirm presumptive cysts and

distinguish between algal cells and the

cysts by contrast microscopy, was found

very applicable in testing of the environmental water samples.

Based on the above results, it is recommended that the alternate method should

be used in evaluating water treatment process efficiencies using high concentrations

of seeded parasites. The ASTM method

is recommended in analyses of environmental samples where the confirmation

step is essential.

A hybrid method, combining the most efficient steps from the two methods, should be

investigated. The membrane filtration for low

turbidity samples, which far outperformed the

polypropylene yarn sampling method in terms

of recovery efficiency of seeded organisms,

shows promise in sample collection. Similarly,

Percoll/Percoll step gradient, used in the alternate method, had better recoveries of seeded

organisms then the Percoll/sucrose gradient.

Since 15 mL tubes were used in place of 50

mL tubes used in the ASTM method, the

procedure of the step gradient flotation was

more economical. On the other hand, staining

on cellulose acetate membranes used in the

ASTM method, had the advantage since the

gradients could be cleared and the internal

structure of the organisms could be visualized

under the phase- or differential- interference

contrast microscopy.

The results of the testing of IFA method

effectiveness have indicated that Giardia cysts

and especially Cryptosporidium oocysts are

lost during the gradient flotation steps of both

methods. Recovery efficiencies increased dramatically in samples, did not contain much

debris, and could be processed without the

flotation steps and stained directly on membranes. Therefore, it is recommended that the

flotation step should be avoided when processing treated (filtered) water samples whenever possible.

Removal of Giardia and

Cryptosporidium through

Conventional Treatment and

Direct Filtration

The following conclusions were formed

from the pilot- and full-scale study on Giardia and Cryptosporidium cysts removal

through conventional treatment and direct

filtration:

• In a properly operated treatment plant

effectively removing turbidity to 0.1-

0.2 NTU, either conventional treatment or direct filtration can result in a

3-log removal of Giardia.

• Cryptosporidium oocysts are more difficult to remove than Giardia cysts,

Figure 5. Relationship between removal of cysts and heterotrophic bacteria.

0 0.5 1

Log removal of HPC

Log removal of Giardia

0 0.5 1

Log removal of HPC

y = 0.7586x + 2.2524

R = 0.0753 2 y = 0.7675x + 2.5245

R = 0.0841 2

Log removal of Cryptosporidium

4

3.5

3

2.5

2

1.5

1

0.5

0

4

3.5

3

2.5

2

1.5

1

0.5

0
9

both in a conventional plant and

through direct filtration (up to 1.0-log

difference).

• Removals of cyst-size particles and

removal of turbidity can be used as

indicators of cyst removal effectiveness.

A general observation about removal

of seeded Giardia and Cryptosporidium

cysts was made that was valid in both

pilot- and full-scale plant throughout the

entire seeding studies. Both effectiveness

and consistency of removal of seeded

Giardia and Cryptosporidium cysts depended primarily on the effectiveness and

consistency of the removal of turbidity.

When treatment by direct filtration consistently produced low turbidity effluent

(0.1-0.2 NTU), the resulting Giardia and

Cryptosporidium cyst removals were consistent and comparable to these achieved

from conventional treatment. As soon as

the plant’s performance changed, and resulting filtered water turbidity fluctuated,

a high variability in cyst concentrations

was detected in collected samples. When

raw water turbidity was high, and it could

not be removed by direct filtration, resulting removals of seeded cysts were low

and inconsistent.

A high correlation coefficient was calculated for the relationship between Giardia cyst

removal and removal of particles ranging between 7 μm and 14 μm, and similarly, for the

relationship between Cryptosporidium oocyst

removal and removal of particles of 4 μm to 7

μm in size. Much lower correlation was established between removals of Giardia and

Cryptosporidium and removal of turbidity. Heterotrophic plate count was not shown to be a

surrogate in evaluation of cyst removals, with

no correlation found between log removal of

seeded cyst and log removal of HPC.

A combination of particle counting and

turbidity measurement was shown to be

an effective tool in water treatment plant

performance evaluation in terms of predicting removals of Giardia and Cryptosporidium. Effective removal of Giardia and

Cryptosporidium from the water would,

however, require treatment plants to consistently produce very low turbidity (0.1-

0.2 NTU)—much lower than the levels

currently required. The results of the study

indicate that the removal of particulates,

measured through particle counting and

turbidity monitoring, should be a critical

factor used in the evaluation of plant performance in Giardia and Cryptosporidium

removal. Continuous and consistent removal of particulates should be monitored

by continuous particle counting and turbidity monitoring.

The results of the project imply that the

credits given for Giardia cyst removal in

direct filtration plants, may be similar to

credits obtained in conventional treatment

plants, and also may be higher than the

credits applicable under the current regulations. Since Cryptosporidium is more difficult to remove than Giardia, and it is also

more resistant to disinfection than Giardia, new requirements need to be developed to control this pathogen. Finally, due

to the need of further defining the credits

given to treatment plants for physical removal of Giardia and Cryptosporidium, a

study on evaluation of removal of these

pathogens through pre-sedimentation

should be conducted.

The full report was submitted in fulfillment of CR818895-010 by the Utah Department of Environmental Quality under

the sponsorship of the U.S. Environmental Protection Agency.
10

Eva C. Nieminski is with the Utah Department of Environmental Quality, Salt Lake

City, UT 84114.

Kim Fox is the EPA Project Officer (see below).

The complete report, entitled "Removal of Cryptosporidium and Giardia through

Conventional Water Treatment and Direct Filtration," (Order No. PB97-162507;

Cost: $35.00, subject to change) will be available only from:

National Technical Information Service

5285 Port Royal Road

Springfield, VA 22161

Telephone: 703-487-4650

The EPA Project Officer can be contacted at:

National Risk Management Research Laboratory

U.S. Environmental Protection Agency

Cincinnati, OH 45268

United States

Environmental Protection Agency

Center for Environmental Research Information

Cincinnati, OH 45268

Official Business

Penalty for Private Use $300

EPA/600/SR-97/025

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POSTAGE & FEES PAID

EPA

PERMIT NO. G-35