PROTOZOA IN WATER

COLLECTING FINISHED
WATER SAMPLES

If Giardia or Cryptosporidium concentrations
in your source water samples exceed 1 per li-
ter during the first 12 months of sampling,
then you must monitor finished water as well
as source water. If you are required to collect
samples from both, collect the finished water
sample first, then the source water sample.

Receiving and verifying the contents of your
sampling kit are addressed in STEPS 1
and 2 of the source water sampling section.

When you are ready to collect your finished

water protozoa sample, bring the following

items with you to the sampling location:

\4 Shipping container sent by the laboratory

Sampling apparatus

Q Fluid proportioning injector (for adding
2% thiosulfate solution to neutralize
effects of chlorination or other disinfectant
treatments)

Plastic sample bags

Sample labels

(4 Frozen ice packs

(2 Several pairs of new latex gloves

Q Approximately 2 gal (4 L) of 2% sodium
thiosulfate solution

(2 Sterile, 250- or 500-mL graduated cylinder
Q Thermometer

FINISHED WaTER —19


ENTERIC VIRUSES IN WATER

Connect the Cartridge Housing Module
& < >| containing the 1-MDS filter to the Pre-
filter Module. Then, reconnect the Dis-
charge Module to the outlet end of the Car-
tridge Housing Module.

42— Source WaTeR


ENTERIC VIRUSES IN WATER

‘Turn off the water at the tap and decide
| whether you need to insert additional
modules into the sampling train.

For source water sampling, you may need to
use the Single Injector Module and/or the Pre-
filter Module.

First, determine if you need to use the Single
Injector Module.

If your pH value is greater than 8.0, you need
to insert the Single Injector Module between
the Regulator and Discharge Modules before
proceeding.

a
&}t—

Single
Injector
Module

tL.

3B— Source warer


Return the Regulator Module and the Injector
Module to the laboratory for cleaning and
sterilization.

Place the Sample Data Sheet in a plastic bag
and pack it on top of the sampling apparatus.

You may need to use additional
packing material to ensure that the
contents of the box will not shift
during transport.

Seal the container and ship it by over-

night courier to the laboratory. Call the

laboratory and notify them of the
sample shipment.

ENTERIC VIRUSES IN WATER

Source Water —47


PROTOZOA IN WATER

Put on new latex gloves to prevent con-

yy) tamination from outside sources. Ster-

ile technique must be used when sam-

pling for Giardia and Cryptosporidium. Any

contamination of the sampling apparatus may
bias the final results.

Assemble the sampling apparatus as shown
below and connect the inlet end of the sam-
pling apparatus to the sampling tap or to an
extension hose connected to the tap.

Inlet End

! Be sure that the filter housing does not
contain the filter.

Note the water meter reading, then slowly turn
on the water.

12— Source Water


ENTERIC VIRUSES IN WATER

minute

‘How ) gallons

Flow
Rate

SB— FINISHED WaTER

10mIThiosulfate _ / Thiosulfate ml
1 gallon water ~ te}

Pour the 2% thiosulfate into a graduated cyl-
inder.

Next, using the formula below, calculate the
rate of thiosulfate injection and adjust the
thiosulfate injector to deliver 10 mL of thiosul-
fate per gallon of flow.

Injection Rate} ‘Trinute

After the thiosulfate flow rate is adjusted,
transfer the injector tube to the carboy of thio-
sulfate.

Monitor the thiosulfate flow rate visually
throughout sampling.

] If there is no suction visibly drawing
down the thiosulfate, or if too much is
flowing, adjust the water bypass screw further
to increase or decrease the pressure differen-

tial between the two gauges, until the flow is
regulated properly.
Connect the Cartridge Housing Mod-
ule. Then reconnect the Discharge
Module to the outlet end of the Car-
tridge Housing Module.

Slowly, start the water flowing through the
sampling apparatus.


ENTERIC VIRUSES IN WATER

Flow } ‘minute

( ‘Bow ) gallons
Rate

56— FINISHED WaTER

10 ml Thiosulfate _ Thiosulfate mi
1 gallon water ~ te,

from the second injector into the thiosulfate
solution.

If there is no suction visibly drawing down

the 2% thiosulfate or the HCl, or if too
much is flowing, adjust the water bypass screws
further to increase or decrease the pressure dif-

ferential between the two gauges, until the flow
is regulated properly.

Adjust the smaller injector screw on the hy-
drochloric acid injector to add sufficient hy-
drochloric acid to maintain a pH of 6.5 to 7.5.

After adjusting the injector, transfer the injec-
tor tube to the carboy of 0.1-molar hydrochlo-
ric acid. As sampling proceeds, periodically
check the pH to ensure that it remains be-
tween 6.5 and 7.5.

Record the adjusted pH on the Sample Data Sheet.
Next, using the formula below, calculate the
rate of thiosulfate injection and adjust the
thiosulfate injector to deliver 10 mL of thiosul-
fate per gallon of flow.

Injection Rate minute

After the thiosulfate flow rate is adjusted,
transfer the injector tube to the carboy of thio-
sulfate.

Monitor the thiosulfate flow rate visually
throughout sampling.

Disregard the next section and proceed to step
7 (page 58).


ENTERIC VIRUSES IN WATER

Ship the container by overnight courier
BRg| © the laboratory Call the laboratory and
notify them of the sample shipment.

62— FINISHED WaTER


ENTERIC VIRUSES IN WATER

‘Turn off the water at the tap and decide
Check whether you need to use the Single In-

pH . .
jector or Double Injector Module.

Insert pubs

li
Ispx ses Eetween || Adust Hcl |_| THe le
> 8 Regulator and Injection

‘Sischarge Injection
Modules

No

et ode r your pH value is greater than 8.0, you need
Regulatorand| © inject the hydrochloric acid and 2% thiosul-
Discharge | fate solution simultaneously. If your pH value
q is less than 8.0, you need to inject only the 2%
Adjust thiosulfate solution.
Timeaton Disregard the next two pages and proceed to
page 57 if your pH value is less than 8.0.

54— FINISHED WaTER


Reassemble the filter housing, and reconnect
the inlet and outlet hoses.

Slowly, start the water flowing through the
sampling apparatus.

Using the pressure regulator, adjust the pres-
sure on the upstream pressure gauge to no
more than 30 psi. Using the water bypass
screw, readjust the downstream pressure
gauge to read 35% less than the upstream
gauge, if necessary.

Record the following information on the
sample label:

+ Time sampling started

+ Initial water meter reading (including
units)
+ Turbidity

PROTOZOA IN WATER

Stop Time: __ Meter Reading: ‘Turbidity
‘Start Time: ‘Meter Reading: Turbidity:
Operator Name: Total Volume Filtered:

Date: Sampling Location:

Place the filter housing in an upright position.

Monitor the water meter to ensure that the
flow rate does not exceed | gallon/min (ap-
proximately 4 liters/min).

FINISHED WaTER —23


ENTERIC VIRUSES IN WATER

Record the following information on the
Sample Data Sheet:

J Date sampling started
(4 Time sampling started

(Q Initial water meter reading (including
units)

SAMPLE DATA SHEET

SAMPLE NUMBER:

WATE! NTU

WATER pH: TEMPERATURE: ac ‘TURBIDITY:

INIT. METER READING: (CHECK UNITS) ft __gallons
date: time:

FINAL METER READING: (CHECK UNITS) ft __gallons
date:

‘TOTAL SAMPLE VOLUME: liters

(Fina Intil meter readings x 28.316 (for readings inf)
__01 3.7454 (or readings in gallons)

CONDITION ON ARRIVAL:

COMMENTS:

Source Water —43


COMMONLY ASKED QUESTIONS

QUESTIONS COMMONLY ASKED BY
DRINKING WATER UTILITIES

What is the purpose of the
ICR rule?

What pathogens are moni-
tored under the ICR rule?

The ICR rule was developed by EPA to collect
occurrence, exposure, and treatment data on
drinking water pathogens and disinfectant by-
products. The pathogen data are needed to de-
termine whether current Surface Water Treat-
ment Regulations should be revised to include
new or more stringent treatment levels for
some microbes. The disinfectant by-product
data are needed to determine whether to regu-
late the chemical by-products that form when
disinfectants react with organic chemicals in
source water.

Although drinking water utilities will be in-
volved in collecting both disinfectant by-prod-
uct and waterborne pathogen data under the
ICR rule, this manual describes the utility's
role in collecting data on drinking water
pathogen occurrence.

The ICR rule requires public water supply sys-
tems to monitor source water (and finished wa-
ter in some cases) for the following pathogens:

+ Giardia cysts

+ Cryptosporidium oocysts

+ Total culturable viruses

+ Fecal coliform or Escherichia coli bacteria
+ Total coliform bacteria

EPA is considering revising the current Sur-

face Water Treatment Regulations because ex-
isting treatment levels for Giardia and viruses


ENTERIC VIRUSES IN WATER

While the water is flushing the sampling appa-
ratus, begin completing your sample data
sheet. Record the following information:

Sample number
@ System location
Q Sampler’s name

SAMPLE DATA SHEET

SYSTEM LOCATION:
SAMPLER'S NAME:
WATER NTU
WATER p) TEMPERATURE: i TURBIDITY:
INIT. METER READING: (CHECK UNITS) ft __gallons
}_ dat _ time: -
FINAL METER READING: ft __gallons
date:

‘TOTAL SAMPLE VOLUME: liters

(Pinal nial meter readings x 28.316 (for readin

{or 3.78S4 (for readings in gallons)) :
CONDITION ON ARRIVAL:
COMMENTS:

36— SourCE WaTER


PROTOZOA IN WATER

Turn on the water at the tap and allow

beg) the water to flow for 2 to 3 minutes or

L until any debris that has accumulated in

the sampling line has cleared or the turbidity
in the water becomes uniform.

‘Turn the water off at the tap

Put on new latex gloves to prevent con-

#24) tamination from outside sources. Ster-

ile technique must be used when sam-

pling for Giardia and Cryptosporidium. Any

contamination of the sampling apparatus may
bias the final results.

Pressure Regulator

Assemble the sampling apparatus by inserting
the fluid proportioning injector
module between the first pres-
sure gauge and the filter hous-
ing, as shown.

Fluid Proportioning
Injector Module

Water Meter
Effluent Hose
Fiter
Holder Flow Control Valve

Connect the inlet end of the sampling appara-
tus to the sampling tap or to an extension hose
connected to the tap.

20— FINISHED WaTER


PROTOZOA IN WATER

Seal the container and follow the labor-
atory’s instructions related to the

cleaning, storage, and return of sam-
pling equipment.

Ship the container by overnight courier
to the laboratory.
16

Call the laboratory and notify them of
the sample shipment.

26— FINISHED WaTER


PROTOZOA IN WATER

22— FINISHED WATER

Pour the 2% sodium thiosulfate solution into
a graduated cylinder. Place the injector tube in
the thiosulfate solution, and adjust the smaller
injector screw, located on the bottom of the in-
jector, so that the flow rate of the 2% thiosul-
fate solution is approximately 10 milliliters
per minute.

If there is no suction visibly drawing
down the thiosulfate solution, or if too
much is flowing, adjust the water bypass
screw further to increase or decrease the pres-
sure differential between the two gauges. A
greater differential between the upstream and
downstream gauges increases the flow rate; a
smaller differential decreases the flow rate.

After the thiosulfate flow rate is adjusted
properly, transfer the injector tube to a carboy
of thiosulfate. You will need to monitor this
rate visually throughout sampling to ensure
that an adequate amount of thiosulfate is be-
ing added to neutralize all of the disinfectants.

Turn off the water at the tap and empty the
water in the filter housing.

Open the filter packaging as aseptically
by | 2s possible and carefully drop the filter
into the filter housing.

i" Hold the loose gasket in place.



Measure the pH, temperature, and tur-

bidity of the source water flowing from

the effluent hose. Record the readings
on the sample data sheet.

SAMPLE DATA SHEET

ENTERIC VIRUSES IN WATER

SYSTEM LOCATION:

SAMPLER'S NAME:

‘WATER
WATER pH: ‘TEMPERATURE: 3
INIT. METER READING: (CHECK UNITS)
date: time:
FINAL METER READING: (CHECK UNITS)
date: time:

TOTAL SAMPLE VOLUM!

(inal Initial meter readings x 28.316 (for readings inf)
fo 3.7454 (for readings t gallons)

liters

CONDITION ON ARRIVAL:

COMM

Source Water —37


PROTOZOA IN WaTER

Disconnect the sampling apparatus
from the water tap.

Be sure to hold the inlet hose above the

level of the outlet hose opening while the
water drains from the housing. This will pre-
vent backwash and loss of particulate matter
from the filter.

Disconnect the inlet and outlet hoses from the
filter housing.

fj Put on fresh latex gloves.
e

As aseptically as possible, remove the
" filter from the housing and put it into a

plastic sample bag.
Pour all of the water remaining in the
12 filter housing into the same plastic bag.

inside the second plastic sample bag.
Transfer the label or label information
to the outside of the outer bag.

a Seal the plastic sample bag and place it

1@— Source WaTER


PROTOZOA IN WaTER

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(Gare peysiuly 404)
@INpoW s0;eLu}

Buluoodoig pini.4

soyeinBey eunsseig

20Z0}01q Buly99]}05 404) urea, Buljdwies


ENTERIC VIRUSES IN WATER

34— Source Water

Turn on the water at the tap and allow
bq the water to flow for 2 to 3 minutes or

until any debris that has accumulated
in the sampling line has cleared or the turbid-
ity in the water becomes uniform.

Put on new latex gloves to prevent con-

tamination from outside sources. Ster-

ile technique must be used when sam-
pling for enteric viruses. Any contamination
of the sampling apparatus may bias the final
results.

Turn off the water at the tap.

Remove the foil from the backflow regulator
on the Regulator Module and connect it to the
water tap or to an extension hose connected to
the tap.

Remove the foil from the other end of the
Regulator Module and from the Discharge
Module. Connect the Discharge Module to the
Regulator Module.

Place the end of the Discharge Module, or an

extension hose connected to the Discharge
Module, into a 1-liter plastic bottle.

Note the water meter reading, then slowly turn
on the water.

Using the pressure regulator, adjust the water
pressure to no more than 30 psi.


ENTERIC VIRUSES IN WATER

|

30

Q Cartridge Housing Module:

+ I-MDS Zetapor Virosorb filter inside a
filter holder

Q Discharge Module:
+ Water meter
+ Flow control valve

The laboratory will also ship three additional
modular sections, as required by your facility.
These may include:

Q Single Injector Module:
+ Fluid proportioning injector
+ Pressure gauge

O Double Injector Module:
+ Two fluid proportioning injectors, in
parallel
+ Pressure gauge


ComMoNty ASKED QUESTIONS

ceptible to environmental stress and disinfection
than protozoa and viruses, and would be elimi-
nated by any system that eliminated more resis-
tant pathogens. However, the ICR rule requires
drinking water utilities to submit coliform moni-
toring data as general indicators of water quality.
Monitoring procedures for fecal coliform, E. coli,
and total coliform densities have been estab-
lished and are not addressed by this manual.

Which drinking water Public water supply systems that serve be-

utilities have to collect pro- tween 10,000 and 100,000 people and use sur-

tozoan and virus samples? face water (or groundwater under the influ-
ence of surface water) are required to monitor
their source water for Giardia cysts and
Cryptosporidium oocysts.

Public water supply systems that serve more
than 100,000 people and use surface water (or
groundwater under the influence of surface
water) are required to monitor their source
water for Giardia cysts, Cryptosporidium oo-
cysts, and total culturable viruses. If pathogen
densities in the source water exceed 1 patho-
gen per liter during the first 12 months of
monitoring, then public water supply systems
also must sample finished water for the re-
maining months.

How often must samples be Public water supply systems that serve be-
taken? tween 10,000 and 100,000 people must collect
samples every two months for 12 months.

Systems that serve more than 100,000 people
must take samples every month for 18 months.


COMMONLY ASKED QUESTIONS

may not be adequate to protect public health
for systems supplied by poor-quality source
water and because of the new threat posed by
Cryptosporidium.

Giardia cysts in drinking water cause more
reported waterborne disease outbreaks than
any other single known pathogen. They also
are more resistant to environmental stresses
and disinfection than almost all other known
waterborne pathogens.

Cryptosporidium oocysts in drinking water have
caused major waterborne disease outbreaks in
the U.S. and other countries and are even more

resistant to disinfection than Giardia.

Several enteric viruses have caused waterborne
disease and may be responsible for many, if not
most, of the outbreaks where a causative agent
was not identified (about half of all reported
outbreaks). Adequate analytical methodology
is not yet available for routine analysis
enteric viruses, so EPA has required monitoring
of total culturable viruses. Total culturable vi-

for many

ruses are a group of enteric viruses commonly
found in poor-quality waters and which EPA
believes are at least somewhat representative of
other pathogenic viruses. Monitoring for total
culturable viruses is useful because this group
contains pathogens and is a potential indicator
of other viral pathogens.

Fecal coliforms, E. coli, and total coliforms have
been used for decades to as: source water
quality. Coliform bacteri

are much more sus-



Put the bags containing the filter into
‘al the shipping container. Place the ice
14 packs around, but not on, the sample
bag to prevent freezing the sample. You may
want to insert several inflated, empty sample
bags between the sample and the ice packs.

(gy) Seal the container and follow the lab-

oratory’s instructions related to the
[15] cleaning, storage, and return of sam-
pling equipment.

to the laboratory. Call the laboratory and

Ship the container by overnight courier
i6i .
notify them of the sample shipment.

PROTOZOA IN WATER

Source Water —17


SAMPLE COLLECTION PROCEDURES
FOR DETECTING PROTOZOA IN WATER



PROTOZOA IN WATER

Allow at least 264 gallons/1000 liters of

f=}} water to pass through the filter. Ata flow
rate of approximately | gallon/minute,

this will require about 4 hours and 45 minutes.

Volume In Volume In Volume In
Sampling Step GALLONS LITERS FT?
Protozoa Flow Rate | _1 per minute 4 per minute .13 per minute
Protozoa Finished
Water Sample 264 1000 36

When the water meter indicates that

fo} 264 gallons/1000 liters of water have

passed through the filter, turn off the
water at the tap.

Record the following information on the
sample label:

+ Time sampling stopped
+ Final water meter reading (including units)
+ Final turbidity

+ Total volume filtered

Start Time: Meter Reading: Turbidity
Operator Name: ‘Total Volume Filtered:
Date: Sampling Location:

Disconnect the sampling apparatus
from the water tap.

' Be sure to hold the inlet hose above the
level of the outlet hose opening while the
water drains from the housing. This will pre-

24— FINISHED WATER


United States Office of Ground Water EPA/814-8-95-001
Environmental and Drinking Water June 1995
Protection Agency Washington, DC 20460

Information Collection
Requirements Rule—
Protozoa and Enteric Virus
Sample Collection Procedures



ENTERIC VIRUSES IN WATER

50— FINISHED WaTeR

(1 Approximately 2 gal (4 L) of 2% sodium
thiosulfate solution

G Approximately 2 gal (4 L) of 0.1-molar
hydrochloric acid solution (for adjusting
pH, if necessary)

2 sterile, 250- or 500-mL graduated
cylinders

Q Plastic sample bags

Sample data sheet

Qi Frozen ice packs

O Several pairs of new latex gloves

Q pH meter

O Thermometer

Turn on the water at the tap and allow

he water to flow for 2 to 3 minutes or

until any debris that has accumulated
in the sampling line has cleared or the turbid-
ity in the water becomes uniform.

Turn off the water at the tap.

ut on new latex gloves to prevent con-
‘amination from outside sources. Ster-
ile technique must be used when sam-
pling for enteric viruses. Any contamination
of the sampling apparatus may bias the final
results.

Remove the foil from the backflow regulator
on the Regulator Module and connect the
module to the water tap or to an extension
hose connected to the tap.


Protozoa IN WATER

Plastic sample bags

Q Ice packs for shipping the collected
samples

G Sample labels

If you are missing any items, contact your labo-
ratory immediately. Do not attempt to collect
the samples without a complete sampling kit.

Once you have verified the contents of the
sampling kit, place the ice packs in the
freezer and repack the box for later use.


PROTOZOA IN WATER

you all of the equipment needed to col-

lect samples for Giardia cyst and Crypto-
sporidium oocyst analyses. When you receive
the sampling kit, check the contents of the car-
ton. The sampling kit should contain the
following items:

1 | Each month, your laboratory will send

(4 Sampling train for collecting protozoa (left):

~Inlet hose

~ Pressure regulator with pressure gauge

-Fluid proportioning injector module,
including an injector and pressure
gauge*

=1-pm nominal porosity filter and holder
made by Parker Hannifan or Filterite

~ Water meter

~ Effluent hose and flow control valve

“Needed for finished water sample collection only.


CREDITS AND
ACKNOWLEDGMENTS

The use of Manufacturer Trade Names in the
production does not constitute endorsement
by the U.S. Environmental Protection Agency.

This video was prepared for the U.S. Environ-
mental Protection Agency, Office of Ground
Water and Drinking Water by DynCorp Viar
and HP Productions, Inc. under contract to
Wade Miller Associates, Inc. (Contract Num-
ber: 68-C2-0113, Subcontract Number: 0113-
02)

U.S. EPA Staff:

Jim Walasek, P.E., Work Assignment Manager
Shay Fout, Ph.D., Technical Advisor

Frank Schaefer, Ph.D., Technical Advisor
Fred Williams, Graphics Advisor

Special thanks to the management and staff of
the Fairfax County Water Authority.

63


SAMPLE NUMBER:

ENTERIC VIRUSES IN WATER

Record the following information on the
Sample Data Sheet:

Q Date sampling ended
Q Time sampling ended
Q Final water meter reading (including units)

SYSTEM LOCATION:

SAMPLER'S NAME:

WATER
WATER pH: TEMPERATURE: °c TURBIDITY:
INIT. METER READING: (CHECK UNITS) ft? —__ gallons
date: time:
FINAL METER READING: (CHECK UNITS) _f —_ gallons
date: time:
‘TOTAL SAMPLE VOLUME: liters
(Pinal nia meter readings x 28.316 (or readings inf)
a (0 3.7454 (or readings in galloos))
CONDITION ON ARRIVAL:
COMMENTS:

Source Water —45


ENTERIC VIRUSES IN WATER

COLLECTING SOURCE
WATER SAMPLES

When you are ready to collect your virus
sample, bring the following items with you to
the sampling location:

Shipping container sent by the laboratory
O Regulator Module

Q Cartridge Housing Module

Q Discharge Module

Single Injector Module (for adding 0.1-molar
hydrochloric acid to adjust pH, if necessary)

Q Prefilter Module (for filtering sediment
from highly turbid water, if necessary)

Q Approximately 2 gal (4 L) of 0.1-molar
hydrochloric acid solution (for adjusting
pH, if necessary)

{2 Sterile, 250- or 500-mL graduated cylinder
O Plastic sample bags

Sample data sheet

Q) Frozen ice packs

Q Several pairs of new latex gloves

Q pH meter

Q Thermometer

Q Turbidimeter

Source Water —33


ENTERIC VIRUSES IN WATER

Remove the foil from the other end of the
Regulator Module and from the Discharge
Module and connect the Discharge Module to
the Regulator Module.

Place the end of the Discharge Module, or an
extension hose connected to the Discharge
Module, into a 1-liter plastic bottle.

Virus Sampling Apparatus with
Regulator and Discharge Modules

Regulator
Module

Discharge
}~ Module

Note the water meter reading, then slowly turn
on the water.

Using the pressure regulator, adjust the water
pressure to no more than 30 psi.

FINISHED WaTER —51


( Prefilter Module:

+ 10 uum polypropylene filter inside a filter
holder

The ends of each module should be

wrapped in foil to ensure that the equip-
ment remains free of contamination. If your
modules are unprotected or compromised,

please contact your laboratory immediately
for further instructions.

If you are missing any items, contact your
laboratory immediately. Do not attempt to col-
lect the samples without a complete sampling
kit.

Once you have verified the contents of the
sampling kit, place the ice packs in the freezer
and repack the box.

ENTERIC VIRUSES IN WATER

Fitter

‘iter Holder

31


COMMONLY ASKED QUESTIONS

Where should samples be
collected?

Who will analyze the
samples?

However, these systems may discontinue moni-

toring if:

+ Viruses are not detected in the source
water during the first 12 months of
monitoring, or

+ Source water has been tested for either
total coliforms or fecal coliforms at least
five times per week for four months before
and two months after the effective date of
the ICR and the total coliform density is
less than 100 colonies/100 mL or the fecal
coliform density in 90 percent of all
samples is less than 20 colonies/100 mL.

Samples must be taken at the intake of each
treatment plant. If a plant has several sources
of water, the system must sample the blended
water from all sources. If this is not possible,
the source with the highest expected pathogen
concentration should be sampled.

EPA has approved several laboratories to ana-
lyze the protozoan and virus samples. Before
collecting samples, you must arrange to have
them analyzed by an EPA-approved laboratory.

If you have not already located an approved
laboratory, notify:
ICR Laboratory Coordinator
EPA Office of Ground Water & Drinking Water
26 West Martin Luther King Drive
Cincinnati, Ohio 45268.
EPA will provide you with a list of approved
laboratories or other appropriate guidance.


PROTOZOA IN WATER

Be sure that the filter housing does NOT
CH) contain the filter.

Note the water meter reading, then slowly turn
on the water.

Using the pressure regulator, adjust the

f=¥| water pressure on the first pressure
gauge to no more than 30 psi. Ss
Flush the sampling apparatus with 20 gallons/

76 liters of water by allowing the water to flow
through the system and out the effluent hose.

While the water is flushing the sampling appa-
ratus, begin completing your sample label.
Record the following information:

+ Sampler’s name
+ Date
+ Sample location

Stop Time: Meter Reading: ‘Turbidity:

Start Time: Meter Reading: —__ Turbidity: __
Operator Name: Total Volume Filtered: =
Date: ‘Sampling L 7

|] Now, you must adjust the thiosulf
6 injector.

m~ First, using the water bypass screw,
the larger top screw in the injector, adjust
the pressure on the downstream pressure
gauge to be at least 35% less than the pres-
sure shown on the upstream gauge. For
ample, if the upstream gauge reads 30}
then the second gauge should read no m
than 19psi.

FINISHED WaTeR —21


Put on fresh latex gloves.

Carefully, disconnect the sampling apparatus
from the water tap.

Disconnect the Cartridge Housing Module
from the sampling train, Turn the filter hous-
ing upside down and allow excess water to
flow out as waste water,

‘Turn the housing upright, and cover the mod-
ule ends with sterile foil.

q Do not attempt to open the filter housing.

The filter and filter housing are
shipped to the laboratory intact. The
Discharge Module may be retained at
the utility and reused.

Place the filter housing into an insu-
lated shipping box.

Set the ice packs around the housing.

Return the Regulator Module and the Injector
Module to the laboratory for cleaning and
sterilization,

Place the Sample Data Sheet in a plastic bag
and pack it on top of the sampling apparatus.
Seal the container.

You may need to use additional
packing material to ensure that the
contents of the box will not shift
during transport.

ENTERIC VIRUSES IN WATER

FINISHED WaTeR —61


ENTERIC VIRUSES IN WaTER

40— Source Water

OPH = 651075

Adjust the smaller injector screw, located on
the bottom of the injector, so that the flow rate
of the HCI is sufficient to maintain a pH of 6.5
to 7.5.

7] If there is no suction visibly drawing
down the HCl, or if too much HCl is

flowing, adjust the water bypass screw fur-
ther to increase or decrease the pressure dif-
ferential between the two gauges. A greater
differential between the upstream gauges in-
creases the flow rate; a smaller differential
decreases the flow rate.

After the HCI flow rate is adjusted properly,
transfer the injector tube to a carboy of HCl.
Periodically check the pH to ensure that suf-
ficient HCI is being added to maintain a pH of
6.5 to 7.5.


Record the adjusted pH on the Sample Data
Sheet.

Next, determine if you need to use the Prefil-
ter Module.

Turn off the water at the tap, and note the tur-
bidity. If the turbidity is greater than 75 NTU,
or for conditions where the 1-MDS filter is ex-
pected to clog before sampling is completed,
you will need to use the Prefilter Module.

Disconnect the Discharge Module and connect
the Prefilter Module to the Regulator Module
or the Injector Module, if it is being used.

ENTERIC VIRUSES IN WATER

_A

Prefilter
Module

Source Water —41


ENTERIC VIRUSES IN WATER

pH < If your pH value is less than 8.0,
|X  | it does not need to be adjusted,
and you can use the Single Injector Module to
inject the 2% sodium thiosulfate solution.

Insert the Single Injector Module between
the Regulator and Discharge Modules before

proceeding.
Single Injector
lodule
: 1
=

‘Turn on the water at the tap and adjust the
water bypass screw so that the pressure on
the downstream pressure gauge is at least
35% less than the pressure shown on the
Regulator Module gauge.

FINISHED WaTER —57


SAMPLE COLLECTION PROCEDURES FOR
DETECTING ENTERIC VIRUSES IN WATER



ENTERIC VIRUSES IN WATER

Collect 317 - 396 gallons or 1200 to
1500 liters of finished water.

Volume In Volume In Volume In
Sampling Process | GALLONS LITERS FT?
Virus Finished
Water Sample 317 - 396 1200 - 1500 43-53

jj When the water meter indicates that 317
- 396 gallons / 1200 - 1500 liters of wa-
ter have passed through the filter, turn

off the water at the tap.

Record the following information on the
Sample Data Sheet:

Date sampling ended

Time sampling ended

( Final water meter reading (including units)

SAMPLE DATA SHEET

SAMPLE NUMBER:

M LOCATION:

R NTU
TEMPERATURE: “c TURBIDITY:
(CHECK UNITS) nv gallons
(CHECK UNITS) ft _ gallons
TOTAL SAMPLE VOLUMI liters
(Pati 128316 (for readings in)
z allows)

60— FINISHED WaTER


PROTOZOAN AND ENTERIC VIRUS SAMPLE
COLLECTION PROCEDURES AS DEFINED BY
THE INFORMATION COLLECTION
REQUIREMENTS RULE

This manual describes the procedures for col-
lecting source water and finished water sam-
ples for protozoan and enteric virus monitor-
ing under the Information Collection
Requirements (ICR) rule. This manual and
the accompanying video comprise a two-part
set of instructional materials that provide
public water supply systems with the informa-
tion needed to properly collect samples for
protozoan and virus monitoring. All water
utility personnel involved with ICR monitor-
ing should watch the video and review this
manual before collecting any samples.

The protozoan collection procedures de-
scribed in this manual and in the video are
based on the procedures in the ICR Protozoan
Method for Detecting Giardia_cysts and
Cryptosporidium Oocysts_in Water_by a
Fluorescent Antibody Procedure. The total
culturable virus collection procedures
described in this manual and in the video are
based on the procedures in the Virus Monitor-
ing Protocol for the Information Collection
Rule. Both of these methods can be requested
by calling the Safe Drinking Water Hotline, at
(800) 426-4791.



ENTERIC VIRUSES IN WATER

COLLECTING FINISHED
WATER SAMPLES

If the concentration of any pathogen in

your source water samples exceeds 1

per liter during the first 12 months of
sampling, then you must monitor finished
water as well as source water.

Sampling of finished water begins in the same
manner as sampling of source water de-
scribed previously, as follows:

When you are ready to collect your finished
water virus sample, bring the following items
with you to the sampling location:

(2 Shipping container sent by the laboratory
O Regulator Module

(4 Cartridge Housing Module

1 Discharge Module

( Single Injector Module (for adding 2% thio-
sulfate solution to neutralize effects of chlo-
rination or other disinfectant treatments)
Double Injector Module (for adding 2%
thiosulfate solution to neutralize effects of
chlorination or other disinfectant treat-
ments while adding 0.1-molar hydrochloric
acid to adjust pH, if necessary)

FINISHED WATER —49


ENTERIC VIRUSES IN WATER

46— Source WaTER

f Put on fresh latex gloves.

Carefully, disconnect the sampling apparatus
from the water tap.

Disconnect the Cartridge Housing
Module from the sampling train.

Turn the filter housing upside down and allow
excess water to flow out as waste water.

Turn the housing upright, and cover the mod-
ule ends with sterile foil.

q Do not attempt to open the filter housing.

If you are using the Prefilter Module, discon-
nect it from the sampling train, repeat the
draining procedure, and cover the module
ends with sterile foil.

The filters and filter housings are
shipped to the laboratory intact.
The Discharge Module may be re-
tained at the utility and reused.

Place the filter housings into an insulated
shipping box.

Set the ice packs around the housings.


Using the pressure regulator, adjust the
water pressure to no more than 30 psi.
Flush the sampling apparatus with 20

gallons/ 76 liters of water by allowing the wa-
ter to flow through the system and out the
effluent hose.

PROTOZOA IN WATER

at

Volume In Volume In Volume In
Sampling Step GALLONS LITERS FT?
System Flush 20 76 27

While the water is flushing the sampling appa-
ratus, begin completing your sample label.
Record the following information:

+ Sampler’s name
+ Date

+ Sample location

Stop Time: Meter Reading: Turbidity: __
Start Time: Meter Reading: Turbidity
Operator Name: ______ Total Volume Filtered:

Date: L "

ter flowing from the effluent hose.

Record the readings on the sample la-
bel. If the turbidity is greater than 160
Nephelometric Turbidity Units (NTU), sam-
pling should be rescheduled for a day when
the turbidity is lower.

6 Measure the turbidity of the source wa-

Turbidity
>160

Source Water —13


Push the red vent button on top of the filter
housing to expel air in the filter. When the air
is totally expelled from the filter, release the
button and open the water tap completely.

Using the pressure regulator on the Regula-
tor Module, adjust the pressure to no more
than 30 psi.

Using the water bypass screw on the injector,
adjust the pressure gauge on the Single Injector
Module to be at least 35% less than the pres-
sure shown on the Regulator Module gauge .

Record the following information on the
Sample Data Sheet:

(4 Date sampling started

( Time sampling started

{2 Initial water meter reading (including units)

SAMPLE NUMBER:

ENTERIC VIRUSES IN WATER

Vent Button

SYSTEM LOCATION:

SAMPLER'S NAME:

TOTAL SAMPLE VOLUME:

(inal-Intal meter readings x 28.316 (or readings inf)
(1 37454 (or readings in gallons)

WATER NTU
WATER pH: ‘TEMPERATURE: °c TURBIDI
INIT. METER READING: (CHECK UNITS) —fe  _galllons
date: time:
FINAL METER READING: (CHECK UNITS) ft __ gallons
date: time:

liters

{ON ON ARRIVAL:

FINISHED WaTER —59


Monitor the water meter to ensure that the
flow rate does not exceed | gallon/min (ap-
proximately 4 liters/min).

Allow at least 26 gallons/100 liters of wa-
ter to pass through the filter. At a flow
(MB) rate of approximately | gallon/minute,
this will require about 30 minutes.

PROTOZOA IN WATER

Volume In Volume In Volume In
Sampling Step GALLONS LITERS FT?
Protozoa Flow Rate |_| per minute 4 per minute .13 per minute
Protozoa Source
Water Sample 26 100 35

When the water meter indicates that

26 gallons/100 liters of water have

passed through the filter, turn off the
water at the tap.

Record the following information on the
sample label:

+ Time sampling stopped

+ Final water meter reading (including units)
+ Final turbidity

+ Total volume filtered

Start Time: Meter Reading: Turbidity;
Operator Name: ‘Total Volume Filtered:
Date: ‘Sampling Location: :

Source Water —15


CONTENTS

CONTENTS

The Information Collection
Requirements Rule ....

Questions Commonly Asked by
Drinking Water Utilities ....

Sample Collection Procedures
for Detecting Protozoa in Water ...

Collecting Source Water Samples.....

Collecting Finished Water Samples......

Sample Collection Procedures for
Detecting Enteric Viruses in Water .

Collecting Source Water Samples

Collecting Finished Water Samples .

Credits and Acknowledgements ...


Using aseptic technique, connect the sterile
tubing to the injector. Fill the sterile graduated
cylinder with 0.1-molar HCI and place the
tube in the graduated cylinder.

Turn on the water at the tap.

Using the water bypass screw—the larger top
screw in the injector—adjust the pressure on
the downstream pressure gauge to be at least
35% less than the pressure shown on the
Regulator Module gauge. For example, if the
Regulator Module gauge reads 30psi, then the
downstream gauge should read no more than
19psi.

ENTERIC VIRUSES IN WATER

Source Water —39


vent backwash and loss of particulate matter
from the filter.

Disconnect the inlet and outlet hoses from the
filter housing.

Hi Put on fresh latex gloves.
e

1] As aseptically as possible, remove the
filter from the housing and put it into a
(Ma) plastic sample bag.

"| Pour all of the water remaining in the
12 filter housing into the same plastic bag.

ly Seal the plastic sample bag and place it
13 inside the second plastic sample bag.

t_— |

‘Transfer the label or label information
to the outside of the outer bag.

fy] Put the bags containing the filter into

the shipping container.
i pping

Place the ice packs around, but not on,
the sample bag to prevent freezing the sample.
You may want to insert several inflated, empty
sample bags between the sample and the ice
packs.

PROTOZOA IN WATER

FINISHED WaTER —25,


e)4 Printed om Recycled Paper

ABOUT THIS MANUAL

This manual is designed to be brought into the
field by drinking water utility personnel when col-
lecting source and finished water samples for pro-
tozoa and viruses. The sample collection steps in
this manual are consistent with those demon-
strated in the accompanying video. To further as-
sociate the steps in this manual with the sampling
demonstration on the video, the photos for each
step are taken directly from the video.

Several graphic conventions are used through-
out the manual to differentiate steps or denote
special actions:

A step icon is used at the beginning of
pra cach step. These steps are parallel to
those in the accompanying video.

Actions denoted by this icon are critical

to ensuring that the sample will be valid

and uncontaminated, such as putting on
fresh latex gloves before handling the filter.

Text denoted by this icon provides ad-
ditional information to the samplers,
but may not be part of the actual col-
lection procedure.

Collecting protozoan and virus samples correctly
under the Information Collection Requirements
Rule can be challenging. Please watch the dem-
onstration video before collecting the samples,
and be sure to follow each step in this manual
when in the field.


ENTERIC VIRUSES IN WATER

Flush the sampling apparatus with 20

721m gallons / 76 liters of water by allowing the

water to flow through the system, out the
effluent hose into the 1-liter plastic bottle.

Volume In Volume In Volume In
Sampling Step GALLONS LITERS FT}
System Flush 20 76 27

SAMPLE DATA SHEET

SAMPLE NUMBER:

While the water is flushing the sampling appa-
ratus, begin completing your sample data
sheet. Record the following information:
Sample number

Q System location

 Sampler’s name

SYSTEM LOCATION:

SAMPLER'S NAME:

WATER pH:
INIT. METER READING:
date:

FINAL
date:

52— FINISHED WaTER

R READING:

TOTAL SAMPLE VOLUME:

Fnaltn

TURBIDITY:

(CHECK UNITS) fe

gallons

time:

(CHECK UNITS) ft’ _ gallons

tin
liters



ENTERIC VIRUSES IN WATER

Virus Sampling Apparatus with
Regulator and Discharge Modules

Regulator
Module

Flush the sampling apparatus with 20
gallons / 76 liters of water by allowing the

water to flow through the system, out the
effluent hose into the 1 -liter plastic bottle.

Volume In Volume In Volume In
Sampling Step GALLONS LITERS ET?
System Flush 20 76 27

Source Water —35


ENTERIC VIRUSES IN WATER

Vent Button

a TM“

Slowly, start the water flowing through
he sampling apparatus.

Push the red vent buttons on top of the
filter housings to expel air in the filters. When
the air is totally expelled from the filters, re-
lease the button and open the water tap com-
pletely.

Using the pressure regulator on the Regulator
Module, adjust the pressure regulator to no
more than 30 psi.

Using the water bypass screw on the injector,
adjust the pressure regulator on the Single In-
jector Module to be at least 35% less than the
pressure shown on the Regulator Module
gauge.

Allow 53 - 80 gallons / 200 - 300 liters of wa-
ter to pass through the filter.

Sampling Step

Volume In Volume In Volume In
GALLONS LITERS FT?

Sampling Source
Water

53-80

200 - 300 7-41

44— Source water

If the virus filter clogs before 53 gallons/
f 100 liters are collected, contact the ap-
proved analyst at your laboratory for further
instructions.

When the water meter indicates that
| 53 - 80 gallons / 200 - 300 liters of wa-

ter have passed through the filter, turn
off the water at the tap.


ENTERIC VIRUSES IN WATER

Each month, your laboratory will send
you all of the equipment needed to col-
lect samples for enteric virus analyses.
When you receive the sampling kit, immedi-
ately check the contents of the carton. The
sampling kit will be shipped as three modules,
and should contain the following items:

(2 Plastic sample bags

Qi Ice packs for shipping the collected
samples

(4 Sample data sheet
O Regulator Module (below):
+ Backflow control valve
+ Swivel female insert
+ Inlet hose
+ Pressure regulator with pressure gauge

Intes tome
Hisar Backflow Control Vah
Regulator ackflow Control Valve

Swivel Female Insert
Pressure Gauge

29


PROTOZOA IN WATER

COLLECTING SOURCE
WATER SAMPLES

When you are ready to collect your protozoa

sample, bring the following items with you to

the sampling location:

(1 Shipping container sent by the laboratory

(Sampling apparatus

\Q Plastic sample bags

‘4 Sample labels

“4 Frozen ice packs

(1 Several pairs of new latex gloves

(2 pH meter

\4 Thermometer

Q Turbidimeter

If you will be collecting samples from both
source water and finished water on the

same day, perform the finished water sampling

first. Using the sampling apparatus on source

water first may cause false positives for finished
water sample analyses.

water to flow for 2 to 3 minutes or until

any debris that has accumulated in the
sampling line has cleared or the turbidity in the
water becomes uniform.

3 Turn on the water at the tap and allow the

‘Turn off the water at the tap.

Source Water —11


Measure the pH, temperature, and tur-
Fy bidity of the source water flowing from
the effluent hose. Record the readings

on the sample data sheet.

ENTERIC VIRUSES IN WATER

SAMPLE NUMBER:

SYSTEM LOCATION:

nani meter readings x 28.316 (fr readings in 1)
rt 3.7AS4 (for readings gallos)

CONDITION ON ARRIVA!

COMMENTS:

SAMPLER'S NAME:
WATER
WATER pH: ‘TEMPERATURE: *c ‘TURBIDITY:
INIT, METER READING: (CHECK UNITS) {gallons
date: time: -
FINAL METER READING (CHECK UNITS) ft _ gallons
liters

FINISHED WATER —53


ENTERIC VIRUSES IN WATER
BH > Bo ]lnsertthe Double Injector Module
between the Regulator and Dis-
charge Modules before proceeding.

Ensure that both injectors are completely
closed before proceeding.

iyi

Adjust the water bypass screws on each injec- ,
tor clockwise as far as possible.

‘Turn on the water.

Next, turn each of the screws one half turn
counterclockwise.

iyi

Continue opening the water bypass screws in
half-turn increments until the reading on the
second pressure gauge is approximately 35%
less than that shown on the Regulator Module
pressure gauge.

Using aseptic technique, connect the sterile
tubing to the injectors.

Pour the 0.1-molar hydrochloric acid solution
into a sterile graduated cylinder and place one
of the injector tubes into it.

Pour the 2% thiosulfate solution into a second,
sterile graduated container. Place the tube

FINISHED WaTER —55.


PROTOZOA IN WATER

After the system has been flushed with
20 gallons / 76 liters of water, turn off
the tap and disconnect the inlet and
outlet hoses from the filter housing.

Using the filter wrench, open and drain the fil-
ter housing.

Open the filter packaging as aseptically as
possible and carefully drop the filter into the
filter housing.

Be sure to hold the loose gasket in place
sing aseptic technique.

Reassemble the filter housing, and reconnect
the inlet and outlet hoses. Place the filter
housing in an upright position.
Slowly, turn on the tap and start the water
flowing through the sampling apparatus.
Using the pressure regulator, adjust the pres-
sure to no more than 30 psi.
Record the following information on the
sample label:
Time sampling started
Initial water meter reading (including
units)
+ Turbidity

Stop Time: Meter Reading: Turbidity
‘Start Time: ‘Meter Reading: Turbidity:
‘Operator Name: Total Volume Filtered:

Date: ‘Sampling Location:

14— Source Water