This document outlines a validation protocol to establish a 72-hour hold time for various water quality parameters in collected water samples. The protocol involves testing samples initially and after 24, 48, and 72 hours of storage at 2-8°C for parameters like pH, conductivity, heavy metals, nitrates, TOC, microbial counts, and bacterial endotoxins. Acceptance criteria are based on meeting specifications for each water type. A final report will determine the validated hold time based on conclusion from the study results.

1. Validation Protocol for Hold Time Study of Collected Water Samples
1.0 OBJECTIVE
Objective of this protocol is to provide documented evidence through the scientific data
to establish and verify that on holding water for over a period of 72 hours after
collection and subsequent storage does not affect its results for different parameters
such as Description, pH, Conductivity, Heavy metals, Nitrates, TOC, Total count and BET.
2.0 SCOPE
The scope of this protocol is to evaluate the hold time of water samples upon holding
water sample containers for 72 hours while monitoring water quality.
3.0 REFERENCE DOCUMENT
Following documents are referred during preparation of the protocol.
Water sampling procedure
Procedure for analysis of Water
4.0 RESPONSIBILITY
4.1 Quality Control
Preparation, review and execution of protocol
4.2 Quality Assurance Review and approval of protocol
5.0 ACCOUNTIBILITY Head of Department
6.0 PROCEDURE
6.1 Pre-Requisite
• Pre incubated media/ molten media.
• Testing accessories
• Water testing reagents
• Availability of sterilized glassware. (Where ever applicable)
• Availability of pyrogen free glassware. (Where ever applicable)
• Testing environment
6.2 Procedure for Hold Time Study
6.2 Procedure for Hold Time Study Test Matrix
Raw
water
Soft
water
Potabl
e
water
Purifie
d
water
Water
for
injecti
on
Pure
steam
Drinki
ng
water

2. Descri
ption
Descri
ption
Descri
ption
Descrip
tion
Descrip
tion
Descrip
tion
Descri
ption
pH pH pH pH
Conduct
ivity pH
Hardne
ss
Hardne
ss TDS
Conduct
ivity
Conduct
ivity TOC
TAMC TAMC TAMC TOC TOC BET TAMC
Pathog
ens
Pathog
ens
Pathog
ens
Heavy
Metals
Heavy
Metals
TAMC
Pathog
ens
Colifo
rms
Colifo
rms
Nitrate Nitrate Colifo
rms
TAMC TAMC
BET
Note: Parameter selected in dark shade is considered for hold time study from particular
type of water sample.
6.3Test Procedure
• Collect samples as per SOP collection of water sample and store the samples up to 3 days
at 2-8°C.
6.3.1 Description
• Examine the water physically such as Color, Odor.
6.3.2 Heavy Metals
• In a glass-evaporating dish evaporate 150 ml of sample to 15 ml on a water bath.
Method: Into a small Nessler Cylinder pipette 10.0 ml of lead standard solution (1ppm
Pb). Into another Nessler Cylinder pipette 12 ml of test solution as prepared above. To
the cylinder containing the standard solution add 2.0 ml of the test solution and mix.
To each cylinder add 2 ml of acetate buffer pH 3.5, mix, add 1.2 ml of thioacetamide
reagent, allow to stand for 2 minutes and view downwards over a white surface, the color
produced with the test solution is not more intense than that produced with the standard
solution.
6.3.3 Nitrate
• To 5 ml sample in a test tube immersed in ice add 0.4 ml of a 10% w/v solution of
Potassium chloride, 0.1 ml of diphenylamine solution and drop wise with shaking 5 ml of
sulphuric acid. Transfer the tube to a water bath at 50°C to allow standing for 15
minutes. Any blue color in the solution is not more intense than that in a solution

3. prepared at the same time and in the same manner using a mixture of 4.5 ml of nitrate
free water and 0.5 ml of nitrate standard solution (2 ppm N03).
6.3.4 Conductivity
• Take the 100 ml sample in a suitable container, and stir the test sample by maintaining
the temperature 25°C ± 1°C, measure the conductivity with the help of calibrated
conductivity meter.
6.3.5 pH
• Take 100 ml of sample and add 0.3 ml of saturated KCI solution. Mix the solution well
and then measure the pH with the help of Calibrated pH meter.
6.3.6 Total Organic Carbon
•Analyze the sample for TOC in a calibrated TOC Analyzer as per the Method for TOC
analysis.
6.3.7 Total microbial Count (Pour Plate Method)
• Dispense one ml of sample into two petridishes. Approximately add 15-20 ml of R2A /
Plate count Agar into each petridishes. Cool the media approximately 45°C (feel on the
dorsal side of the hand, it should be bearable). Cover the petridish, mix the sample with
the agar by tilting or rotating the dishes and allow the contents to solidify at room
temperature. Invert the petridishes and incubate at 30-35°C for 5 days. After incubation,
examine the plates for growth, count the number of colonies and express the average for
the two plates in terms of the number of colony forming units per ml.
6.3.8 Total microbial Count (Filtration Method)
• The procedure gives the use of a single disposable/ autoclaveable filtration funnels and
filter holder, using suitable filtration system.
•Use sample size as specified in the specification for filtration through the 0.45 m
filter.
•After completion of filtration of sample, rinse the filter with 100 ml sterile water
remove the filter using sterilised forceps and transfer it immediately to the previously
prepared petri-dish with appropriate medium (R2A agar/Plate count agar).
• Place the membrane carefully so that the air should not be trapped inside the filter, as
this will prevent nutrient medium from reaching the entire membrane surface. Replace the
lid. Invert the petri dish and incubate at 30-35°C for 5 days. Count the number of
colonies on the membrane and express the results as per specification.
6.3.9 Bacterial Endotoxin Test
• Refer the SOP for Bacterial Endotoxin Testing.

4. • Perform the analysis on 0 hours, 24 hours, 48 hours and 72 hours
Related: Sampling, Preservation and Storage Procedure of Water Sample
7.0 ACCEPTANCE CRITERIA
Its shall meets the specification of its particular water type.
8.0 CONCLUSION
After complete evaluation of the Hold time study for collected water samples a final Hold
time study summary report shall be prepared which should essentially contain discussion
and conclusion which clearly determine the hold time period for water samples.
9.0 ABBREVIATIONS
SOP - Standard operating procedure ° C - Degree Celsius TOC - Total organic carbon Pb -
Lead
w/v - Weight per volume BET - Bacterial endotoxin test ppm - Parts per million ml -
Millilitre
TDS - Total dissolved solids %- Percentage
TAMC - Total aerobic microbial count
N03 - Nitrate
KCL- Potassium chloride