Water Sample Analysis

Microbiological Analysis

PHO’s laboratory performs microbiological testing of water samples for the detection of bacterial indicators of contamination and specific pathogens in water. Each water sample is tested for specific indicators according to the water source.

If testing is required on a water source that is not listed in PHO’s laboratory water testing menu below, or testing is required to identify specific etiological agents in water related to a laboratory confirmed clinical case or outbreak investigation, boards of health are asked to consult with the microbiologist, or designate, overseeing the water testing program prior to sample submission.

Sample collection requirements, sample handling, shipping conditions, test information including testing frequency, turnaround times and reporting limits are dependent on the specific water source. For disinfected water sources, ensure the sodium thiosulfate preservative in the collection bottle has not expired prior to sampling. Samples will not be processed if the requisition is not completely and accurately filled in when received at the laboratory; and a new sample and completed form will be required to be submitted. [Click on individual testing links for test directory details – see below.]

Drinking water (including bottled water)
Drinking water – Private citizen
Ice – Treated
Public Beach Water
Recreational Water Facilities, Public pools/Spas
Suspected Sewage Contamination – Water

For specific etiological agent requests, please contact the PHO's laboratory Customer Service Centre at 416-235- 6556 or toll free 1-877-604-4567.

For general information related to sample collection, labelling, storage, transportation and chain-of- custody of drinking water samples that fall under the Drinking Water Systems Regulation (Ontario Regulation 170/03) and Small Drinking Water Systems (Ontario Regulation 319/08), refer to the Ministry of Environment, Conservation and Park’s Practices for the Collection and Handling of Drinking-Water Samples.

Drinking Water Analysis

Important Information Related to Drinking Water Analysis

Type of Drinking Water System

As part of an investigation under the Health Protection and Promotion Act (HPPA), public health inspectors may collect drinking water samples in accordance with the following sections of the Health Protection and Promotion Act (HPPA): Section 13, Section 41, Subsections (1), (2), (4), and (5)²⁸. This may include collecting samples at drinking water systems regulated under O. Reg. 170/03 and O. Reg. 319/08 as well as unregulated systems where the public has access to the water (e.g., campground with fewer than 6 connections). Consult with Ministry of Health Environmental Health Protection Policy and Partnerships Branch (MOH-EH) or legal counsel for additional information on the classification of the drinking water systems.

All drinking water testing must be conducted by a licensed laboratory using licensed drinking water testing methods.

The type of drinking water system identified on the requisition triggers specific notification requirements. Laboratories have an obligation to report test results as outlined in section 18 and 18.1 of the Safe Drinking Water Act, 2002 for drinking water samples submitted under O. Reg. 170/03 Drinking Water Systems or O. Reg. 319/08 Small Drinking Water Systems

The details are outlined in the Drinking Water – Official Agencies Test Information Sheet.

Completing the requisition and submitting the sample according to Instructions For Official Agencies Submitting Water Samples to the Public Health Ontario Laboratory will help to ensure samples are accepted.

Chain of Custody

The chain of custody must be maintained for drinking water samples collected under O. Reg. 170/03 Drinking Water Systems or O. Reg. 319/08 Small Drinking Water Systems

For drinking water samples indicated as “HPPA O. Reg 319/08” or “SDWA O. Reg 170/03”, there are two ways to maintain the chain of custody:

Table 7: Options for Maintaining a Drinking Water Sample Chain of Custody

Situation	Action
Courier - Indirect delivery of the sample to the laboratory (cannot deliver the sample directly to the laboratory)	For samples delivered by courier: Place a signed and dated "Regulated Water Seal" (refer to Appendix B: Laboratory Submission Forms) up and over the cap of the sample in such a way that the two ends of the seal are attached to the shoulder of the bottle, and the midpoint of the seal is stuck to the top of the cap. The sample is sent to the laboratory with a completed requisition according to the sample transport instructions in the test directory – test information sheet.
In person - Direct delivery of the sample to the laboratory	Complete the following fields in the “For Regulated Drinking Water or Legal Samples” section of the requisition: The printed name of the person relinquishing the sample to laboratory personnel The signature of the person relinquishing the sample Date and time the sample(s) is relinquished The sample is sent to the laboratory with a completed requisition according to the sample transport instructions in the test directory – test information sheet.

Compliance Samples

Drinking water systems under O. Reg. 170/03 or O. Reg. 319/08 are required to follow the requirements under those regulations. If sampling is performed to demonstrate compliance with the required monitoring frequency, they are to use a licensed commercial laboratory for their testing.

The exception is O. Reg. 319/08 small drinking water systems that supply Ontario Parks; routine compliance samples are analyzed by the PHO’s laboratory.

Audit Samples

Public health inspectors may sample regulated drinking water systems as part of an audit these are considered audit samples. Routine monitoring of these drinking water systems by Board of Health staff is not recommended.

Drinking Water Sample Hold Time

Drinking water must be analyzed in accordance with a licensed test method. PHO’s laboratory uses modified versions of the MECP methods; namely, E3407 Membrane Filtration Method Using DC Agar for the Simultaneous Detection and Enumeration of Total Coliforms and Escherichia coli in Drinking Water, and E3408: The Spread Plate Method for the Enumeration of Aerobic, Heterotrophic Bacteria in Drinking Water. These methods include a sample hold time of 48 hours. For results to be valid for the purposes of the Safe Drinking Water Act, 2002, samples must be tested within the hold time.

Etiological Agent Testing

A Public Health Inspector or designate under the authority of the Medical Officer of Health may collect water samples for identification of an etiological agent (e.g., Legionella spp., Salmonella spp., etc.) from drinking water systems if required for investigational purposes or outbreak situations. On the Bacteriological Analysis Water - Multiple Sample Requisition for Official Agencies, these samples must be identified as “Non-Regulated”, “HPPA Regulated Premises”, or “Private Residence”, and marked as an “Outbreak Investigation” with the outbreak/investigation number, etiological agent and clinical case identification (e.g., iPHIS, PHO’s laboratory barcode identification). Alternatively, complete all sections of the Environmental Microbiology Investigation Requisition with appropriate information to ensure accurate tracking of the sampling and complete all fields as outlined on page 2 of the form.

Drinking Water Submissions under a Provincial Officer’s Order or Approval (Directive)

Environmental officers and public health inspectors have the legal authority to enter a facility at any time to ensure compliance (i.e., MECP Provincial Officers for Ontario’s environmental laws and public health inspectors under the HPPA). If it is determined that a serious non-compliance has occurred, an Order can be issued that sets out obligations for a specific person or persons in relation to a specific operation.

Analysis for a bacteriological health related parameter not included in Ontario Drinking Water Quality Standards Ontario Regulation 169/03; e.g., Heterotrophic Plate Count (HPC) or Background, contact PHO’s laboratory Customer Service Centre and ask to speak to the Program Coordinator Drinking Water Testing or designate.

Drinking Water Research

If board of health personnel wish to engage in a study involving drinking water (e.g., to identify trends associated with ground water contamination in various geographic areas, etc.) contact PHO’s laboratory Customer Service Centre and ask to speak to the Program Coordinator, Drinking Water Testing.

Research projects must be reviewed by the microbiologist overseeing PHO’s laboratory Environmental Microbiology programand approval must be granted by the PHO's laboratory Research and Project Review Committee before research begins.

The Drinking Water Testing Services regulation O. Reg. 248/03 outlines the requirements related to drinking water quality research (e.g., scientific studies, including special audits or surveys to assess drinking water quality).

A Notification Form for Drinking Water Testing Research and Method Development must be submitted to the Ministry of the Environment, Conservation and Parks.

Laboratory Acceptance Criteria

The accuracy of the test results may be affected by collection, handling and shipping. Submit water samples in a timely manner to PHO’s laboratory. Water samples that do not meet the acceptance criteria may not be accepted by the laboratory. A new sample may be submitted with a new requisition. The acceptance criteria are outlined below.

Table 8: Acceptance Criteria for Water Sample Submissions

Description	Acceptance Criteria
Submitter	Water samples must be submitted by an authorized submitter: Official Agencies: Local board of health or an Ontario Park under the Ministry of Natural Resources and Forestry (MNRF). Requests for testing by any other organization (e.g., other public health partners or Ontario Ministries) will require approval from the Director Quality, Regulatory Affairs and Customer Service and microbiologist overseeing the water testing program, or designate, prior to submission. Private drinking water systems (e.g., well water): Private citizen submissions of samples collected from a “single household” and serves fewer than 6 private residences but not the public.
Sample Type	The water sample must be collected from within the province of Ontario. The following types of water sources are analyzed at the PHO's laboratory. For specific analyses, refer to the PHO's laboratory test directory – test information sheet: Local board(s) of health: Drinking Water – Official Agency Ice – Treated Public Beach Water Recreational Water Facilities, Public Pools/Spas Suspected Sewage Contamination - Water Specific etiological agent MNRF Ontario Parks: Drinking Water – Official Agency Public Beach Water Private Citizen Drinking Water – Private Citizen Refer to the acceptance criteria
Sample Requisition	The sample must be received with a requisition that is complete and accurate. Submitters will be required to submit clarification in writing when there is conflicting information on the form (e.g., a drinking water system is listed as a “HPPA regulated premise”, but the requisition includes a regulated drinking water system number). A unique identifier is used to link the sample with the requisition: For Official Agency water samples, the sample barcode or barcode number must be present on both the requisition and the sample when it is received at the laboratory. For treated ice samples, a unique identifier is written on the sample bag and the requisition; they must match. For Private Citizen drinking water samples, a unique identifier, e.g., the submitter’s name can be used as the unique identifier to link the requisition and the sample; however, samples are accepted without a unique identifier when the sample is received at the laboratory with the requisition secured around the bottle. Water samples for identification of an etiological agent (e.g., Legionella spp., Salmonella spp., etc.): These samples must be identified as “Non-Regulated”, “HPPA Regulated Premises”, or “Private Residence”, and marked as an “Outbreak Investigation” with the outbreak/investigation number, etiological agent and clinical case identification (e.g., iPHIS, PHO's laboratory barcode identification).
Sample Collection	Water samples must be collected in PHO's laboratory water collection bottles. Drinking water samples must be collected from the cold water tap and not the hot water tap. Treated ice samples must be collected in a sterile plastic bag with wire closure. Sufficient amount of sample for the analyses is required (i.e., 200 mL unless otherwise specified in the test directory – test information sheet). Drinking water samples must be aesthetically acceptable; i.e., clear, colourless and odourless, and does not contain debris or substances that interfere with the test. For Private Citizen drinking water submissions, only requisitions with a version number of 3743-44 (01/01) or newer are accepted.
Sample Transport	The sample container must be secure and not broken or damaged when received at the laboratory. Samples should be stored and transported at temperatures between 2 and 8 °C following collection. Drinking water samples must be less than 25 °C (preferably between 2 – 10 °C) but not frozen when received at the laboratory. For details on sample transport for public beach, recreational water facility or public pool/spa refer to the test directory – test information sheet.
Sample Hold Time	Drinking water samples must be sent to the laboratory as soon after collection as possible since they must be tested within the hold time for the test as specified in the test directory – test information sheet for the analysis required. Non potable water (e.g., public beach, public pool/spa, recreational water facility, sewage, etc.) must be tested within one calendar day of collection.

Reporting and Interpretation of Test Results

The following tables outline the reporting and acceptable limits for potable and non-potable water testing. Refer to Appendix D : Water Testing Fact Sheets for interpretation and additional details on Background or Non-target bacteria, Campylobacter spp., Coliforms, Escherichia coli, Heterotrophic Plate count (HPC), Legionella spp., Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus.

POTABLE WATER

OFFICIAL AGENCY – POTABLE / ICE-TREATED

Table 9: Reporting and Acceptable Limits for Potable Treated Ice Samples

Testing Type	Reporting Limit	Acceptable Limit
Background or Non-Target Bacteria (CFU / 100 mL)	0 to *NDOGN/ NDOGT	At the discretion of the MOH. Only reported on regulated drinking water samples submitted under a “Provincial Officer’s Order” or “Approval (Directive)”.
Total Coliform^a (CFU / 100 mL)	0 to > 80	0
Escherichia coli^a (CFU / 100 mL)	0 to > 80	0
Heterotrophic Plate Count (HPC) (CFU / 1 mL)	< 10 to > 3.0 x 10³, **NDOGHPC	Acceptable Limit < 500 ***

*NDOGN - No Data Overgrown with non-target/NDOGT - No Data Overgrown with target

**NDOGHPC - No Data Overgrown heterotrophic plate count

*** The standard for HPC under O. Reg. 169/03 was revoked June 6, 2006 (O. Reg. 248/06) and therefore, HPC is not reportable under the Safe Drinking Water Act. The acceptable limit listed is based on United States Environmental Protection Agency standards³⁵.

OFFICIAL AGENCY – BOTTLED WATER

Table 10: Reporting and Acceptable Limits for Bottled Water Samples

Testing Type	Reporting Limit	Acceptable Limit
Total Coliform^a (CFU / 100 mL)	0 to > 80	0
Escherichia coli^a (CFU / 100 mL)	0 to > 80	0
Heterotrophic Plate Count (HPC) (CFU / 1 mL)	< 10 to > 3.0 x 10³, *NDOGHPC	< 500
Pseudomonas aeruginosa	(CFU / 100 mL): 0 to > 100 (CFU / 50 mL): < 2 to > 200^b	0
Staphylococcus aureus	(CFU / 100 mL): 0 to > 100 (CFU / 50 mL): < 2 to > 200^b	0

*NDOGHPC - No Data Overgrown heterotrophic plate count

PRIVATE CITIZEN – PRIVATE DRINKING WATER SYSTEMS

Table 11: Reporting and Acceptable Limits for Private Drinking Water Samples

Testing Type	Reporting Limit	Acceptable Limit
Total Coliform^a (CFU / 100 mL)	0 to > 80	≤ 5
Escherichia coli^a (CFU / 100 mL)	0 to > 80	0

^aAn overgrown sample; that is, crowding and/or confluent and/or non-identifiable microbial growth on a plate, is considered an adverse result. When there is an overgrown condition and there is no evidence of Total Coliform or E. coli, NDOGN - No Data Overgrown with non-target is reported. The water may be unsafe to drink. When there is an overgrown condition and there is evidence of Total Coliform and/or E. coli, NDOGT - No Data Overgrown with target is reported. The water is unsafe to drink.

^bWhen 50 mL is analyzed.

NON POTABLE WATER

OFFICIAL AGENCY – PUBLIC BEACH WATER

Table 12: Reporting and Acceptable Limits for Public Beach Water Samples

Testing Type	Reporting Limit	Acceptable Limit
Escherichia coli (CFU / 100 mL)	< 10 to > 1.0 x 10³	≤ 200

OFFICIAL AGENCY – SUSPECTED SEWAGE CONTAMINATION

Table 13: Reporting and Acceptable Limits for Suspected Sewage Contamination Water Samples

Testing Type	Reporting Limit	Acceptable Limit
Escherichia coli^a (CFU / 100 mL)	< 1.0 x 10³ to > 1.0 x 10⁶	Not Applicable

OFFICIAL AGENCY – RECREATIONAL WATER FACILITIES, PUBLIC POOLS/SPAS

Table 14: Reporting and Acceptable Limits for Recreational Water Facilities and Public Pools/Spas Samples

Testing Type	Reporting Limit	Acceptable Limit
Background or Non-target bacteria (CFU / 100 mL)	Only reported if NDOGN/NDOGT	Not Applicable
Total Coliform^a (CFU / 100 mL)	< 2 to > 160	< 2
Escherichia coli^a (CFU / 100 mL)	< 2 to > 160	< 2
Heterotrophic Plate Count (HPC) (CFU / 1 mL)	< 10 to > 3.0 x 10³, *NDOGHPC	Appendix D: Water Testing Fact Sheets
Pseudomonas aeruginosa	(CFU / 100 mL): 0 to > 100 (CFU / 50 mL): < 2 to > 200^b	< 10
Staphylococcus aureus	(CFU / 100 mL): 0 to > 100 (CFU / 50 mL): < 2 to > 200^b	< 50

^a An overgrown sample; that is, crowding and/or confluent and/or non-identifiable microbial growth on a plate, is considered an adverse result. When there is an overgrown condition and there is no evidence of Total Coliform or E. coli, NDOGN - No Data Overgrown with non-target is reported. When there is an overgrown condition and there is evidence of Total Coliform and/or E. coli, NDOGT - No Data Overgrown with target is reported.

^b When 50 mL is analyzed.