Joint Test Plan for Verification of HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer
January 2010
Prepared by
Amy Dindal
Mary Schrock
Zachary Willenberg
Battelle Memorial Institute
505 King Avenue
Columbus, OH 43201-2693
and
Mette Tjener Andersson
Christian Gran
DHIDANETV Water Centre
Agern Alle 5,
DK-2970
H0rsholm, Denmark
and
Mona El-Hallak
John Neate
ETV Canada
2070 Hadwen Road Suite 201 A
Mississauga, Ontario L5K 2C9
Canada
and
John McKernan
Evelyn Hartzell
Michelle Henderson
U.S. Environmental Protection Agency
26 Martin Luther King Drive
Cincinnati, OH 45268
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FOREWORD
DHIDANETV Water Test Centre (DHI) which operates a Danish verification scheme,
DANETV, supported by the Danish Ministry for Science, Technology and Innovation; the
United States Environmental Protection Agency Environmental Technology Verification
Program's Advanced Monitoring Systems (U.S. EPA ETV AMS) Center operated by Battelle
(Battelle AMS Center) under a Cooperative Agreement with the U.S. EPA; and Ontario Centre
for Environmental Technology Advancement (OCETA) through ETV Canada which operates the
Canadian ETV Program on behalf of Environment Canada are conducting an international joint
verification test of the HACH-LANGE GmbH water toxicity technologies named LUMIStox 300
Bench Top Luminometer and ECLOX Handheld Luminometer.
This document, which is a compilation of three separate documents, will be used by
DHI, the U.S. EPA ETV AMS Center, and ETV Canada to jointly verify the performance of
these technologies. It is composed of a process document, a verification protocol, and a test plan
for joint verification. Combined, these three documents satisfy the requirements of a U.S. EPA
ETV approved verification test/QA plan and ensure that the requirements of all three programs
are met. The verification protocol and test plan were developed by DHI with input from Battelle,
U.S. EPA, ETV Canada, and other stakeholders. Together these documents satisfy DANETVs
programmatic requirements. The process document was developed by the Battelle AMS Center
with input from DHI, ETV Canada, and U.S. EPA. It was developed as a supplement to the DHI
documents, to ensure that all of the U.S. EPA ETV programmatic requirements are met. All three
documents were reviewed and approved via the ETV process prior to the start of testing.
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TABLE OF CONTENTS
Process Document:
Section Page
1 INTRODUCTION 6
2 QUALITY SYSTEMS 12
3 VERIFICATION PLANNING 13
4 VERIFICATION TEST IMPLEMENTATION 14
5 ASSESSMENT AND RESPONSE 16
6 DOCUMENTATION AND REPORTING 22
7 REFERENCES 25
Appendix A ROLES OF KEY PERSONNEL 26
Appendix B KICK OFF MEETING CHECKLIST 27
Appendix C REVIEW REPORT FORM 29
Appendix D JOINT VERIFICATION TESTING DOCUMENT DEVIATION FORM 31
Appendix E ASSESSMENT REPORTING FORM 33
Appendix F EXAMPLE JOINT VERIFICATION STATEMENT 35
Appendix G STATISTICAL EVALUATIOM OF TEST PLAN TESTS A AND C 38
Verification Protocol:
Section Page
1 TABLE OF CONTENTS ii
2 INTRODUCTION 1
2.1 Name of product 1
2.2 Name and contact of vendor 1
2.3 Name of center/verification responsible 1
2.4 Verification test organization 2
2.5 Technical experts 3
2.6 Verification process 3
3 DESCRIPTION OF THE TECHNOLOGY 4
4 DESCRIPTION OF THE PRODUCTS 5
4.1 LUMIStox 300 5
4.2 ECLOX 5
5 APPLICATION AND PERFORMANCE PARAMETER DEFINITIONS 6
5.1 Application definition 6
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5.2 Performance parameters for verification 6
5.3 Additional parameters 8
6 EXISTING DATA 9
6.1 Summary of existing data 9
6.2 Quality of existing data 9
6.3 Accepted existing data 10
7 TEST PLAN REQUIREMENTS 10
7.1 Test design 10
7.2 Reference tests and analysis 11
7.3 Data management 12
7.4 Quality assurance 12
7.5 Test report 12
8 EVALUATION 12
8.1 Calculation of performance parameters 12
8.2 Evaluation of test data quality 14
8.3 Compilation of additional parameters 14
8.3.1 User manual 14
8.3.2 Product costs 15
8.3.3 Occupational health and environment 16
9 VERIFICATION SCHEDULE 17
10 QUALITY ASSURANCE 17
Appendix 1 - Terms and definitions used in the verification protocol 19
Appendix 2 - References 23
Appendix 3 -Application and performance parameter definitions 26
Test Plan:
Section Page
1 TABLE OF CONTENTS ii
2 INTRODUCTION 1
2.1 Verification protocol reference 1
2.2 Name and contact of vendor 1
2.3 Name of center/test responsible 1
2.4 Expert group 2
3 TEST DESIGN 2
3.1 Test sites 6
3.1.1 Types 6
3.1.2 Addresses 6
3.2 Tests 6
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3.2.1 Test methods 7
3.2.2 Test staff 8
3.2.3 Test schedule 8
3.2.4 Test equipment 9
3.2.5 Type and number of samples 9
3.2.6 Operation conditions 10
3.2.7 Operation measurements 10
3.2.8 Product maintenance 11
3.2.9 Health, safety and wastes 12
4 REFERENCE TESTS AND ANALYSIS 12
4.1 Analytical laboratory 13
4.2 Analytical parameters 13
4.3 Methods of test and analysis 13
4.4 Analytical performance requirements 14
4.5 Preservation and storage of reference samples 15
5 DATA MANAGEMENT 15
5.1 Data storage, transfer and control 15
6 QUALITY ASSURANCE 16
6.1 Test plan review 16
6.2 Performance control - reference test and analysis 16
6.3 Test system control 18
6.4 Data integrity check procedures 19
6.5 Test system audits 19
6.6 Test report review 20
7 TEST REPORT 20
7.1 Test site report 20
7.2 Test data report 20
7.3 Amendment report 20
7.4 Deviations report 21
Appendix 1 - Terms and definitions used in the test plan 22
Appendix 2 - References 26
Appendix 3 - Reference methods 28
Appendix 4 - In-house test methods 30
Appendix 5 - In-house analytical methods 50
Appendix 6 - Data reporting forms 52
Appendix 7 - Data management 67
Appendix 8 - Deviations and amendments 70
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EXECUTIVE SUMMARY
DHIDANETV Water Test Centre (DHI) which operates a Danish verification scheme,
DANETV, supported by the Danish Ministry for Science, Technology and Innovation; the
United States Environmental Protection Agency Environmental Technology Verification
Program's Advanced Monitoring Systems (U.S. EPA ETV AMS) Center operated by Battelle
(Battelle AMS Center) under a Cooperative Agreement with the U.S. EPA; and Ontario Centre
for Environmental Technology Advancement (OCETA) through ETV Canada which operates the
Canadian ETV Program on behalf of Environment Canada are jointly verifying the HACH-
LANGE GmbH water toxicity technologies named LUMIStox 300 Bench Top Luminometer and
ECLOX Handheld Luminometer.
Under this joint effort, DHI was responsible for developing the verification
protocol, preparing the test plan including quality assurance (test/QA), testing, and generating
the verification report/verification statement in accordance with their requirements. The Battelle
AMS Center provided technical and quality assurance oversight throughout the DHI process
to ensure U.S. EPA ETV requirements were also met, and facilitated reviews and audits by QA
personnel, U.S. EPA Program management, and stakeholders of the verification protocol, test
plan, testing, and verification report. The Battelle AMS Center also developed a process
document to supplement the protocol and test plan developed by DHI to ensure that all of the
U.S. EPA ETV programmatic requirements were met. The process document, combined with the
protocol and test plan, satisfy the requirements of a U.S. EPA approved verification test/QA
plan. This document is a compilation of these three documents. ETV Canada also provided
technical and quality assurance oversight throughout the DHI process to ensure that the Canadian
ETV program requirements were met.
The purpose of this verification is to evaluate the performance of a wastewater rapid
toxicity technology that could be used to monitor industrial or domestic wastewater. The
verification protocol covers two products from the same vendor, both are acute toxicity tests with
luminescent bacteria. In the bioluminescence testing with LUMIStox and ECLOX, a strain of
naturally occurring luminescent bacteria, Vibrio fischeri, is used. Vibrio fischeri is a non-
pathogenic, marine, luminescent bacterium which is sensitive to a wide range of toxicants. When
properly grown, luminescent bacteria produce light as a by-product of its cellular respiration.
Any inhibition of cellular activity results in a decreased rate of respiration and a corresponding
decrease in the rate of luminescence. The light emission/luminescence can be measured with a
LUMIStox or ECLOX luminometer. Inhibition of the light emission in the presence of a sample
is determined against a non-toxic control. Verification parameters will include determination of
the criterion of detection, the range of application, precision as measured by repeatability and
reproducibility, agreement with accepted values, and robustness. Other parameters such as an
evaluation of the user manual, product costs, and environmental and occupational health and
safety aspects will also be included.
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The Business of Innovation ( WATER«ENVIRONMENT«HEALTH
Environmental Technology
Verification Program
Advanced Monitoring
Systems Center
Process Document for
U.S. EPA ETV AMS Center
DHI DANETV Water Centre
and
ETV Canada
Joint Verification of the
HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer
and
ECLOX Handheld Luminometer
-------
Process Document for
U.S. EPA ETV AMS Center
DHI DANETV Water Centre
and
ETV Canada
Joint Verification of the
HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer
and
ECLOX Handheld Luminometer
January 12,2010
Prepared by
Battelle
505 King Avenue
Columbus, OH 43201-2693
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US ETV AMS Center, DHIDANETV Water Centre and ETV Canada
Joint Verification Process Document
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Date: 01/12/10
TABLE OF CONTENTS
Section Page
1 INTRODUCTION 6
2 QUALITY SYSTEMS 12
3 VERIFICATION PLANNING 13
4 VERIFICATION TEST IMPLEMENTATION 14
5 ASSESSMENT AND RESPONSE 16
6 DOCUMENTATION AND REPORTING 22
7 REFERENCES 25
Appendix A ROLES OF KEY PERSONNEL 26
Appendix B KICK OFF MEETING CHECKLIST 27
Appendix C REVIEW REPORT FORM 29
Appendix D JOINT VERIFICATION TESTING DOCUMENT DEVIATION FORM 31
Appendix E ASSESSMENT REPORTING FORM 33
Appendix F EXAMPLE JOINT VERIFICATION STATEMENT 35
Appendix G STATISTICAL EVALUATION OF TEST PLAN TESTS A AND C 38
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List of Figures Page
Figure 1. Organization Chart for the Joint Verification Test of HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer 7
List of Tables
Table 1.0 DANETV Responsibilities Concerning Reviews and Audits 11
Table 2.0 Assessments for the Joint Verification 19
Table 3.0 Document and Reporting Responsibilities for the Joint Verification* 24
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Date: 01/12/10
DISTRIBUTION LIST
John McKernan
Michelle Henderson
Evelyn Hartzell
U.S. EPA
26 West Martin Luther King Drive
Mail Code: 208
Cincinnati, OH 45268
Christian Gran
Mette Tjener Andersson
DHI DANETV Water Centre
Agern Alle 5,
DK-2970
H0rsholm, Denmark.
Raymond Klicius
Environment Canada
200 Sacre-Coeur
Gatineau, Quebec K1A OH3
Canada
John Neate
Richard Smith
Mona El Hallak
ETV Canada
2070 Hadwen Road Suite 201 A, Mississauga,
Ontario L5K2C9
Canada
Elmar Grabert
Vendor: HACH-LANGE GmbH
Willstatterstrasse 11
40549 Diisseldorf
Germany
Amy Dindal
Mary Schrock
Zachary Willenberg
Battelle
505 King Ave.
Columbus, OH 43201
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Joint Verification Process Document
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Date: 01/12/10
1 INTRODUCTION
DHI DANETV Water Test Centre (DHI) which operates a Danish verification
scheme, DANETV, supported by the Danish Ministry for Science, Technology and
Innovation; the United States Environmental Protection Agency Environmental Technology
Verification Program's Advanced Monitoring Systems (U.S. EPA ETV AMS) Center
operated by Battelle (Battelle AMS Center) under a Cooperative Agreement with the U.S.
EPA; and Ontario Centre for Environmental Technology Advancement (OCETA) through
ETV Canada which operates the Canadian ETV Program on behalf of Environment Canada
will jointly verify the HACH-LANGE GmbH (Vendor) water toxicity technologies named
LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer. This
document establishes a process to ensure that the requirements of all three programs will be
met for the joint verification to be successful.
The purpose of all three ETV programs is to provide objective and quality-assured
performance data on environmental technologies, so that users, developers, regulators, and
consultants can make informed decisions about purchasing and applying these technologies.
Figure 1 describes the organizational relationships for this joint verification. DHI will
perform the verification protocol preparation, the test plan preparation, including quality
assurance (QA), testing, and verification report/test report/verification statement preparation
in accordance with DANETV requirements. To ensure U.S. EPA ETV program acceptance
of the verification, the Battelle AMS Center will provide technical and quality assurance
oversight throughout DHI's process to confirm that each step meets the U.S. EPA ETV
program requirements. The Battelle AMS Center will facilitate the necessary reviews and
audits, coordinate stakeholders and QA personnel for the verification protocol, the test plan,
the testing activities, and the verification reports and statements. This will provide the
necessary oversight for the U.S. ETV program to ensure a quality process of evaluating, data
collection, and reporting for this project, which was deemed an EPA QA Level III project by
the EPA ETV AMS Center Project Officer. ETV Canada will facilitate the necessary reviews
and audits by Environment Canada program management, stakeholders, and QA personnel
for the verification protocol, the test plan, the testing activities, and the verification report.
This will provide the necessary oversight for the Canadian ETV program to ensure a quality
process of evaluating, data collection, and reporting. Because DHI is performing the
technical work and preparing the drafts of the documents, it is assumed that DHI will follow
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Joint Verification Process Document
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Date: 01/12/10
the necessary process to ensure a Danish ETV verification from their actions. Therefore, this
document is focused on what Battelle, ETV Canada and DHI must do to ensure that the
verification fulfills the requirements of the U.S. EPA ETV program and the Canadian ETV
program. The efforts of DHI in testing and verification are described in a verification
protocol and a test plan prepared in compliance with the DANETV Centre Quality Manual
(CQM)1. Together the process document, verification protocol, and test plan are the
equivalent of the U.S.EPA ETV AMS Center test/QA Plan and an ETV Canada technology
specific test/QA plan. The roles of each participant shown in Figure 1 are described in the
following section. The names of the key personnel and their roles during this verification are
presented in Appendix A.
United States
Environmental Protection
Agency, Environmental
Technology Verification
Program (U.S. EPA ETV)
Battelle and U.S.
EPA ETV Advanced
Monitoring Systems
Center
U.S. ETV AMS
Center Water
Stakeholder
Committee
Expert
Group/External
Peer Reviewers
HACH-LANGE
GmbH
Technology Vendor
Environment Canada
Ontario Centre for
Environmental Technology
Advancement (OCETA),
ETV CANADA
DHI DANETV
Water Centre
DHI
Figure 1. Organization Chart for the Joint Verification Test of HACH-LANGE GmbH
LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer
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Joint Verification Process Document
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Date: 01/12/10
1.1 HACH-LANGE GmbH Involvement
HACH-LANGE GmbH, the technology vendor, has entered into a Verification Contract with
DHI and a joint verification agreement with all three parties, DHI, Battelle and ETV Canada. The
vendor will provide the following support during joint verification of the vendor's technology:
*J* A person from vendor's organization to be Battelle's, ETV Canada's, and DHI's technical
points of contact and to lead vendor's participation in verification of vendor's technology;
and
* Review and comment on the process, plan, and report documents which will jointly
satisfy documentation requirements of the U.S., Canadian, and Danish ETV programs;
and other documents pertaining to verification of vendor's technology as requested by
Battelle and/or ETV Canada and/or DHI; and
* Permission to post/cite information about vendor's technology, including the Verification
Protocol, Test Plan, Verification Report, and Joint Verification Statement, on the U.S.
ETV website (www.epa.gov/etv), ETV Canada website (www.etvcanada.ca), DANETV
website (www.etv-denmark.com) and in other program publications; and
* At no cost to DHI, Battelle or ETV Canada, vendor's technology and associated
equipment/materials for testing, appropriate training in its operation, and on-site support
on an as-needed basis; and
»«» Written descriptions, diagrams, and/or photographs of vendor's technology, as input for
the process, plan, and report documents.
1.2 DHI Involvement
During the verification of vendor's technology, DHI will:
*J* Provide a person from DHI's organization to be Battelle's, ETV Canada's and the
vendor's point of contact and to lead DHI's participation in verification of the vendor's
technology;
* Prepare and revise the Verification Protocol, Test Plan, Verification Report, Test Report,
Verification Statement, and other documents pertaining to the verification of the vendor's
technology and allow Battelle, U.S. EPA, ETV Canada, Environment Canada, and the
Expert Group/Peer Reviewers the opportunity to review and comment on these
documents;
* Provide input, review, and comment on the Process Document to be prepared by Battelle;
*J* Assemble a team of qualified technical staff to conduct the verification test in accordance
with the Verification Protocol, Test Plan, and this document;
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Date: 01/12/10
*J* Ensure that all quality procedures specified in the Verification Protocol, Test Plan,
DANETV CQM1, and this document are followed;
*J* Participate in a (virtual) joint kick-off meeting, led by Battelle and including
representatives from ETV Canada, (using checklist in Appendix B) prior to test initiation;
*J* Provide a summary of the QA activities performed by the DHI DANETV Water Centre
Internal Auditor during the verification testing; and
*J* Provide permission to post/cite information about DHI's involvement in the joint
verification, including the Joint Verification Protocol, Test Plan, Verification Report, Test
Report and Verification Statement, on the U.S. ETV website and the Canadian ETV
website and in other program publications.
1.3 Battelle Involvement
During the ETV verification of the vendor's technology, Battelle will:
*J* Provide a person from Battelle to be DHI's, ETV Canada's and the vendor's point of
contact;
* Prepare a procedural document (Process Document) outlining the process of the vendor's
technology verification for acceptance by the U.S. EPA (this document);
*J* Provide input, review, and comment on the Verification Protocol, Test Plan, Verification
Report, Test Report, Verification Statement, and other documents pertaining to
verification of the vendor's technology;
*J* Coordinate review of the Verification Protocol, Test Plan, Verification Report, Test
Report, Verification Statement, and other documents pertaining to verification of the
vendor's technology with U.S. EPA and U.S. expert reviewers and consolidate these
comments for submission to DHI;
*J* Lead a (virtual) joint kick-off meeting with participants from DHI and ETV Canada
(using checklist in Appendix B) prior to test initiation;
*J* Strive to obtain U.S. EPA approval for the final Verification Protocol, Test Plan, Test
Report, Verification Report, including a Verification Statement; and
*J* Comply with all quality procedures and program requirements specified in the
Verification Protocol, Test Plan, ETV AMS Center QMP2, and U.S. ETV Program
QMP3, as follows:
o Prepare and get U.S. EPA ETV approval of an audit checklist and provide the
checklist to DHI prior to the audit;
o Conduct a technical systems audit once during the verification test;
o Audit at least 10% of the verification data;
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o Prepare and distribute an assessment report for each audit;
o Verify implementation of any necessary corrective action; and
o Provide a summary of the quality assurance/quality control (QA/QC) activities and
results for the verification reports.
1.4 ETV Canada Involvement
During the ETV verification of the vendor's technology, ETV Canada will:
*J* Provide a person from ETV Canada to be DHI's, Battelle's and the vendor's point of
contact;
*J* Provide input, review, and comment on the Process Document, Verification Protocol,
Test Plan, Verification Report, Verification Statement, and other documents pertaining to
verification of the vendor's technology;
* Coordinate review of the Verification Protocol, Test Plan, Verification Report, Test
Report, Verification Statement, and other documents pertaining to verification of the
vendor's technology with Environment Canada and Canadian expert reviewers as needed,
and consolidate these comments for submission to DHI;
*J* Participate in a (virtual) joint kick-off meeting, led by Battelle and including
representatives from DHI, (using checklist in Appendix B) prior to test initiation;
*J* Inform Environment Canada of the final Verification Protocol, Test Plan, Verification
Report, including the Verification Statement; and
*J* Comply with all quality procedures and program requirements specified in the
Verification Protocol, Test Plan, and ETV Canada General Verification Protocol (GVP)4,
as follows:
o Review Test Plan;
o Review test data;
o Provide summary of review; and
o On site audit - might not be required by the Canadian ETV Program.
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1.5 DANETV Involvement
DANETV responsibilities are based on the requirements stated in the DANETV CQM1 and
include the following:
Table 1.0 DANETV Responsibilities Concerning Reviews and Audits.
DHI External
Internal Expert
Function
Tasks
Plan document with
verification protocol and test
plan
Test system
Report document with test
report and verification report
Technical
expert
Review
-
Review
Trained auditor
QA staff
Audit
~
Technical
expert
Review
-
Review
1.6 U.S. EPA Involvement
A complete list of U.S. EPA's responsibilities for the AMS Center are based on the
requirements stated in the AMS Center ETV QMP.2 The U.S. EPA will provide technical and quality
review for this joint verification activity to ensure the test planning and reporting documents are in
compliance with the U.S. ETV program requirements.
1.7 Environment Canada Involvement
The Canadian ETV program is delivered by ETV Canada under a license agreement from
Environment Canada. ETV Canada will consult with Environment Canada as required.
1.8 Stakeholder Committee and Expert Group/Peer Reviewer Involvement
The AMS Center's Water Stakeholder Committee is made up of buyers and users of
water monitoring technologies. This committee assists in prioritizing the types of
technologies to be verified and in specific cases, provides testing support. It also has
representatives that assist in review of the Test/QA plans, Verification Reports, Test Report
and Verification Statements. The AMS Center Water Stakeholder Committee provided
concurrence for the Center to proceed with testing in this area. The stakeholders have been
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kept apprised of progress throughout the planning process for this test and have provided
input during progress meetings on the test design.
In addition, the U.S. ETV AMS Center obtains the peer review of at least one external
peer reviewer and one EPA peer reviewer who are not directly involved with the verification
test. ETV Canada uses an independent technical expert to perform the peer review of
documents and test results. Reviews will be documented using Review Report Form
(Appendix C). Battelle will consolidate comments from the U.S. reviews into one Review
Report form and ETV Canada will consolidate comments from Canadian reviews into one
Review Report form, to ease DHI implementation of comments.
The DANETV program uses an Expert Group to perform the external peer review of
the documents and give input on the verification. For this test, the Expert Group is made up
of three individuals to fulfill the requirements of all three programs. These individuals are
named with their affiliations in the Verification Protocol5 and Test Plan6.
2 QUALITY SYSTEMS
The DHI, Battelle, and ETV Canada quality systems to be implemented for this joint
verification will conform with the specifications listed in:
• ANSI/ASQ E4-2004, "Specifications and Guidelines for Quality Systems for
Environmental Data Collection and Environmental Technology Programs"7
• International Standards Organization (ISO) 90018.
• Laboratory accreditation ISO 17025.
Per the U.S. EPA ETV Program QMP3, verification organization quality systems,
such as DHI's quality systems, are to be reviewed and approved by verification organization
management, the U.S.EPA AMS Center Manager, and the U.S. EPA AMS Center QA
Manager. This process document will serve to define the specific quality activities that will
be performed by Battelle, DHI, and ETV Canada for this joint verification and clearly
identify the processes required to comply with the quality manuals for the three
organizations.
Per the Canadian ETV Program, DHI's quality systems are to be reviewed and
approved by ETV Canada.
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3 VERIFICATION PLANNING
In performing the verification test, DHI, Battelle and ETV Canada will follow the
technical and QA procedures specified in the Verification Protocol5, Test Plan6, and this
process document. Because DHI is preparing the Verification Protocol, Test Plan,
Verification Report, Test Report and Verification Statement, as well as conducting the testing
activities, the procedures and expectations of the U.S. EPA ETV program and Canadian ETV
program need to be clarified in a document that explains the process and requirements (this
document).
3.1 Planning the Test Design
The verification test design will produce a Verification Protocol5 and Test Plan6 based
upon the DANETV, U.S. EPA ETV, and ETV Canada processes. These two documents,
together with this process document, represent the equivalent of a U.S. EPA ETV AMS
Center Test/QA plan and ETV Canada's technology-specific test/QA plan. The protocol
includes an Application and Performance Parameter Definition Document (Appendix 3 in the
Verification Protocol5) that developed relevant parameters and ranges for verification
considering the vendor stated performance, government standards, and other technologies and
methods in the market. The Application and Performance Parameter Definition Appendix
was not jointly produced and is a specific process within the DANETV program; therefore, it
was not reviewed by the U.S. EPA ETV program or ETV Canada. In designing this
verification test, DHI staff used consensus-accepted test design, a U.S. EPA ETV protocol for
similar products, a standard for verification of online equipment (ISO 158399) and a protocol
from TESTNET10. All relevant activities pertaining to environmental data operations have
been identified, as well as performance specifications and the appropriate controls.
Additionally Battelle conducted a statistical review of the test plan design for calculating
EC50 and EC20 generated by analyzing various sets of solutions with ranges between 10 and
90% inhibition which is included in Appendix G.
Finally, a process document (this document) was produced by Battelle to address the
process and differences between the programs to ensure a successful joint verification.
Collectively, these three documents (the Verification Protocol5, Test Plan6, and Process
Document for the U.S. EPA ETV AMS Center, DHI DANETV Water Centre and ETV
Canada Joint Verification of the HACH-LANGE GmbH LUMIStox 300 Bench Top
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Luminometer and ECLOX Handheld Luminometer) are referred to as the "testing
documents".
The U.S. EPA ETV process utilized its Water Stakeholder Committee to guide the
test design process. The Water Stakeholder Committee provided concurrence for the Center
to proceed with testing in this area. The stakeholders have been kept apprised of progress
throughout the planning process for this test and have provided input during progress
meetings on the test design. A U.S. EPA peer reviewer, who is also an ETV AMS Center
Water Stakeholder, was identified to review the Verification Protocol and Test Plan. ETV
Canada will review the Test Plan and Verification Protocol (an external expert will be
involved if required). The DANETV program uses an Expert Group to perform the external
peer review of the Verification Protocol and Test Plan documents and give input on the
verification. For this test, the Expert Group is made up of individuals to fulfill the
requirements of all three programs. It includes up to three individuals that have extensive
experience in the field of water toxicity monitoring, one U.S. EPA reviewer, one European
reviewer, and one Canadian reviewer. These reviewers have no direct involvement in the
verification test beyond providing their reviews. The comments from the reviews performed
by the Expert Group of the Verification Protocol, Test Plan, Verification Report, Test Report
and Verification Statement will be reconciled by DHI. The review process will utilize the
Review Report Form produced by DHI which is included in Appendix C.
4 VERIFICATION TEST IMPLEMENTATION
This technology performance verification will be implemented according to the
Verification Protocol5 and the Test Plan6 (including technical procedural documents)
prepared during planning. Generation of verification test data will not be initiated until the
Verification Protocol and Test Plan are approved by all three programs. System control data
can be produced before approval. Any data generated before the required documents are
approved will have to be repeated. In performing the verification test, DHI will perform an
internal audit of the data collection and handling that follows the technical and QA
procedures specified in these documents, as well as, the DANETV CQM1. Battelle and ETV
Canada will perform technical systems audits (TSA) as appropriate to be sure that these
requirements are being met.
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A virtual joint kick-off meeting will be held prior to the start of the verification test to
review procedures for the test with all verification testing staff. The joint kick-off meeting
checklist is provided in Appendix B.
Test personnel will have access to the approved testing documents, approved changes
to testing documents, and all referenced documents. When a prescribed sequence for the
work is defined in the testing documents, work performed shall follow that sequence.
Changes to that sequence need to be documented by preparing a deviation (Appendix D). All
verification test activities will be documented. Suitable documents are bound notebooks (e.g.
laboratory record books, or LRBs), laboratory data sheets, spreadsheets, computer records,
and output from instruments (both electronic and hardcopy). All documentation is
implemented as described in the testing documents. All implementation activities are
traceable to the testing documents and to the test personnel. The responsibilities of specific
test personnel listed in these testing documents that leave the project before it is completed
will be reassigned.
When work cannot be implemented according to the approved testing documents,
DHI shall be responsible for providing a written deviation report for the test records.
Substantial changes to the way work is being implemented compared to the way work is
described in the testing documents will be discussed by a teleconference of representatives of
the three programs or through an email exchange between the three program representatives,
before substantial changes are implemented. As soon as Battelle becomes aware of a
deviation, Battelle will also notify the U.S. EPA AMS Center Project Officer and QA
Manager. A deviation report is produced for any changes to the testing document that
occurred during the test. Deviation reports must be retained in the verification test records
and summarized in the Verification Report. Frequent deviations from established procedures,
which indicate that testing cannot be performed as written, should result in a retrospective
review of the written document(s) and possible revision. Deviations will include all the
information displayed on the forms shown in Appendix D.
All persons responsible for performing verification testing and HACH-LANGE
GmbH will receive copies of the final versions of the Verification Protocol5 and the Test
Plan6 and associated documentation provided by DHI. Current versions of the Verification
Protocol5 and the Test Plan6 and any applicable methods and SOPs are required to be
physically in place at the technology verification testing sites. Battelle oversight and
inspection of the verification test will be provided by the Battelle AMS Center Quality
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Manager or designee, ETV Canada oversight and inspection of the verification test will be
provided by the Quality Manager. On site oversight and inspection activities by Battelle will
occur during one week. An audit checklist for Battelle to use will be prepared by Battelle and
approved by the U.S.EPA AMS Center Project Officer and U.S. EPA AMS Center QA
Manager. If required, an audit checklist for ETV Canada will be prepared and approved by
the ETV Canada Program Manager. The audit checklists will be provided to DHI prior to the
audit. The audit will begin with an "In Briefing" conducted by the Battelle AMS Center
Quality Manager and, if applicable, the ETV Canada Quality Manager to specify and clarify
the necessary points of the audit. Testing during laboratory activities will be observed along
with viewing the external laboratory performing the reference analyses. To verify full
implementation of the testing documents, the inspection will include the testing process and
any documentation associated with the process, such as sample chain of custody transfers,
instrument maintenance and calibration, sample preparation and analysis, and data records.
At the conclusion of the audit there will be an "Exit Briefing" held to discuss the findings and
corrective actions necessary. The Battelle AMS Center Quality Manager and, if applicable,
ETV Canada Quality Manager will also provide a written audit report, verify the completion
of any corrective actions needed, and retain a copy of the report with permanent Battelle
AMS Center Quality Manager records and a copy in the ETV project records. The audit
report will be sent to DHI within 10 working days after the end of the audit. The audit report
will be commented on by DHI and comments addressed before it is distributed. The
Assessment Reporting Form is presented in Appendix E. The U.S. EPA AMS Center Project
Officer and ETV Canada Program Manager will be included in the routing of the inspection
results and a written copy provided to the U.S. EPA AMS Center Project Officer, the U.S.
EPA AMS Center QA Manager, the ETV Canada Program Manager and Quality Manager,
and DHI.
5 ASSESSMENT AND RESPONSE
Assessments will be planned, scheduled, conducted, and reported in order to measure
the efficacy of the DHI, Battelle and ETV Canada quality procedures and verification
execution. The testing will be audited internally by the DHI Internal Auditor in accordance
with the Verification Protocol5 and Test Plan6. The DHI Document reviewer will perform the
technical review of the Test Plan and Verification Report documents. The DHI Internal
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Auditor will perform an audit based upon identified critical points. The DHI Document
Reviewer and Internal Auditor equate to the Battelle AMS Center Quality Manager and ETV
Canada Quality Manager. The procedure includes two main steps:
• Check that the protocol/plan is prepared and followed in accordance with the DANETV
CQM (horizontal audit).
• Check verification/test parameters and data at the identified critical points, i.e., a vertical
audit in laboratory, office.
Data from the testing will be controlled by the DHI Verification Responsible and Test
Responsible when received (see Appendix A for definition of roles of key personnel and an
explanation of the parallel roles between DANETV, U.S. EPA ETV, and ETV Canada
programs). Data integrity will be controlled by the Test Responsible (transfer of raw data to
spreadsheets) and Verification Responsible (calculations as part of evaluations) as spot
checks (5% of the data).
Monitoring of the work process to be conducted by the Battelle AMS Center Quality
Manager and ETV Canada Quality Manager will be done to:
• Ensure satisfactory performance based on requirements,
• Ensure required actions (as specified in implementation documents) are performed so that
routine measurements meet specifications,
• Ensure preventive maintenance is performed and documented as specified in facility and
study records,
• Ensure calibrations are performed as planned and prescribed,
• Ensure corrective actions are implemented and documented as planned in response to
items of nonconformance.
Assessment types, responsibility, and schedule for this joint verification will be as
shown in Table 2.0, and are defined below. Battelle will conduct one technical systems audit
(TSA) and one audit of data quality (ADQ) for this program. Battelle completed a quality
systems audit (QSA) of DHI in February of 2009.
Quality Systems Audit (QSA), an on-site review of the implementation of the DHI quality
procedures. This review is used to verify the existence of, and evaluate the adequacy of, the
internal quality system. This review will be done in conjunction with the Technical Systems
Audit.
Technical Systems Audit (TSA), a qualitative on-site evaluation of sampling and/or
measurement systems associated with a particular verification test. The objective of the TSA
is to assess and document the acceptability of all facilities, maintenance, calibration
procedures, reporting requirements, sampling, and analytical activities, and quality control
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procedures in the test. Conformance with the testing documents and associated methods
and/or Standard Operating Procedures is the basis for this assessment. The Battelle AMS
Center Quality Manager will prepare and use an audit checklist that is approved by the EPA
AMS Center Quality Manager. If necessary, the ETV Canada Quality Manager will prepare
an audit checklist and ETV Canada Program Manager will review. The checklists will be
available to DHI before the audit takes place. This review will be done in conjunction with
the QSA.
Performance Evaluation Audits (PE), a quantitative evaluation of the measurement systems
used. The type and frequency of performance evaluation self-audits are specified in the Test
plan for the joint verification test. The value or composition of reference materials must be
certified or verified prior to use, and the certification or verification must be adequately
documented. The Battelle AMS Center Quality Manager and ETV Canada Quality Manager
will review results of PE audits during the TSA; however, it is most preferable for the PE
results to be shared with Battelle and ETV Canada as soon as they are available, so that any
issues can be resolved.
Audits of Data Quality (ADQ), an examination of the verification data after they have been
collected and verified by project personnel. The Battelle Verification Test Coordinator will
review 100% of the data and the Battelle AMS Center Quality Manager will audit at least
10% of all verification data, including equations and calculations. The ETV Canada Quality
Manager will audit all test data including calculations. DHI will provide technology test data
and associated records (e.g., data sheets; notebook records) from the first day of testing within
one day and thereafter on a weekly basis to ETV Canada and Battelle. The first set of
reference laboratory data will also be sent within one day of receipt by DHI, then on a weekly
basis. Battelle will immediately forward these data to US.EPA upon receipt (i.e., for
simultaneous review). The goal of this data delivery schedule is prompt identification and
resolution of any issues.
Note that if it is determined by DHI, Battelle, U.S. EPA, or ETV Canada during any
of the assessments that test objectives of acceptable quality cannot be achieved, a stop work
order should be considered and discussed among all parties. See Section 5.2 for more
information on stop work orders.
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Table 2.0 Assessments for the Joint Verification
Assessment Subject of Minimum Reason for
Tool Assessors Responders Assessment Frequency Assessment Report Reviewed by
Quality
Systems
Audit
Technical
Systems
Audits
Performance
Evaluation
Audits
Audits of
Data Quality
Battelle AMS
Center Quality
Manager and
ETV Canada
Quality Manager
Battelle AMS
Center Quality
Manager, ETV
Canada Quality
Manager and DHI
Internal Auditor
Battelle AMS
Center Quality
Manager, ETV
Canada Quality
Manager and DHI
Internal Auditor
Battelle AMS
Center Quality
Manager, ETV
Canada Quality
Manager and DHI
Internal Auditor
DHI
DHI
DHI
DHI
DANETV
CQM
Verification
Protocol, Test
Plan, and
Process
Document
Verification
Protocol, Test
Plan, and
Process
Document
raw data and
summary data
Once*,
thereafter as
requested
Once
Once
At least 10%
of the
verification
data
Assess quality
management
practices of
verification
collaborators
Assess
technical
quality of
verification
tests
Assess
measurements
performance
Assess data
calculations
and reporting
U.S. EPA directors of
quality assurance,
U.S. EPA AMS Center
Project Officer,
Battelle AMS Center
Manager and Test
Coordinator, ETV
Canada Program
Manager and Quality
Manager, DHI
Verification
Responsible
U.S. EPA AMS Center
Project Officer,
U.S. EPA AMS Center
QA Manager, Battelle
AMS Center Manager
and Test Coordinator,
ETV Canada Program
Manager and Quality
Manager, DHI
Verification
Responsible
U.S. EPA AMS Center
Project Officer,
U.S. EPA AMS Center
QA Manager, Battelle
AMS Center Manager
and Test Coordinator,
ETV Canada Program
Manager and Quality
Manager, DHI
Verification
Responsible
U.S. EPA AMS Center
Project Officer,
U.S. EPA AMS Center
QA Manager, Battelle
AMS Center Manager
and Test Coordinator,
ETV Canada Program
Manager and Quality
Manager, DHI
Verification
Responsible
*QSA of DHI completed by Battelle in February 2009.
5.1 Assessment Reports
Each assessment must be fully documented. Battelle completed a QSA of DHI in
February 2009 and this report is available. Within 10 working days of the TSA and ADQ, the
Battelle AMS Center Quality Manager will provide a draft audit report to the U.S. EPA AMS
Center Project Officer and QA Manager. The draft audit report will summarize any potential
issues or findings noted during the audits. The draft audit report may require additional
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editorial review prior to submitting to testing staff for corrective actions and corrective
actions will not be documented in the draft; however, the goal is to provide immediate
information to the U.S. EPA AMS Center Project Officer and QA Manager on the types of
corrective actions being requested as part of the assessments. The PE audits will be reviewed
as part of the ISA. Also, DHI has been asked to share PE results with Battelle and ETV
Canada as soon as they are available so that any issues can be resolved. If information on the
PE audits is provided to Battelle outside of the TSA, Battelle will forward that information to
the U.S. EPA AMS Center Project Officer and QA Manager within 10 days of receipt. The
Battelle AMS Center Quality Manager, the ETV Canada Quality Manager and the DHI
Verification Responsible will archive all assessment reports generated for this verification
test.
Each assessment must be responded to by the appropriate level of management. The
Battelle quality assessment reports shall require a written response by the person performing
the inspected activity, and acknowledgment of the assessment by the Battelle AMS Center
Verification Test Coordinator. The Assessment Reporting Form is provided in Appendix E.
The audit report will be based on the forms in Appendix E, but a separate report will be
prepared. An assessment report will also be prepared by the DHI Internal Auditor and
provided to the Verification Center Test Coordinator for archive.
Corrective action must be documented and approved on the original assessment
report, with detailed narrative in response to the assessor's finding. Initials and date are
required for each corrective action response. Acknowledgment of the response will be
provided by the Battelle AMS Center Verification Test Coordinator.
Implementation of corrective actions must be verified by the Battelle AMS Center
Quality Manager or the DHI Internal Auditor to ensure that corrective actions are adequate
and have been completed. This will be done in real-time if corrective actions can be
immediately performed and signed off on the assessment report. Alternatively, should the
corrective action require additional approvals not immediately available on-site, the DHI
Internal Auditor may need to repeat the inspection, as the designee of the Battelle AMS
Center Quality Manager, in order to corroborate the implementation and effectiveness of the
corrective action.
Implementation of corrective actions will be verified by the ETV Canada Quality
Manager to ensure that corrective actions are adequate and have been completed. This will be
done during data review.
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5.2 Stop Work
Assessor responsibility and authority to stop work during a verification test for quality
considerations is delegated to DHI, Battelle, and ETV Canada. DHI must ensure compliance
with all applicable Danish federal, state, and local safety policies during the performance of
verification testing.
Should it be determined by Battelle, U.S. EPA, or ETV Canada during an assessment
that test objectives of acceptable quality cannot be achieved during performance of
verification testing, Battelle and ETV Canada are responsible for immediately notifying the
DHI Verification Responsible of the need to consider a stop work order. The DHI
Verification Responsible will then direct the staff accordingly. The U.S EPA AMS Center
QA Manager will notify the U.S. EPA AMS Center Project Officer if work of inadequate
quality is discovered.
During verification, the ETV Canada Quality Manager will notify the ETV Canada
Program Manager if work of inadequate quality is discovered.
Documentation is required of any stop work order and the corrective action
implemented and shall be maintained as part of the Battelle quality records, with a copy
provided to the U.S. EPA AMS Center Project Officer and U.S. EPA AMS Center QA
Manager and as a part of ETV Canada quality records, with a copy provided to the ETV
Canada Quality Manager, and DHI.
5.3 Response
Responses to TSA adverse findings should be addressed within 10 working days after
the TSA report is completed. However, it is expected that findings that have a direct impact
on the conduct of a verification test will be corrected immediately following notification of
the finding.
Responses to each adverse finding will be documented in the assessment report.
Ideally, assessment reports will provide space after each adverse finding for a response to be
recorded. The response will indicate the corrective action taken or planned to address the
adverse finding. The response should be signed and dated by the staff responsible for
implementing the corrective action.
Any corrective action that cannot be immediately implemented will be verified
following completion by the Battelle AMS Center Quality Manager or designee and by the
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ETV Canada Quality Manager. Once all corrective action associated with an assessment
report has been taken, the Battelle AMS Center Quality Manager or designee and the ETV
Canada Quality Manager will initial the corrective action in the assessment report thus
documenting verification of the corrective action. Any impact that an adverse finding had on
the quality of verification test data should be addressed in the verification report.
The TSA report, with responses to adverse findings recorded within, will be sent to
U.S. EPA within 10 working days after the Battelle AMS Center Quality Manager has
verified all corrective actions.
6 DOCUMENTATION AND REPORTING
6.1 Documentation and Reporting Responsibilities
Responsibilities for activities concerning documentation and reporting are
summarized in Table 2.0 and are detailed below.
6.1.1 Preparation
Individual case requirements and this document shall guide document and record
content and/or format. Guidance for content and/or format are derived by the U.S. EPA
ETV, ETV Canada and DANETV directives and the following documents:
• ANSI/ASQ E4-20047.
• U.S. ETV AMS Center QMP2.
• U.S. EPA ETV Program QMP3.
• U.S. EPA document "EPA QA/R-2, EPA Requirements for Quality Management Plans"
March 2001.
• ETV Canada GVP4, February 2007.
• DHICQM1.
• Nordic Water Technology Verification Centers. NOWATECH. Final technical
report. Nordic Innovation Centre Project 06223. August 2009.
6.1.2 Review/Approval
Record review/approval for joint verification testing documents shall be performed by
qualified technical and/or management personnel as described in Table 3.0. The individual
reviewer shall have access to all needed references.
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6.2 Reporting
The end result of the joint verification process will be a Verification Report, Test
Report and Verification Statement for the HACH-LANGE GmbH LUMIStox 300 Bench Top
Luminometer and ECLOX Handheld Luminometer. The review and approval procedures for
the verification report and statement for U.S. EPA ETV program and ETV Canada purposes
are given in Table 2.0. The Verification and Test Report will be reviewed by external
reviewers in the Expert/ Peer Reviewer Group and the Verification Statement will be signed
by an U.S. EPA laboratory director, Battelle management, ETV Canada management
(OCETA), the DHI Director of Research and Quality Management and the DANETV
Steering Committee Head. Appendix F presents a preliminary template for a Verification
Statement. This document will be expanded and organized to meet U.S. EPA ETV, ETV
Canada and DANETV program requirements.
All logos will appear on the Verification Statement. These will include: U.S. ETV,
Battelle, Environment Canada and ETV Canada, DANETV, DHI, and U.S. EPA logos. All
logos except the U.S. EPA logo will appear on the cover page of all other joint testing
documents (Test Plan, Verification Protocol, Test Report, Verification Report). All of these
testing documents will be made publicly available on the U.S. EPA ETV Web site
(www.epa.gov/etv), the DANETV Web site (www.etv-denmark.com), and ETV Canada Web
site (www.etvcanada.ca) regardless of the technology's performance.
The vendor will comply with the DANETV, U.S. EPA ETV and ETV Canada policies
on referencing the verification documents of their technology.
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Table 3.0 Document and Reporting Responsibilities for the Joint Verification*
Record Preparation/
Type Updating Review Approval Finals Distributed to:
Verification
Protocol and
Test Plan
(including SOPs,
amendments and
deviations)
Raw data
Verification
Report
ETV Verification
Statement
Audit Reports
Audit Reports
Audit Reports
DHI
DHI
DHI
DHI
DHI Internal
Auditor
Battelle AMS
Center Quality
Manager
ETV Canada
Quality
Manager
Battelle AMS Center Manager,
Battelle AMS Center Quality Manager,
U.S. EPA AMS Center QA Manager,
U.S. EPA AMS Center Project Officer,
ETV Canada Program Manager,
ETV Canada Quality Manager,
ETV Canada External Technical Expert if
required,
DHI Verification Responsible,
DHI Document Reviewer,
Stakeholders/Expert Group,
Vendor
DHI Internal Auditor
Battelle AMS Center Manager,
Battelle AMS Center Quality Manager,
U.S. EPA AMS Center QA Manager,
U.S. EPA AMS Center Project Officer,
ETV Canada Program Manager,
ETV Canada Quality Manager,
ETV Canada External Technical Expert if
required,
DHI Verification Responsible,
DHI Document Reviewer,
Stakeholders/Expert Group,
Vendor
Battelle AMS Center Manager,
Battelle AMS Center Quality Manager,
U.S. EPA AMS Center QA Manager,
U.S. EPA AMS Center Project Officer,
ETV Canada Program Manager
ETV Canada Quality Manager
ETV Canada External Technical Expert if
required
DHI Verification Responsible,
DHI Document Reviewer,
Vendor,
U.S. ETV Program Director
Stakeholders/Expert Group
DHI Test Responsible
DHI Verification Responsible
Battelle AMS Center Manager,
Battelle Verification Test Coordinator,
DHI Verification Responsible
DHI Test Responsible
U.S. EPA AMS Center QA Manager
ETV Canada Program Manager
ETV Canada Quality Manager
U.S.EPA AMS Center
Project Officer,
U.S. EPA AMS Center QA
Manager,
U.S. ETV Program Director
ETV Canada Program
Manager,
ETV Canada Quality
Manager,
DHI Verification
Responsible
N/A
U.S.EPA AMS Center
Project Officer,
U.S. EPA AMS Center QA
Manager,
U.S. ETV Program Director
ETV Canada Program
Manager,
ETV Canada Quality
Manager
DHI Verification
Responsible
U.S. EPA Laboratory
Director,
U.S. ETV Program
Director, Battelle
Management,
U.S. EPA AMS Center
Project Officer,
U.S. EPA AMS Center QA
Manager,
ETV Canada Program
Manager,
ETV Canada Quality
Manager
DHI Director R&D and
Quality Management
DANETV Steering
Committee Head
N/A
N/A
Testing Staff,
Vendor,
U.S. EPA AMS Center Project
Officer,
U.S.EPA AMS Center QA Manager,
ETV Canada Program Manager,
ETV Canada Quality Manager
DHI Verification Responsible
U.S.EPA can request copies
U.S. ETV Program Director,
U.S. EPA AMS Center Project
Officer,
U.S. ETV Webmaster,
ETV Canada Program Manager,
ETV Canada Quality Manager
Vendor,
DHI Verification Responsible
U.S. ETV Program Director,
U.S. EPA AMS Center Project
Officer,
Battelle AMS Center Manager,
ETV Canada Program Manager,
ETV Canada Quality Manager
DHI Verification Responsible,
U.S. ETV Webmaster,
Vendor
DHI Verification Responsible,
Battelle AMS Center Quality
Manager,
Battelle AMS Center Manager,
Battelle Verification Test Coordinator,
U.S. EPA AMS Center Project
Officer,
U.S. EPA AMS Center QA Manager,
ETV Canada Program Manager,
ETV Canada Quality Manager
U.S.EPA AMS Center Project Officer,
U.S.EPA AMS Center QA Manager,
Battelle AMS Center Manager,
Battelle Verification Test Coordinator,
DHI Verification Responsible
ETV Canada Program Manager,
ETV Canada Quality Manager
*See Appendix A for the roles and names of the individuals filling these roles.
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REFERENCES
1. ETV Test Centre and Test Organization. Centre Quality Manual - Water technology. Version 2.
October 2009.
2. Battelle. Quality Management Plan for the ETV Advanced Monitoring Systems Center. Version
7.O.November 2008.
3. United States Environmental Protection Agency. Environmental Technology Verification
Program Quality Management Plan. Version 3.0. January 2008.
4. ETV CANADA General Verification Protocol (GVP). February 2007
5. DHI. HACH-LANGE GmbH LUMIStox 300 Bench Top Luminometer and ECLOX Handheld
Luminometer, Joint Verification Protocol. Review Version. November 2009.
6. DHI. HACH-LANGE GmbH LUMIStox 300 Bench Top Luminometer and ECLOX Handheld
Luminometer, Joint Test Plan. Review Version. November 2009.
7. American Society for Quality. ANSI/ASQ E4-2004. Quality systems for environmental data and
technology programs - Requirements with guidance for use. 1-4-2004.
8. International Standardization Organization. ISO 9001. Quality management systems -
Requirements. 11-15-2008.
9. International Standard Organization. ISO 15839. Water quality-On-line sensors/analyzing
equipment for water-Specifications and performance tests. First Edition 2003-10-15.
10. TESTNET Evaluation Report Test case la:TOXcontrol BioMonitor for Surface water
Workpackage 3. 01-03-2008.
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APPENDIX A
ROLES OF KEY PERSONNEL
DANETV Role:
DANETV person who signs the Verification Statement: DANETV Steering Committee Head
- Christian Gran
DHI DANETV Water Centre (DHI) Roles:
DHI person who signs the Verification Statement: Director of Research & Development and
Quality - J0rn Rasmussen
DHI Verification Responsible: Mette Tjener Andersson
DHI Test Responsible: Claus J0rgensen
DHI Document Reviewer: Margrethe Winter-Nielsen
DHI Internal Auditor: Bodil Mose Pedersen
DHI Verification and Test staff: several - see protocol and plan
U.S. EPA ETV Roles:
U.S. EPA person who signs the Verification Statement: National Risk Management Research
Laboratory (NRMRL) Director: Sally Gutierrez
U.S. EPA ETV Program Director, Teresa Harten
U.S. EPA AMS Center Project Officer: John McKernan
U.S. EPA AMS Center QA Manager: Michelle Henderson
Battelle Advanced Monitoring Systems (AMS) Center Roles:
Battelle person who signs the Verification Statement: Chemical, Environmental and Materials
Operations Manager: Lisa McCauley
AMS Center Manager: Amy Dindal
AMS Center Quality Manager: Zack Willenberg
AMS Center Verification Test Coordinator: Mary Schrock
Environment Canada Roles:
Environment Canada person who oversees the Canadian ETV Canada Program: Head of
Technology Program: Raymond Klicius.
ETV Canada Roles:
Ontario Center of Environmental Technology Advancement person who signs the
Verification Statement: President and CEO: Kevin Jones
ETV Canada Program Manager: John Neate
ETV Canada Quality Manager: Mona El Hallak
Parallel Roles between the DANETV, U.S. EPA ETV and ETV Canada programs:
Verification Responsible = Center Manager = Program Manager
Test Responsible = Center Test Coordinator = Quality Manager
Internal Auditor + Document Reviewer = Center Quality Manager = Quality Manager
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APPENDIX B
KICK OFF MEETING CHECKLIST
ETV JOINT VERIFICATION TEST KICK-OFF MEETING
PURPOSE
To prepare verification testing staff for the DANETV, U.S. EPA ETV, ETV Canada joint verification
test and review critical logistical, technical, and administrative aspects of the test. The kick-off
meeting will be scheduled prior to the start of testing. It should be near the start of the test but allow
time for DHI to address any lingering issues.
FORM
The kick off meeting will be virtual, i.e. based upon phone and WebEx sharing of documents.
STAFF TO ATTEND
• Verification test coordinator/responsible (DHI, Battelle)
• Verification Test quality staff (DHI, Battelle)
• Representative of ETV Canada approved by ETV Canada
• U.S. EPA ETV. Environment Canada, and DANETV program staff (invited but optional)
All testing staff involved in all phases of test will subsequently have a kick-off meeting on-site with
the DHI Verification Responsible. The external laboratory is informed through requisitions of
analyses only.
PROJECT MANAGEMENT
• Review roles/responsibilities of all staff attending meeting
• Review test schedule, review notifications of testing schedule (e.g., U.S. ETV stakeholders,
others?)
• Documentation of all pertinent forms.
o Vendor-Collaborator agreement
o Peer review forms on Verification Protocol and Test Plan. Must include one U.S.
EPA reviewer/two non-U.S. EPA peer reviewers (preferably one from Canada and
one from Europe).
o Final Test Plan approved by vendor.
o Documentation that the vendor is satisfied that the staff operating the technology are
proficient in its use.
QUALITY ASSURANCE
• Copies of all standard methods cited or included in the Test Plan, the final Test Plan, and the
final Joint Verification Protocol are available to verification testing staff and in the laboratory
where test will be performed?
• U.S. EPA QA staff pre-notified of test start date and ETV Canada Quality Manager.
• Remind testing staff to sign and date everything.
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US ETV AMS Center, DHIDANETV Water Centre and ETV Canada
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Date: 01/12/10
• If samples are to be transported between labs, DHI should bring an example chain-of-custody
form to the meeting, review how to complete, and where to obtain form.
• Review deviation/amendment procedures at meeting - what to do in the middle of a test if
testing document cannot be followed - who to notify/what forms to file.
• Review testing document at meeting - identify key testing procedures and critical steps to
ensure no ambiguity or questions.
• Are or will there be copies of the certificates of analysis in the verification test records
(documentation from vendor attesting to the quality and concentration of stock standard
solutions used in the test)?
• If applicable, discuss when performance evaluation (PE) audit(s) will be performed? Who
will perform? Have materials/equipment been purchased or obtained for the PE audit? What
are quality control (QC) limits will be considered acceptable? What should be done if QC
limits are not met? Who should be contacted?
• Will regular communication between DHI, Battelle and ETV Canada be maintained? If so,
how? Daily/weekly email updates?
TECHNICAL
• Emphasize to testing staff to document anything and everything that is observed about the
technologies, particularly if there are unusual sample results (e.g., sample color).
• Are provisions made to handle daily preparation of solutions/standards, if necessary?
• Take digital photos of all test activities.
DATA/REPORTING
• Review data recording forms or sheets at meeting or discuss how/where will data be recorded
for each testing activity
• How are data going to be converted electronically? Are data saved in technology undergoing
verification and then exported to Excel? Or will data be recorded manually by the operators?
If so, how will transcription errors be avoided?
• Data review - can data be sent out every two weeks as collected for review? Who will be
doing review for each data set sent out? If Battelle or ETV Canada staff are not on-site, how
will data be transmitted to them?
• Distribute and review report schedule. Reporting should begin at the same time as testing.
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Date: 01/12/10
APPENDIX C
REVIEW REPORT FORM
Review report
Document title:
Reviewer
name:
Name:
Organization:
Address:
Telephone:
E-mail
Document date:
Review date:
Review results
Rate items
Contents
Scope
Organization
Data quality
Method validity
Conclusions
Other (specify)
Satisfactory
Unsatisfactory
Overall recommendation
Acceptable as is
Minor revisions
Major revisions
Not acceptable
Reason
-------
Revision details
Topic
Report chapter,
section, page
Revision
required
Reason
Revision action (to be filled in by
document owner during revision after
review)
Add additional rows, if pertinent.
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US ETV AMS Center, DHIDANETV Water Centre and ETV Canada
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Date: 01/12/10
APPENDIX D
JOINT VERIFICATION TESTING DOCUMENT
DEVIATION FORM
DEVIATION REPORT
TESTING DOCUMENT TITLE AND DATE:
DEVIATION NUMBER:
DATE OF DEVIATION:
DESCRIPTION OF DEVIATION:
CAUSE OF DEVIATION:
IMPACT OF DEVIATION ON THE TEST:
CORRECTIVE ACTION:
ORIGINATED BY:
DHI Test Responsible, Battelle AMS Center Verification Test Coordinator or ETV Canada
DATE
ACKNOWLEDGED BY:
DHI Quality Manager Battelle AMS Center Quality Manager
DATE DATE
ETV Canada Quality Manager
DATE
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Date: 01/12/10
Required Distribution with documentation - All individuals/organizations listed below:
Battelle AMS Center Manager
DHI Verification Responsible
Battelle AMS Center Quality Manager
DHI Internal Auditor
ETV Canada
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Date: 01/12/10
APPENDIX E
ASSESSMENT REPORTING FORM
Quality Assurance Routing Sheet
Verification Test:
Audit Type:
Test Coordinator:
Vendor:
Auditor: Date:
Test Coordinator, please complete the attached form indicating CORRECTIVE
ACTION TAKEN (IF NEEDED), sign and date this Routing Sheet in the space
provided beside your name, and return the entire set when completed to the Battelle
AMS Center Quality Manager no later than .
Route To Signature Date
DHI Test Responsible
Battelle AMS Center Test
Coordinator
ETV Canada
Approval
Battelle AMS Center
Manager
Battelle AMS Center
Quality Manager
DHI Verification
Responsible
ETV Canada Quality
Manager
-------
Audit Comment Sheet
Instructions: The Battelle AMS Center Quality Manager or ETV Canada Quality Manager will fill out the first column for the audit indicated above. The
Verification Test Coordinator (or assigned responder) will respond to the comments and initial and date the response in column three. The Battelle AMS
Center Quality Manager or ETV Canada Quality Manager will verify and document that the response/corrective action has been completed by initialing and
dating the final column.
QA Comment
Testing Coordinator
Response/Corrective Actions
Responder
Initials/ Date
QA Initials/
Date
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Joint Verification Process Document
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Date: 01/12/10
APPENDIX F
EXAMPLE JOINT VERIFICATION STATEMENT
THE ENVIRONMENTAL TECHNOLOGY VERIFICATION
PROGRAM
This is a preliminary template of a Verification Statement that may be expanded and organized to meet
DANETV, U.S. EPA ETV, and ETV Canada program requirements.
DANETV
U.S. Environmental Protection Agency
Battelle
The Business of Innovation
WATER'ENVIRONMENT-HEALTH
ETV Joint Verification Statement
TECHNOLOGY TYPE:
APPLICATION:
TECHNOLOGY NAME:
COMPANY:
ADDRESS: PHONE:
FAX:
WEB SITE:
E-MAIL:
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Date: 01/12/10
• Description of EV and the organizations involved in this joint verification.
• Name technology category and technology (product) that was jointly verified.
VERIFICATION AND TEST DESCRIPTION
• Describe the verification test- when, how
• Describe the performance parameters
• Describe the QA performed
TECHNOLOGY AND PRODUCT DESCRIPTION
• Describe the technology (product)
VERIFICATION RESULTS
• Summary of results by performance parameters
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Signature blocks for:
DANETV Steering Committee Head - Christian Gran
DHI Director of Research and Quality - J0rn Rasmussen
Battelle Chemical, Environmental and Materials Operations Manager - Lisa McCauley
U.S. EPA National Risk Management Research Office of Research and Development -
Sally Gutierrez
OCETA, President and CEO- Kevin Jones
NOTICE: ETV verifications are based on an evaluation of technology performance under specific,
predetermined criteria and the appropriate quality assurance procedures. U.S. EPA, Battelle, DANETV, DHI,
Environment Canada and ETV Canada make no expressed or implied warranties as to the performance of the
technology and do not certify that a technology will always operate as verified. The end user is solely
responsible for complying with any and all applicable federal, state, and local requirements. Mention of
commercial product names does not imply endorsement.
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Date: 01/12/10
APPENDIX G
STATISTICAL EVALUATION OF TEST PLAN
TESTS A AND C
Testing is designed to follow Hach Lange operating manual procedures for testing under ISO 11348-3.
The 10-90% inhibition range used in the Hach Lange protocol is specified in the ISO 11348-3 method in
Section 9.2 Determination of EC-values. This section of the ISO method also specifies calculating and
reporting EC20 and EC50, which is why these two EC values are used as the reported result. The EC
values are generated in the Hach Lange protocol using a nine dilution scheme from 1:2 to 1:32 of the
highest inhibition solution. This dilution scheme (discussed in Test Plan section 3.2.1) is also specifically
recommended in ISO11348-3 Annex B , Section B.2, on sample preparation (shown in the ISO Method
Annex B Table B.I).
Additionally, a Battelle statistician reviewed the test design where EC20 and EC50 are calculated with
various solutions in the 10-90% inhibition range to provide additional information on reasonableness of
this approach. The results of this evaluation are discussed below:
Objective
Throughout the test design, scenarios are provided for EC50 and EC20 values to be calculated based on
between three and nine samples. The calculation algorithm for EC50/EC20 is applied only to samples
which result in fluorescence inhibition between 10 and 90 percent. The upper limit of nine samples is
achieved when the planned nine point dilution series (32, 24, 16, 12, 8, 6, 4, 3, and 2 fold dilutions) all
result in inhibition results between 10 and 90 percent. Fewer than nine samples may be used when a
specific test calls for it or when the standard nine point dilution series produces inhibition results outside
the 10 to 90 percent range. In these cases, the concentrations with results outside the 10 to 90 percent
range are excluded from the EC50/EC20 calculation. A question of interest is whether a nine point
dilution curve is adequate to accurately estimate the EC50/EC20 and to what extent fewer than nine
dilutions may negatively impact the accuracy.
To evaluate the concerns about EC50/EC20 accuracy, a statistical simulation study was performed.. Data
were randomly generated from an assumed statistical model under different scenarios for numbers of test
dilutions and corresponding expected percent inhibition results. The EC50/EC20 values were calculated
at each iteration of the simulation analysis and the entire set of values was summarized to provide 95
percent confidence limits on the expected accuracy of the responses.
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Date: 01/12/10
Results
For Test A which will involve a nine point curve with the lowest concentration expected to yield 10%
inhibition and the highest concentration expected to yield 90% inhibition using the nine dilution scheme
provided in the test plan the EC50 would be estimated, with 95% confidence to (0.92, 1.09) times the true
value if the true variability in measured results at 50 percent inhibition for a particular sample is a
standard deviation of 10 percent. For EC 20, the estimated value shouldbe within (0.89, 1.11) times the
true value.
When less than nine points are used, because the planned dilution series did not perfectly span the 10 to
90 percent inhibition range, a penalty in accuracy is seen. As an example, instead of (0.92, 1.09) times
the true EC50, a test where only five of the values are within the 50 to 90 percent inhibition range would
be (0.79, 1.18). For EC 20, it can be seen that missing values in the upper range (I. e., only five points
ranging from 0.1 to 0.5) does not negatively impact accuracy (0.88, 1.12).
Test C proposes to estimate EC50 with values of 60 percent and below. In this case, it is likely only about
five points will be usable, but the EC 50 at (0.88, 1.20) times the true value and the EC20 at (0.84, 1.18)
times the true value have about 50% more uncertainty. A test to estimate EC20 with values of 30percent
and below may yield only three usable test points in the nine dilution series, and the EC20 would be
estimated at 95 percent confidence to (0.86, 1.19) times the true value, not too dissimilar to the (0.89,
1.11) times for the full nine point curve.
All the results were also calculated under two other assumed variability models: one with the 50 percent
inhibition having a lower variance estimate of 5percent (i.e., two standard deviation would be 45 to
55 percent inhibition, and one with the 50 percent inhibition having a higher variance estimate of
20 percent (i.e., two standard deviation would be 30 to 70percent inhibition). The lower variance
resulted in less uncertainty in EC50 and EC20 estimation, while the higher variance resulted in greater
uncertainty.
However, the comparative results for the different testing scenarios were similar.
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Date: 01/12/10
All results
Dilution si
Scenarios
dilution
32
24
16
assume Id-fold dilution series cowers from ID to 90 percent inhibition
2ries is 32, 24, 16, 12, 8, 6, 4, 3, and 2 fold
for Evaluation
I/dilution
0.03
0.04
0.06
12 OLOS
8 0.13
G
4
3
2
0.17
0.25
0.33
0.50
% Std@5D% Inhibition
5%
10%
20%
%Std@50% Inhibition
5%
10%
20%
full 9
0.10
0.15
0.25
0.34
0.50
0.61
0.75
O.S3
0.90
"PperS
0.50
0.61
0.75
0.83
0.90
0.93
a96
a98
a99
lower 5
0.01
0.02
0.04
aoe
0.10
0.15
0.25
0.34
0.50
max 60
0.02
ao3
ao5
0.08
0.14
0.21
0.33
0.44
0.60
max 30
aoi
aoi
ao2
0,03
0.05
0.07
0.13
0.1S
0.30
95% Confidence Interval of Ratio Curve Fit EC to True EC
EC 20
(0.94,1.06]
(0.89,1.11]
(0.76,1.23]
(a81,:L13]
(0.62,1.36]
(0.26,1.68]
(0.94,1.06)
(0.88,1.12)
(0.73,L25)
(0.93,1,07)
(0.34,1.14)
(0.64,1,27)
(0.93,1-08)
(0.86,1.19]
(0.53,2.07)
EC 50
(0.96,1.04]
(0.92,1.09]
(0.84,1.19]
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US ETV AMS Center, DHIDANETV Water Centre and ETV Canada
Joint Verification Process Document
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Date: 01/12/10
e_i is the error between the observed log(p/(l-p)) response and the regression predicted bO+bl *log(c)
value.
The errors are assumed to be independent and normally distributed with mean 0 and variance a2
Note 1: This logistic regression model appears to be appropriate based on the documentation found in
the LUMIStox 300 Operating Manual of January 2008. This is based on Section 4.4, "Calculating the EC
Value ", which references calculating % inhibition divided by (1 -% inhibition) values and using the
associated concentration values and plotting them on a two dimensional logarithmic coordinate system. It
is presumed that data have been collected from this instrument and shown to exhibit the desired linear
relationship in the logarithmic coordinates. However, no such data are presented for reference.
Note 2: The assumed constant variance a2 in the model is an important one. The implication of the
assumption is that in the natural scale (%inhibition vs concentration), the variability of results is greatest
for the concentration corresponding to 50% inhibition and diminishes toward zero as concentrations lead
to %inhibition results of either 0% or 100%. If this assumption is not appropriate, a different model may
be preferable.
From the model, the desired quantities ofEC50 and EC20 are calculated as follows:
EC50: Since log(0.5/(l-0.5)) = log(l) = 0, the EC50 is the concentration given by exp(-bo/bl)
EC20: Since log(0.2/(l-0.2)) = log(l/4), the EC20 is the concentration given by exp((log(l/4)-bo)/bl)
In order to perform the statistical simulation analysis, several assumptions needed to be made. For the
statistical model provided, the bO, bl, and a are unknown values that jointly define the "true "
relationship between % inhibition and concentration. To determine the bO andbl values, "Table 6.1
Results from HACH-LANGE test of LUMIStox and ECLOX" in the LUMIStox 300 Bench Top
Luminometer ECLOXHandheld Luminometer Joint verification protocol was consulted. This table
showed 10 and 90 percent inhibition concentrations as well as the EC50 for several different compounds
identical or related to those to be evaluated in the upcoming tests. From Table 6.1, the p-Cresol result
was chosen as typical. With an approximate 90% inhibition of 6.0 mg/L and an EC50 (50% inhibition) of
1.5 mg/L, it was calculated that the bO would be -0.64 and that the bl would be 1.58. Since the difference
between 0.38 and 6.0 is approximately 16-fold, it can be assumed that a perfectly selected nine point
dilution series as specified in the test plan will produce percent inhibition results ranging from 10 percent
for the highest dilution and up to 90 percent for the lowest dilution. The results for many of the
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US ETV AMS Center, DHIDANETV Water Centre and ETV Canada
Joint Verification Process Document
Page 42 of 42
Date: 01/12/10
compounds in Table 6.1 were similar. The 10 to 90 percent inhibition range was as low as 6-fold in a
couple cases (corresponding to larger bl values) and as wide as 18 fold in one case (corresponding to
smaller bl values), and the entire analysis completed here could easily be repeated with values derived
from other assumed true relationships, but the p-Cresol result seemed satisfactory as a start. To
determine the variance estimate, it would be best to have a single run in which the same dilution was
evaluated in all nine points, and a corresponding mean and standard deviation of the percent inhibition
values calculated. This should be done across a range of average percent inhibition values, with multiple
compounds, and perhaps under different test conditions to assure consistency. Lacking any of these data,
a simple assumption had to be made and it was decided that in the original units of percent inhibition,
repeated measurement of the same sample that averages 50 percent inhibition might reasonably have a
standard deviation of 10 percent of the 50 percent (i.e., two standard deviations would be anywhere from
40 percent inhibition to 60 percent inhibition). For sensitivity, the analysis was repeated for a standard
deviation of five percent and one of 20 percent.
With the bO, bl, and a2 estimates, the simulation required different dilution series to be defined. A
standard nine point series with dilutions as defined above with the 32-fold dilution producing on average
10 percent inhibition and the 2-fold dilution producing 90 percent inhibition was the starting point. From
the assumed statistical relationship and with the normally distributed variance, one iteration of the
simulation consisted of producing the nine percent inhibition values centered at the corresponding values
of the relationship and randomly perturbated using a normal distribution with the assumed variance. The
nine randomly determined (consistent with the model) points were then fit to a logistic regression
relationship using least squares estimation to get an estimated bO and bl (different from the true bO and
bl assumed, but generally similar). Once the estimated bO and bl were obtained, they were used to
calculate an estimated EC50 or EC20 using the equations above. This entire process was repeated 10,000
times for one scenario. The 10,000 EC 50 or EC 20 values were then divided by the true EC 50 or EC 20 as
determined by the initially assumed bO and bl, and the ratios ranked from smallest to largest. The 250th
smallest and 250th largest were selected to provide a 95 percent confidence interval for the ratio of
calculated EC50 or EC20 that might be expected to occur for a given true relationship between percent
inhibition and concentration within a particular sample. This is variation that does not reflect
circumstances where the underlying true relationship is not the same (i.e., any other sample). All
calculations were performed in Excel.
-------
DANETV
Baielle
CJcUldCXcl Mi*: lUititiutp -V Innnrratic
LUMIStox 300 Bench Top Luminometer
ECLOX Handheld Luminometer
Joint verification protocol
Luminescent bacteria test for use in wastewater
LUMIStox 300
January 2010
Approved
-------
LUMIStox 300 Bench Top Luminometer
ECLOX Handheld Luminometer
Joint verification protocol
Agern Alle 5
DK-2970 H0rsholm
Denmark
Tel: +4545169200
Fax: +4545169292
mta@dhigroup.com
www.dhigroup.com
Vendor
HACH-LANGE GmbH
Vendors representative
Dr. Elmar Grabert
Project
DANETV
Authors
2
1
0
Revision
Mette Tjener Andersson
Approved verification protocol
Verification protocol for approval
Verification protocol for review
Description
Key words
Acute toxicity; EC50; ECLOX; ISO 11348-3;
Luminescent bacteria; LUMIStox; Wastewater
Project No
11800378
Date
January 2010
Approved
by
Sten Lindberg
MTA
MTA
MTA
By
MWN S
MWN S
MWN S
Checked Appr
L 13/01-10
L 11/12-09
L 16/11-09
oved Date
Classification
IEI Open
n Internal
n Proprietary
Distribution
DHI
HACH-LANGE
Battelle
ETV Canada
MTA, CLJ, MVN
Dr. Elmar Grabert
Mary Schrock
Mona EI-Hallak
No of copies
File distribution
only
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1 TABLE OF CONTENTS
1 TABLE OF CONTENTS
2 INTRODUCTION 1
2.1 Name of product 1
2.2 Name and contact of vendor 1
2.3 Name of center/verification responsible 1
2.4 Verification test organization 2
2.5 Technical experts 3
2.6 Verification process 3
3 DESCRIPTION OF THE TECHNOLOGY 4
4 DESCRIPTION OF THE PRODUCTS 5
4.1 LUMIStoxSOO 5
4.2 ECLOX 5
5 APPLICATION AND PERFORMANCE PARAMETER DEFINITIONS 6
5.1 Application definition 6
5.2 Performance parameters for verification 6
5.3 Additional parameters 8
6 EXISTING DATA 9
6.1 Summary of existing data 9
6.2 Quality of existing data 9
6.3 Accepted existing data 10
7 TEST PLAN REQUIREMENTS 10
7.1 Test design 10
7.2 Reference tests and analysis 11
7.3 Data management 12
7.4 Quality assurance 12
7.5 Test report 12
8 EVALUATION 12
8.1 Calculation of performance parameters 12
8.2 Evaluation of test data quality 14
8.3 Compilation of additional parameters 14
8.3.1 User manual 14
8.3.2 Product costs 15
8.3.3 Occupational health and environment 16
9 VERIFICATION SCHEDULE 17
10 QUALITY ASSURANCE 17
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APPENDIX 1 19
Terms and definitions used in the verification protocol 19
APPENDIX 2 23
References 23
APPENDIX 3 26
Application and performance parameter definitions 26
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2 INTRODUCTION
Environmental technology verification (ETV) is an independent (third party) assessment
of the performance of a technology or a product for a specified application, under defined
conditions and quality assurance.
This verification is a joint verification between DANETV, the U.S. EPA ETV Advanced
Monitoring Systems (AMS) Center and the Canadian ETV Program. The objective of the
verification is to evaluate the performance of a wastewater rapid toxicity technology that
could be used to monitor industrial or domestic wastewater.
This verification includes two products from one vendor.
2.1 Name of product
The verification protocol covers two products from the same vendor, both are acute
toxicity tests with luminescent bacteria. The target products are respectively LUMIStox
300 bench top luminometer and ECLOX handheld luminometer. Both can operate in
connection with a LUMIStherm thermostat and the PC software LUMISsoft4.
2.2 Name and contact of vendor
HACH-LANGE GmbH, Willstatterstrasse 11, 40549 Dusseldorf, Germany, phone +49
211 52880.
Contact: Dr. Elmar Grabert, email: elmar.grabert@hach-lange.de, phone +49 211 5288
241.
Web site: www.hach-lange.de
2.3 Name of center/verification responsible
Danish Centre for Verification of Climate and Environmental Technologies, (DANETV),
DHI DANETV Water Centre, DHI, Agern Alle 5, DK-2970 H0rsholm, Denmark.
Verification responsible: Mette Tjener Andersson, email mta@dhigroup.com, phone +45
1691 48.
U.S. EPA ETV Advanced Monitoring Systems Center (Battelle), Battelle Memorial
Institute, 505 King Avenue, Columbus, Ohio 43201-2693, U.S.A.
Verification responsible: Mary E. Schrock, email schrock@battelle.org, phone +1 614 424
4976.
ETV Canada, 2070 Hadwen Road Suite 201 A, Mississauga, Ontario L5K 2C9, Canada.
1
-------
Verification responsible: Mona El- Hallak, email melhallak@etvcanada.ca, phone +1 905
8224133 extension 239.
2.4 Verification test organization
The verification will be conducted as a joint verification between the Danish Centre for
Verification of Climate and Environmental Technologies (DANETV), the Canadian
Environmental Technology Verification Program (ETV Canada) and the U.S.
Environmental Technology Verification (U.S. EPA ETV) program. The verification is
planned and conducted to satisfy the requirements of the ETV scheme currently being
established by the European Union (EU ETV) as well as the Canadian and U.S. ETV
programs. Verification and tests will be performed by DHI as DANETV Water
Technology ETV Center (DHI DANETV Water Centre) under contract with the Danish
Agency for Science, Technology and Innovation. Battelle will participate as the manager
of the ETV Advanced Monitoring Systems (AMS) Center through a cooperative
agreement with the U.S. Environmental Protection Agency (EPA). ETV Canada will
participate as manager of the Canadian ETV Program.
The day-to-day operations of the verification and tests will be coordinated and supervised
by DHI personnel, with the participation of the vendor, HACH-LANGE. The testing will
be conducted in the DHI laboratories, H0rsholm, Denmark. DHI will operate the
luminometers during the verification. HACH-LANGE will provide the luminometers, the
thermostats, bacteria, software, user manuals and operation instructions. They will
participate in development of protocol and plans in cooperation with DHI. Battelle and
ETV Canada will ensure that the verification and tests are planned and conducted to
satisfy the requirements of the U.S. and Canadian ETV programs, including input and
concurrence from their stakeholder groups, as described in the process document /20/
produced to ensure compliance of the verification with the requirements of the U.S. and
the Canadian ETV programs. Battelle and ETV Canada will also participate in the
development of the verification protocol and test plan for the verification and tests and
perform quality assurance of the verification and tests. Verification protocol and test plan
will be reviewed and approved by U.S. EPA ETV AMS Center and ETV Canada.
Three technical experts have been identified to provide independent expert review of the
planning, conducting and reporting of the verification and tests.
The organization chart in Fig 2.1 identifies the relationships of the organization associated
with this verification and tests.
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USEPAETV Environment DANETV
CanadaETV
Programme
Battelle EW Canada Verification
DHIWTC
Test
Battelle EW Canada
stakeholders stakeholders
Technical experts HACH-LANGE
Fig 2.1 Organization of the verification and tests.
2.5 Technical experts
Three technical experts have been appointed. They are:
Dr. Joel Allen, email: allen.joel@epa.gov, phone +1 513 487 2806. U.S.EPA, Office of
Research and Development/National Risk Management Research Lab oratory /Water
Supply and Water Resources Division/Water Quality Management Branch.
Associate Professor Kresten Ole Kusk, email: kok@env.dtu.dk, phone +45 4525 1569.
Technical University of Denmark, Department of Environmental Engineering.
Dr. Ali Amiri, email: aamiri@oceta.on.ca, phone +1 905 822 41 33 ext 222. Ontario
Center for Environmental Technology Advancement (OCETA).
2.6 Verification process
The principles of operation with the role of the verification and test documents and the
different sub-bodies responsible are given in Fig 2.2.
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Fig 2.2
Principles of operation of the DANETV verification scheme for joint verification.
The QA group covers the expert group, Battelle, U.S. EPA ETV and ETV Canada. Audits
will be performed internally by DHI, and optionally by Battelle, U.S. EPA or ETV
Canada. Battelle is expected to perform an audit.
References for the verification process are the Quality Management Plan from Battelle 111,
the General Verification Protocol from ETV Canada /3/ and the Quality Manual for the
ETV operations at DHI following the DANETV Quality Manual Template III.
The final verification protocol, the test plan and the above mentioned process document
shall be seen as one consolidated verification description.
A joint U.S. EPA ETV, ETV Canada and DANETV verification statement will be issued
after completion of the verification. One verification report and verification statement will
cover both the LUMIStox 300 Bench Top Luminometer and the ECLOX Handheld
Luminometer.
DESCRIPTION OF THE TECHNOLOGY
Luminometers such as LUMIStox and ECLOX are in vitro testing systems that use
bioluminescent bacteria to detect toxic compounds in water, e.g. wastewater; river and
lake water; leachate from soil, waste, rubble, etc.; or directly in fluent chemicals.
Bioluminescence tests are metabolic inhibition tests that provide acute toxicity analyses. In
the bioluminescence testing with LUMIStox and ECLOX a strain of naturally occurring
luminescent bacteria, Vibrio fischeri, is used. Vibrio fischeri is a non-pathogenic, marine,
luminescent bacterium which is sensitive to a wide range of toxicants. When properly
grown, luminescent bacteria produce light as a by-product of its cellular respiration. Any
inhibition of cellular activity results in a decreased rate of respiration and a corresponding
decrease in the rate of luminescence. The light emission/luminescence can be measured
with a LUMIStox or ECLOX luminometer.
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Inhibition of the light emission in the presence of a sample is determined against a non-
toxic control. The luminescence is measured after a contact time of 5 (optional), 15 and 30
minutes at 15°C, taking into account a correction factor, which is a measure of intensity
change of control samples during the exposure time.
4 DESCRIPTION OF THE PRODUCTS
4.1 LUMIStox 300
The LUMIStox 300 is a bench top luminometer that has been developed as a measuring
unit for the luminescent bacteria test. In combination with the LUMIStherm incubation
block it conforms to the technical requirements of ISO 11348.
ISO 11348 describes determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri. The ISO standard contains three parts, using freshly prepared
bacteria, liquid-dried bacteria and freeze-dried bacteria respectively. For the LUMIStox
(and ECLOX) freeze-dried bacterium is used (ISO 11348-3, /23/). ISO 11348-3, Section
9.2 specifies determining EC values using solution concentrations with inhibition values
between 10% and 90% following a dilution scheme described in Annex B, Table Bl
involving nine dilutions ranging from 1:2 to 1:32 and reporting the resulting £€20- and
ECso-values.
The LUMIStox 300 has a built-in photometer function and an automatic measuring and
evaluation routine, which enables it to recognize color effects in the luminescent bacteria
test and to take these into account in the test result.
The photometer function also allows the color effect to be estimated in advance, and can
be used to determine the extinction (as OD - optical density) of bacteria suspensions for
the purpose of assessing light extinction.
The LUMIStox 300 can be connected to a PC running LUMISsoft4 that enables the
operator performing and recording luminescent bacteria tests to conduct all of the ISO
11348 requirements. The results from LUMIStox are either LID or ECso-values,
representing Lowest Ineffective Dilution causing less than 20%l inhibition and Effective
Concentration causing 50% inhibition, respectively. The ECso-values are the commonly
used result from toxicity tests, while the LID is used as a standard practice in Germany.
4.2 ECLOX
The ECLOX is a portable instrument designed to provide data appropriate for risk
assessments in the event of environmental releases, emergency situations, preventive
security measures, and regulatory monitoring.
The ECLOX is designed in particular to be used for the Luminescent Bacteria Toxicity
Test and to be used with a Chemiluminescence Toxicity Test. Both tests will give results
1 LID of 20% inhibition is stated in ISO 11348-3, Annex B, Section B.5.
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in a short-term and simple way in the field or in the laboratory. The ECLOX used in the
field gives values of % inhibition.
Additionally the ECLOX can be used to deliver luminescence values. If the ECLOX is
connected to LUMISsoft4, a PC program for the luminescent bacteria test according to
ISO 11348, LID and ECso-values can be calculated and recorded.
5 APPLICA TION AND PERFORMANCE PARAMETER DEFINITIONS
The application is defined as detailed in the application definition appendix, Appendix 3,
in terms of matrix/matrices for use, targets of monitoring and effects.
5.1 Application definition
An overview of matrix, effect, target and technology for the LUMIStox and ECLOX is
given in Table 5.1.
Table 5.1 Description of matrix, effect, targets and technologies for LUMIStox and ECLOX.
Matrix
LUMIStox and
ECLOX are applied
for wastewater; river
and lake water;
leachate from soil,
waste, rubble, etc.; or
directly in fluent
chemicals.
Verification testing
will be conducted on
domestic and
industrial wastewater
effluents
Effect
Measurement of
toxicity as indicated
by inhibition of
luminescent bacteria
by a variety of
compounds including
metal ions, organic
pesticides, inorganic
and organic
pollutants and
surfactants
Additional
parameters:
User manual quality,
product cost,
environmental health
and safety
Targets
The target for the
application is
measurement of
toxicity, specifying
criterion of detection
(CD), range of
application, precision
(repeatability and
reproducibility),
agreement with
accepted values and
robustness
Technologies
ECLOX and
LUMIStox analyses
for inhibition of light
emitting luminescent
bacterium Vibrio
fischeri
5.2 Performance parameters for verification
The performance parameters relevant for the application, as derived in Appendix 3, are
presented in Table 5.2. The ranges presented for these parameters are used for planning
the verification and testing only.
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Table 5.2 Relevant ranges of performance parameters in effluent industrial and domestic waste wafer.
LUMIStox
ECLOX
Criterion of
detection
% inhibition
<10
<10
Range of
application
L/L
>1/2-<1/32
>1/2-<1/32
Precision (RSD)
%
Repeatability
<20
<20
Reproduce-
ability
<30
<30
Agreement
with
accepted
values
%
100 ±50
100 ±50
Robustness
%
100±50
100±50
For toxicity testing it is not possible to determine the limit of detection (LoD). Instead it is
chosen to determine the criterion of detection (CD) based on the standard deviation of
blanks (2% NaCl solution and bacteria suspension, no toxic compound added).
The range of application for a chemical analysis is usually the range of analyte
concentration from the limit of detection to the highest concentration with linear response.
This concept is not meaningful for a toxicity test of a water sample, because the test does
not measure a concentration but an inhibitory effect as a function of the dilution of the
sample. The range of application for determining ECso therefore has to be considered in
terms of dilution. According to the HACH-LANGE manual estimation of an ECso of a
water sample requires a minimum of three measurements where the inhibition is between
10% and 90%. In addition one of the three measurements must be above 50%. If the
standard dilution row is considered as described in the LUMIStox 300 Operation manual
and in Annex B of the ISO 11248-3:2007 with 9 dilutions (2, 3, 4, 6, 8, 12, 16, 24, 32
times dilution in the test suspension) then ECso should be in the range of dilutions > 2 and
< 32 times dilution assuming three measurements with inhibition between 10 and 90%.
Based on test results will be given ranges of concentrations of the compounds tested in this
study, which will give an inhibition within the range of application.
Precision shall be evaluated under repeatability and reproducibility conditions.
Repeatability is defined as the relative standard deviation of measurements done with the
same measurement procedure, same operators, same measuring system, same operating
conditions and same location, and replicate measurements on the same or similar objects
over a short period of time. Reproducibility is defined as the relative standard deviation of
measurements under different conditions such as different locations, operators, measuring
systems, and replicate measurements on the same or similar objects. In laboratory
terminology, repeatability is the within-series precision and the reproducibility the
between-series precision. For reproducibility of luminescent toxicity testing, the difference
in bacteria batches is considered to be the greatest source of deviation. Precision will be
determined as the relative standard deviation of the EC20 and ECso results generated during
testing.
Trueness is generally the closeness of agreement between the (mean) concentrations found
in measurements and the true or accepted concentration. According to ISO 11348-3 the
true or accepted ECso-value of a substance is obtained, as long as the criteria in the ISO
are met. Reference testing with purpose of gaining true toxicities will therefore not be
performed. For this verification it is chosen to determine "trueness", as "agreement with
accepted values". This agreement will be the inhibition results (ECso-values) obtained in
-------
the tests compared to robust literature values for ECso-values, with clear reference to tests
being performed according to the ISO 11348-3 method, for the same compound. The
agreement with accepted values will only be determined for test substances where robust
literature values are available.
The parameters of robustness to be verified are pH change, temperature change, presence
of color or turbid material in sample, difference in start concentration, matrix variation,
and type of cuvette. Robustness is basically the trueness as found for different values of
the robustness parameters.
Samples will be tested with different concentration of color and turbid material, since the
ISO standard specifies that they will cause interference. Correction methods are available
for both LUMIStox and ECLOX, these methods will be verified.
The ISO 11348-3 recommends test to be performed at a pH range of 7±0.2, but states that
pH values of 6.0-8.5 are acceptable. Tests will be performed comparing the three pH-
values.
The ISO 11348-3 specifies that a thermostat or similar shall cool the test vials to 15±1°C.
The thermostat will be monitored. Tests will be performed comparing temperatures of
14°C, 15°C and 16°C. In case the test of the thermostat shows greater variation than 14-
16°C, the minimum and maximum temperatures will be used instead of 14°C and 16°C.
When testing wastewater samples it may not be possible to cover the range from 10-90%
inhibition, which is ideal. Therefore tests are performed with maximum concentrations of
approximately 30% and 60% inhibition (ECso and ECeo), to see how that affects the
determination of EC20 and ECso. The start concentration of causing approximately 30%
and 60% inhibition, are chosen to ensure determination of EC20 and ECso, where the last
will only be possible to determine with the start concentration of approximately 60%
inhibition.
Testing of industrial and domestic effluent wastewater samples is included. These tests
will include testing of the wastewater as received as well as spiked non-inhibitory
wastewater. The tests will be performed to see the effect of the wastewater matrix on the
luminescent test.
Generally glass cuvettes are used in the LUMIStox and plastic cuvettes are used in the
ECLOX. HACH-LANGE has though stated that plastic cuvettes also can be used in
LUMIStox, to ease the testing all test will therefore be performed with use of plastic
cuvettes except for test L where LUMIStox robustness towards cuvettes (glass or plastic)
will be tested.
5.3 A dditional parameters
Besides the performance parameters to be obtained by testing, compilation of parameters
describing user manual, product costs and occupational health & safety issues of the
product are required as part of the verification.
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EXISTING DATA
6.1 Summary of existing data
The vendor has recently performed tests with the LUMIStox and ECLOX instruments for
determination of precision expressed by the relative standard deviation (RSD). The results
are given at a contact time of 15 minutes and are listed in Table 6.1.
Table 6.1
Results from HACH-LANGE test of LUMIStox and ECLOX.
Compound
CrD+
Zn'+
Pbz+
SDS
CTAB
Formaldehyde
Hydroquinone
p-Cresol
CIST
Range 1 0-90%
inhibition
mg/L
1 .7-27
1.5-9.0
0.21-2.5
0.14-2.3
0.33-6.0
4.4-35
0.03-0.20
0.38-6.0
0.51-8.1
LUMIStox
No. of bacteria
batches/ no. of
replicates
3/5
2/4
2/4
3/6
2/4
2/4
2/7
2/4
2/6
EC50
mg/L
6.6
4.3
0.49
0.66
0.84
15
0.093
1.5
2.7
RSD
%
38
25
8.0
16
5.8
9.5
46
33
74
ECLOX
No. of bacteria
batches/ no. of
replicates
1/3
1/3
1/3
1/3
1/3
1/3
EC50
mg/l
8.6
4.2
0.48
0.55
1.1
14
RSD
%
26
15
8.7
2.8
16
5.1
Not tested
1/3
1.6
6.6
Not tested
The range 10-90% inhibition is the measurement interval used for calculating the ECso-
values. 10% inhibition equals to ECio, while 90% equals to ECgo. This range will for
compound included in this verification be used as guidance for test range.
Notice that the test of LUMIStox was performed on 2-3 different bacteria batches, while
the test of ECLOX was performed on one bacteria batch only. This results in higher
relative standard deviations for LUMIStox than for ECLOX.
It should be mentioned that the relative standard deviation is calculated by the vendor with
no reference to number of samples tested in each bacteria batch.
The vendor has made a note on results for cyanide regarding cyanide to be difficult to
work with in the laboratory at a pH of 7.
At pH 7 almost all cyanide is in the volatile and toxic HCN form and evaporation of HCN
can occur.
6.2 Quality of existing data
The tests are performed by the vendor and not by an independent body. Furthermore, the
analyses are not conducted by a laboratory with ISO 17025 accreditation. The data can
therefore only be used for planning of the verification, and not as verification data.
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6.3 Accepted existing data
No existing data are accepted for use as part of this verification test. However, these data
provide useful background for planning the test.
7 TEST PLAN REQUIREMENTS
Based upon the application and performance parameter identification, Section 5, the
requirements for test design have been set up, see below. The detailed test plan is prepared
separately, based upon the test requirements specified below.
7.1 Test design
The outline of the required tests is shown in Table 7.1, more details of the test design are
found in the Test Plan 7257. The principle behind the design is that three test set-ups are
used:
• LUMIStox 300 bench top luminometer with LUMIStherm thermostat and LUMISsoft4
PC software. According to ISO 11348-3.
• ECLOX handheld luminometer with LUMIStherm thermostat and LUMISsoft4 PC
software. Conditions similar to ISO 11348-3.
• ECLOX handheld luminometer with use of firmware.
Three matrices will be used in the testing: spiked 2% NaCl MilliQ water, domestic
effluent wastewater, and industrial effluent wastewater.
10
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Table 7.1 Test design and associated performance parameters.
Test
no.
A
B
C
D
E
F
G
H
I
J+K
L
Performance parameters
Range, Repeatability, Agreement with accepted values
Criterion of detection
Robustness, effect of start cone, on repeatability
Reproducibility
Robustness, sample temperature at field use
Robustness, sample temperature at laboratory use
Robustness, pH
Robustness, color
Robustness, turbidity
Robustness, matrix
Robustness, cuvettes
Equipment
LUMIStox
X
X
X
X
X
X
X
X
X
X
ECLOX incl.
thermostat and
software
X
X
X
X
X
X
X
X
X
ECLOX incl.
firmware
X
Matrix
g
i
o
re
z
sS
cs
X
X
X
X
X
X
X
X
X
X
Waste water
X
Tests will be performed with specific compounds in 2% NaCl MilliQ water to determine
their £€20- and ECso-values. The tests will show the range of responses towards these
specific toxic compounds. Secondly, tests will be performed on effluent wastewater with
and without spiking with a toxic compound. This will show the robustness of the
luminescent tests towards the wastewater matrix. The last test will evaluate the effect on
results between use of glass cuvettes and plastic cuvettes in the LUMIStox Benchtop.
According to ISO 11348-3 three reference substances shall be tested for each batch of
bacteria. These tests will be performed solely on the LUMIStox.
7.2 Reference tests and analysis
The true value of a toxicity test cannot be determined since no bacteria vials are fully
identical, and therefore the test results will react slightly different in every test. The
reference tests will therefore not be used as true values as seen in other verifications, but
will give an identification of the level of toxicity and e.g. false negative tests performed
with the LUMIStox or ECLOX equipment (false negative: no response observed where
there should be a response). A false negative result should be investigated. False negative
results could be caused by improper handling of samples and test equipment, or could be
because test equipment is not responding as expected. As a result the test of reference
samples will be limited.
Luminescent bacteria reference tests must be done under ISO 17025 accreditation 1161
using the ISO 11348-3 Luminescent bacteria test method, e.g. with Microtox® and must be
documented to satisfy performance parameters as derived for LUMIStox and ECLOX in
Table 5.2 and Appendix 3.
11
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Reference analysis of stock solutions must also be done under ISO 17025 accreditation
1161 with appropriate methods by an independent laboratory.
7.3 Data management
Data storage, transfer and control must be done in accordance with the requirements of
ISO 9001 IY1I enabling full control and retrieval of documents and records. The filing and
archiving requirements of the DHI Quality Manual must be followed (10 years archiving).
7.4 Quality assurance
The quality assurance of the tests must include control of the test system at DHI DANETV
Water Centre, control of the reference test and reference analysis performed at external
laboratories and control of the data quality and integrity.
The test plan and the test report will be subject to review by the expert group as part of the
review of this verification protocol and the verification report, see Fig 2.2.
As this verification is a joint verification with the U.S. EPA ETV and ETV Canada, an on-
site audit by Battelle AMS Center is to be included in the quality assurance.
7.5 Test report
The test report must follow the principles of the template of the DHI DANETV
verification center quality manual template III with data and records from the tests
presented. For this joint verification, the principles of the U.S. EPA ETV and ETV Canada
formats must be complied with as well.
One joint test report will be prepared for LUMIStox and ECLOX.
8 EVALUATION
The evaluation includes calculation of the performance parameters, see Section 5.2 for
definition, evaluation of the data quality based upon the test quality assurance, see Section
7.4, and compilation of the additional parameters as specified in Section 5.3.
The calculations behind the EC20 and ECso determination in the software LUMISsoft4 are
not verified as part of this test, but will be spot-checked by a graphical check of the EC 20-
and ECso-values for one dilutions series.
8.1 Calculation of performance parameters
By testing a dilutions series with inhibitions in the range from 10-90%, EC20- and ECso-
values can be calculated according to principles in ISO 11348-3. This is performed by the
PC software LUMISsoft4 connected to the HACH-LANGE instruments. To estimate
ECso-values a minimum of 3 measurements have to be in the range from 10-90%
12
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inhibition. Furthermore, one concentration has to give responses above 50% inhibition and
one concentration has to give response below 50% for determination of valid EC2o-values.
For use of the ECLOX without connection to PC, the results will be recorded as %
inhibition and EC-values cannot be determined directly.
Calculations of EC20- and ECso-values (and in the case of ECLOX using firmware, %
inhibition) are performed according to generally accepted statistical principles such as
those described in 191 and as described in Table 8.1, referring also to the test design shown
in Table 7.1.
Table 8. 1 Calculations used for the test results
Parameter
Calculation
Explanations
Criterion of detection
CD is criterion of detection;
t0 95(f) is the Student's t factor for f where f= n-1
degrees of freedom.
n is number of measurements;
skis a pooled estimate for standard deviation of
luminescent in control glasses
Range of application
Minimum: just above 2*EC50
Maximum: just less than
32*EC50
EC50: Concentration causing 50% inhibition
Precision
(repeatability or
reproducibility), as
relative standard
deviation, RSD
D -
i
x -x
imax imm
X =
n
d =•
m
1.693
DJ is the range at level i;
ximin and ximax are the lowest and highest
measurements at level i;
jq is the average of n measurements;
m is the number of levels;
dj is the relative range at level i;
d is the mean relative range for all m levels used
with three replicates, i=3 in Xj
Agreement with
accepted values, A
Based on robust
literature values
(obtained by use of
ISO 11 348-3)
n
n
t = — x 100%
A =
m
J. is the mean of measurements at level i,
y i is the literature value at level i, y,
Ai is the agreement at level i;
A is the mean agreement for all levels
Robustness, R
R = ™ x 100%
xro is the average of measurement under
conditions of robustness test;
xre is the average of measurements under
reference conditions
13
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Calculations on performance parameters will be performed in Excel 2007 set up for the
purpose with the equations required.
8.2 Evaluation of test data quality
The information of the test report on the reference test and analysis, the test system and
data quality and integrity control will be evaluated against the requirements set in this
protocol and the objectives set in the test plan.
The spreadsheet used for the calculations will be subject to control on a sample basis (spot
validation).
The internal audit report and the external audit report prepared by Battelle AMS Center,
see Section 7.4, will be evaluated and major findings compiled and reported.
8.3 Compilation of additional parameters
8.3.1 User manual
The verification criterion for the user manual is that it describes the use of the equipment
adequately and understandable for the typical laboratory technician and test coordinator.
This criterion is evaluated through evaluation of a number of specific points of
importance; see Table 8.2 for the parameters to include.
A description is complete, if all essential steps are described, if they are illustrated with a
figure or a photo, where relevant, and if the descriptions are understandable without
reference to other guidance.
14
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Table 8.2 Criteria for user manual evaluation.
Parameter
Product
Principle of operation
Intended use
Performance expected
Limitations
Preparations
Unpacking
Transport
Assembly
Installation
Function test
Operation
Steps of operation
Points of caution
Accessories
Maintenance
Trouble shooting
Safety
Chemicals
Power
Complete
description
Summary
description
No description
Not relevant
Cost prices for LUMIStox, ECLOX, thermostat, the software LUMISoft4 and additional
equipment as cuvettes, bacteria and chemicals will be given in verification report.
8.3.2 Product costs
The capital investment costs and the operation and maintenance cost, could be seen as the
sustainability of the product, will be itemized based upon a determined design basis 7287,
see Table 8.3 for the items that will be included.
15
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Table 8.3
List of capital cost items and operation and maintenance cost items per product unit.
Item type
Capital
Site preparation
Buildings and land
Equipment
Utility connections
Installation
Start up/training
Permits
Operation and maintenance
Materials, including chemicals
Utilities, including water and energy
Labor
Waste management
Permit compliance
Item
Number
None
The design basis will be described and the cost items relevant for the LUMIStox and
ECLOX listed. Note that the actual cost for each item is not compiled and reported.
8.3.3 Occupational health and environment
The risks for occupational health and safety and for the environment associated with the
use of the products will be compiled. The compilation will list chemicals used during
product operation and classified as toxic, T, or very toxic, Tx, for human health and/or
very environmentally hazardous (N) according to 7297. The information will be given as
amount used per product unit (sample), see Table 8.4 for format.
Table 8.4 Compilation of classified chemicals used during product operation.
Compound
CAS number
Classification
Amount used per
product unit
Additional risks from installing, operating and maintaining the product will be evaluated,
compiled and reported, if relevant. In particular, risks for human health associated with
power supply and danger of infections will be considered.
16
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VERIFICATION SCHEDULE
The verification is planned for 2009-2010. The overall schedule is given in Table 9.1.
Table 9.1
Verification schedule.
Task
Quick scan
Verification protocol and test plan
Test
Test reporting
Verification
Verification report
Report document review
Verification statement
Timing
October 2009
October to December 2009
January and February 2010
January and February 2010
February 2010
February 2010
March 20 10
March and April 2010
10 QUALITY ASSURANCE
The quality assurance of the verification is described in Table 10.1 and Fig 2.2. The
quality assurance of the tests are described in the test plan but are summarized here, as
well as in the process document prepared by Battelle /20/.
Table 10.1 QA plan for the verification.
Initials
Tasks
Plan document with
verification protocol and
test plan
Test system
Report document with
test report and
verification report
DHI
MWN
Review
-
Review
BOP
Audit
Battelle
AMS
Center
ZW
Review
Audit
Review
U.S. EPA
ETV
JMK, MH
Review
-
Review
ETV
Canada
MEH
Review
-
Review
Expert
Group
KOK, JA, AA
Review
-
Review
An internal review of plan and report documents is conducted by the Head of Innovation,
Margrethe Winther-Nielsen (MWN). A test system audit (see test plan) is conducted
following GLP audit procedures by a trained auditor: Senior Chemical Engineer Bodil
Mose Pedersen (BOP).
The Battelle Quality Manager, Zachary Willenberg (ZW) will perform a technical systems
audit (TSA) during this verification and test.
U.S. EPA staff, John McKernan (JMK) and Michelle Henderson (MH), and Mona El-
Hallak (MEH) from ETV Canada will review the plan and report documents.
17
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The expert group, Kresten Ole Kusk (KOK), Joel Allen (JA) and Dr. All Amiri (AA) will
review the plan and report documents.
Reviews will be done using the DANETV review report template.
18
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APPENDIX 1
Terms and definitions used in the verification protocol
19
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The abbreviations and definitions used in the verification protocol are summarized below.
Where discrepancies exist between DANETV and U.S. EPA ETV terminology, definitions
from both schemes are given.
Word
Agreement
with accepted
values
AMS Center
Analytical
laboratory
Application
CD
CTAB
DANETV ETV
EC
ECLOX
Effect
EN
ETV
EU
Evaluation
Experts
GLP
ISO
LID
Limit of
detection
LoD
LUMISsoft4
LUMIStherm
LUMIStox
DANETV
Here defined as the % agreement between
literature values and test results
Advanced Monitoring Systems Center at
Battelle
Independent analytical laboratory used to
analyze reference samples
The use of a product specified with respect
to matrix, target, effect and limitations
Criterion of detection
Cetyl trimethyl ammonium bromide
The Danish Centre for Verification of
Climate and Environmental Technologies
Effect concentration, e.g. causing 50%
inhibition (EC50)
ECLOX handheld luminometer from
HACH-LANGE
The way the target is affected
European standard
Environmental technology verification
(ETV) is an independent (third party)
assessment of the performance of a
technology or a product for a specified
application, under defined conditions and
adequate quality assurance
European Union
Evaluation of test data for a technology
product for performance and data quality
Independent persons qualified on a
technology in verification or on verification
as a process
Good Laboratory Practice
International Standardization Organization
Lowest ineffective dilution. Often seen as
the dilution in a dilution series causing less
than 20% inhibition
Calculated from the standard deviation of
replicate measurements at less than 5
times the detection limit evaluated.
Corresponding to less than 5% risk of false
blanks
PC software from HACH-LANGE ,
produced for LUMIStox
Thermostat from HACH-LANGE ,
produced for LUMIStox
LUMIStox 300 bench top luminometer from
U.S. EPA ETV
EPA program that develops generic
verification protocols and verifies the
performance of innovative environmental
technologies that have the potential to
improve protection of human health and
the environment
An examination of the efficiency of a
technology
Peer reviewers appointed for a verification
20
-------
Word
Matrix
Method
OD
PC
Performance
claim
Performance
parameters
Precision
(Environmen-
tal) product
QA
Range of
application
Reference
analyses
Repeatability
Reproducibility
Robustness
RSD
SDS
Stakeholder
DANETV
HACH-LANGE
The type of material that the product is
intended for
Generic document that provides rules,
guidelines or characteristics for tests or
analysis
Optical density
Personal computer
The effects foreseen by the vendor on the
target (s) in the matrix of intended use
Parameters that can be documented
quantitatively in tests and that provide the
relevant information on the performance of
an environmental technology product
The relative standard deviation obtained
from replicate measurements, here
measured under repeatability or
reproducibility conditions
Ready to market or prototype stage
product, process, system or service based
upon an environmental technology
Quality assurance
Generally: the range from the LoD to the
highest concentration with linear response.
For this verification the range is based on
range of dilution of a test sample
Analysis by a specified reference method
in an accredited (ISO 17025) laboratory
The precision obtained under repeatability
conditions, that is with the same
measurement procedure, same operators,
same measuring
system, same operating conditions and
same location, and replicate
measurements on the same
or similar objects over a short period of
time
The precision obtained under
reproducibility conditions. Measurement
performed at different locations, operators,
measuring systems, and replicate
measurements on the same or similar
objects
% variation in measurements resulting
from defined changes in matrix properties
Relative standard deviation in %
Sodium lauryl sulphate
U.S. EPA ETV
(Environmental) technology
Buyers and users of technology,
technology developers/vendors, the
consulting engineers, the finance and
export communities, government
permitters, regulators, first responders,
21
-------
Word
Standard
Target
(Environmen-
tal) technology
Test/testing
Trueness
ISA
U.S. EPA
Vendor
Verification
Vibrio fischeri
DANETV
Generic document established by
consensus and approved by a recognized
standardization body that provides rules,
guidelines or characteristics for tests or
analysis
The measurable property that is affected
by the product
The practical application of knowledge in
the environmental area
Determination of the performance of a
product by parameters defined for the
application
The % recovery of true value obtained
either from knowledge on the preparation
of test solutions or from measurements
with reference methods
Technical system audit
United States Environmental Protection
Agency
The party delivering the product or service
to the customer
Evaluation of product performance
parameters for a specified application
under defined conditions and adequate
quality assurance
Light producing bacteria used in
luminescent bacteria test
U.S. EPA ETV
emergency response, disaster planners,
public interest groups, and other groups
interested in the performance of innovative
environmental technologies
An all-inclusive term used to describe
pollution control devices and systems,
waste treatment processes and storage
facilities, and site remediation technologies
and their components that may be utilized
to remove pollutants or contaminants from,
or to prevent them from entering the
environment
The technology developer, owner, or
licensee seeking verification
Establishing or proving the truth of the
performance of a technology under
specific, predetermined criteria, test plans
and adequate data QA procedures
22
-------
A P P E N D I X 2
References
23
-------
1. DANETV Test Center and Test Organization. Center Quality Manual - Water technology. Version 2. October
2009.
2. Battelle. Quality management plan (QMP) for the ETV Advanced Monitoring Systems Center. Version 7.0.
Dated November 2008.
3. ETV Canada General Verification Protocol. February 2007.
4. HACH-LANGE. ECLOX Tox LUMIStox specs and features.
5. HACH-LANGE. LUMIStox workshop.
6. ETV, EPA U.S. Environmental Protection Agency. Environmental Technology Verification Report. Strategic
Diagnostics Inc. Microtox. Rapid toxicity testing system. Battelle. November 2003.
7. ETV, EPA U.S. Environmental Protection Agency. Environmental Technology Verification Report. Strategic
Diagnostics Inc. Deltatox. Rapid toxicity testing system. Battelle. November 2003.
8. ETV, EPA U.S. Environmental Protection Agency. Environmental Technology Verification Report.
CheckLight, Ltd. ToxScreen-II. Rapid toxicity testing system. Battelle. November 2003.
9. TESTNET. D3.la Evaluation report test case la: TOXcontrol BioMonitor for surface water. DHL 01-03-
2008.
10. Machery-Nagel. Rapid Tests. Water Analysis. Page 128-130. Located onwww.mn-net.com, 24-09-2009.
11. HIDEX. New compact three in one personal multilabel tester Triathler. 2002. Located on www.hidex.com,
24-09-2009.
12. Dr Lange. Correcting absorptive inhibition in the luminescent bacteria test by means of a combined
luminometric/photometric procedure. INFO 13. January 1998.
13. Directive 2008/105/EC of the European parliament and the council on environmental quality standards in the
field of water policy. 16 December 2008.
14. Bekendtgerelse om miljekrav for vadomrader og krav til udledning af forurenende staffer til vandleb, seer
eller havet. Bekendtgerelse nr. 1669. Dated 14. December 2006.
15. Miljeministeriet. By- og Landskabsstyrelsen. Oversigt over forslag til kvalitetskriterier for staffer omfattet af
bilag 1 i bekendtgerelse nr. 1669/2006 om miljekrav for vandomrader mv. J. Nr. BLS-489-00051. Dated 7.
October 2009.
16. ISO. General requirements for the competence of testing and calibration laboratories. ISO 17025. 2005.
17. International Standardization Organisation. EN ISO 9001. Quality management systems - Requirements. 15-
11-2008.
24
-------
18. Gavaskar, A. and Gumming, L. Cost Evaluation Strategies for Technologies Tested under the Environmental
Technology Verification Program. 2001. Battelle.
19. European Commission. Commission Directive on classification, packaging and labelling of dangerous
substances. 2001/59/EC. 2001.
20. Environmental Technology Verification Program. Advanced Monitoring Systems Center. Process Document
for U.S. EPA ETV AMS Center, DHIDANETV Water Centre and ETV Canada. Joint Verification of the
HACH-LANGE GmbH LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer.
Battelle. Draft October 2009.
21. Farre, M., Asperger, D. Kantiani, L., Gonzalez, S., Petrovic, M. & Barcelo, D. (2008) Assessment of the
acute toxicity of triclosan and methyl triclosan in wastewater based on the bioluminescence inhibition of
Vibrio fischeri. Anal Bioanal Chem 390:1999-2007.
22. Romanelli, M.F., Moraes, M.C.F., Villavicencio, A.L.C.H. and Borrely, S.I. (2004) Evaluation of toxicity
reduction of sodium dodecyl sulfate submitted to electron beam radiation. Radiation Physics and Chemistry
71:409-411.
23. ISO. Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio
fischeri (Luminescent bacteria test). 11348-3. 2007.
24. Verordnung tiber Anforderungen an das Einleiten vor Abwasser in Gewasser (Abwasserverordnung - Abw).
Dated 21.03.1997
25. DANETV. LUMIStox 300 Bench Top Luminometer, ECLOX Handheld Luminometer. Luminescent bacteria
test for use in waste water. Joint test plan. DHI. 2009.
26. Nelson, S.M. and Roline, R.A. (1998) Evaluation of the sensitivity of rapid toxicity tests relative to daphnid
acute lethality tests. Bull. Contain. Toxicol. 60:292-299
27. Backhaus, T., Froehner, K., Altenburger, R., and Grimme, L.H. (1997) Toxicity testing with Vibrio fischeri:
comparison between the long term (24H) and the short term (30 min) bioassay. Chemosphere 35, 12, 2925-
2938.
28. Gavaskar, A. and Gumming, L. Cost Evaluation Strategies for Technologies Tested under the Environmental
Technology Verification Program. 2001. Battelle.
29. European Commission. Commission Directive on classification, packaging and labelling of dangerous
substances. 2001/59/EC. 2001.
25
-------
A P P E N D I X 3
Application and performance parameter definitions
26
-------
This appendix defines the applications and the relevant performance parameters used to
verify the performance of an environmental technology following the DAKETV Program.
1
Applications
The intended application of the product for verification is defined in terms of the matrix,
the targets and the effects of the product.
The LUMIStox and ECLOX are luminometers which measure light from the light
producing bacteria Vibrio jischeri, as indicator of acute toxicity.
1.1 Matrix/matrices
The luminometers are sold for testing of wastewater; river and lake water; leachates from
soil, waste, rubble, etc.; or directly in fluent chemicals. The matrix in which the
application is being verified is wastewater effluent from both domestic and industrial
sources.
1.2 Effect
The luminometers can measure any acute toxicity that causes an effect on the light
emission from Vibrio fischeri. In the ISO 11348-3 7237 standard, which the LUMIStox is
being tested according to, three compounds are listed as reference substances to be
included in validity testing. These are 3,5-dichlorophenol, zinc(II) as zinc sulphate
heptahydrate and chromium (VI) as potassium dichromate.
The verification will include these reference substances as well as selected metal ions,
organic pesticides, organic toxic compounds, industrial chemicals and surfactants.
1.2.1 Compounds to be tested
The vendor has suggested a list of compound to be included in the verification, these are
listed in Appendix Table 1.
Appendix Table 1 List of compounds suggested by vendor.
Group
Heavy metals
Organic pesticide
Organic pollutants
Industrial pollutant
Surfactants
Compound
Hg-complexes as HgCI2
Pb2+ as Pb(NO3)2
Zn2+ as ZnSO4+7H2O
Cr2O72" as K2Cr2O7
2,4,5 Trichloroanilin
Formaldehyde
p-Crecol
Hydroquinone (benzene-1,4-diol)
Cyanide (CN-) as KCN
SDS (sodium lauryl sulphate)
CTAB (cetyl trimethyl ammonium bromide)
27
-------
The vendor has performed tests on all suggested compounds except HgCl2 and 2,4,5
Trichloroanilin.
Each of the target groups and vendor suggested compounds was evaluated as follows:
Hg is banned in the EU, it is therefore not likely to be found in European domestic
wastewater today. Hg is difficult to work with in the laboratory. For these reasons Hg is
excluded.
Copper is included since it is a good representative for heavy metals in both domestic and
industrial wastewater, and since it is found in wastewater as many different ions.
The ISO 11348-3 uses 3,5-dichlorophenol, Zn2+ (as ZnSO4+7H2O) and Cr6+ (as K2Cr2O7,
in water resulting in Cr2C>72) as reference substances for testing the quality of delivered
bacteria batches. Cr2Oj2 will be included giving the possibility to do some reference to the
standard and the precision test which is described in Appendix Table 7. Zn2+ will be
included since good literature values exist.
Having two positive metals ions (Cu2+ and Zn2+), seems sufficient and Pb2+ has therefore
been excluded from the test program.
2,4,5 Trichloranilin is not a regularly used pesticide. Instead a pesticide produced by the
Danish company Cheminova and included in their standard effluent wastewater analyses is
included. The specific pesticide, flutriafol, has been chosen in cooperation with
Cheminova.
Hydroquinone is not seen as a compound with special relevance for effluent wastewater
and is therefore excluded.
Formaldehyde and p-cresol are easily degradable and relatively volatile. It is therefore
unlikely that they will remain in the wastewater effluent after treatment in the plant.
Instead, triclosan, which is widely used in household products and found in domestic
wastewater, is included. Triclosan is toxic to bacteria.
U.S. EPA ETV has performed verification of similar equipment, but to be used on a
chlorinated drinking water matrix. The selection of compounds for those tests was made
with a different focus than in this verification. However, the U.S. EPA ETV verification
included cyanide, which also is included in the list of compounds suggested by vendor.
The vendor has found cyanide to be difficult to work with at pH 7. Cyanide will be
included as target compound, but special actions will be taken to ensure and monitor loss
of cyanide from test solutions.
In addition to the listed surfactants, nonylphenol ethoxylate will be included in the test
since it is a well know surfactant that is very toxic to aquatic organisms and is unwanted in
the water environment. By including nonylphenol the three surfactants will represent
anionic, cationic and nonionic detergents.
The final list of compounds to be included in the verification is listed in Appendix Table 2.
28
-------
Appendix Table 2 List of compounds to be included in test with notification on whether compound is typical
for domestic or industrial wastewater.
Group
Heavy metals
Organic
pesticides
Organic
pollutants
Industrial
pollutant
Surfactants
Compounds suggested by
vendor
Hg-complexes as HgCI2
Pb2+ as Pb(N03)2
Zn2+ as ZnSO4+7H2O
Cr2O72" as K2Cr2O7
2,4,5 Trichloroanilin
Formaldehyde
p-Crecol
Hydroquinone (benzene-1,4-diol)
Cyanide (CN-) as KCN
SDS (sodium lauryl sulphate)
CTAB (cetyl trimethyl ammonium
bromide)
Chosen compounds
Cu^+ as Cu(NO3)2
CrO72" as K2CrO7
Zn2+ as ZnSO4+7H2O
Flutriafol
Triclosan
Cyanide (CM") as KCN
SDS (sodium lauryl
sulphate)
CTAB (cetyl trimethyl
ammonium bromide)
Nonylphenol ethoxylate
Domestic
X
X
X
X
X
X
X
X
Industrial
X
X
X
X
X
X
X
X
X
X
Appendix Table 3 is a list of ECso-values for the selected compound found in the
literature.
Appendix Table 3 ECso-values from literature for the selected compounds.
Group
Heavy metals
Organic
pesticides
Organic
pollutants
Inorganic
pollutant
Surfactants
CAS no.
7758-98-7
7778-50-9
7733-02-0
7667-21-0
3380-34-5
57-12-5
151-21-3
57-09-0
104-35-8
Compound
Cu2+ (Cupper
sulfate)
Cr20/-
Zn2+
Flutriafol
Triclosan
Cyanide (CN~)
SDS
CTAB
Nonylphenol
ethoxytale
EC50
(Vibrio fischeri)
mg/L
7.1
(0.35-19.5, n=3)
18.7 mg/L ±11% (potassium
dichromate)
2.2 mg/l ± 23%
(zinc sulphate heptahydrate)
no data found
0.28
4
2.09
0.972
no data found
According to
ISO 11 348-3
to be
determined
Yes
Yes
Yes
to be
determined
unknown
Yes
Reference
/26/
/23/
/23/
721 /
/6/
/22/
1211
30 minutes incubation time.
29
-------
1.3 Target(s)
The targets for the application are generally reported in terms of limit of detection (LoD),
precision (repeatability and reproducibility), trueness, range of application and robustness.
For toxicity testing the limit of detection is not possible to determine. Instead it is chosen
to determine the criterion of detection (CD) based on the standard deviation of blanks. The
trueness of the inhibition is difficult to measure, and therefore the verification of trueness
will be replaced a verification of agreement with accepted values, which will be evaluated
by comparing the measured value to available robust literature values obtained by use of
the ISO 11348-3 method, for same compound. The range of the application cannot be
determined directly by identification of linear range as for regular measurements. For this
verification range is based on the inhibitions needed to determine ECso-values, see
description in Section 4 Performance parameter definitions.
The values of the targets claimed by the vendor are given in Appendix Table 4 for the
products.
The vendor has incorporated equipment in the LUMIStox for color correction of
inhibition. With the use of the color correction on colored samples a robustness of 95-
113% was shown. Without color correction, the robustness was 109-148% 7247.
The robustness is the change in results due to defined variations in e.g. concentration level,
temperature, pH, color, turbidity, cuvette types, matrix (pure water versus wastewater).
The ISO 11348-3 standard includes the possibility of testing (marine) saltwater samples;
however, saltwater samples are not included in robustness testing of the products.
Appendix Table 4 Vendor claim of performance /4/.
LUMIStox
ECLOX
Criterion
on
detection3
%
inhibition
(10)
(10)
Precision (RSD)
%
Precision of
instrument
0.7
2
Precision of
test4
<20
<20
Range of
application
(linear screening
range)
% inhibition
10-90
10-90
Agreement with
accepted values
%
Not specified
Not specified
Robustness
%
Not specified
Not specified
The vendor has recently tested selected compounds. The results can be found in Table 6.1,
in Section 6.1 Summary of existing data.
In the ECLOX manual the vendor states the following:
Due to nature of the simplified procedure and that the test is carried out at ambient
temperatures the results may differ if compared directly with results [derived] for
the same sample using the ISO 11348 method.
Given as part of linear range.
1 Is not clearly stated from vendor as repeatability or reproducibility.
30
-------
1.4 Exclusions
The verification is to be performed on one effluent domestic wastewater and one industrial
wastewater, other media are excluded. However, individual test substances are tested in
2% NaCl MilliQ-water.
According to the vendor, samples containing chlorine as a result of drinking water
chlorination will interfere with the test results by affecting the viability of the bacterial
agents. Chlorine containing samples are excluded from the test.
2 General performance requirements
No formal performance requirements for the application have been identified in the
European Union or the U.S. and Canada.
The conventional performance parameters of analytical and monitoring methods and
equipment are limit of detection (LoD), precision (repeatability and reproducibility),
trueness, specificity, linearity and matrix sensitivity. The uncertainty of measurements
may be used to summarize the performance. Parameters may be added to characterize
variations of equipment, e.g. on-line or on-site monitoring instruments.
2.1 Regulatory requirements
No regulatory requirements exist for measurement of luminescent toxicity. The new Water
Framework Directive 2009/90/EC of 31 July 2009 contains a minimum performance
criteria of 25% RSD, applicable for all methods of analysis.
In Germany, wastewater regulations include results from luminescent bacteria tests (LID,
lowest ineffective dilution) as quality criteria for several industries including the chemical
industry, the rubber industry, cooling towers and waste treatment plants 7247. For the
chemical industry a LID = 32 is accepted, meaning that the wastewater has to be diluted a
maximum of 32 times to obtain a toxicity below 20% inhibition towards the luminescent
bacteria.
For a few of the compounds, environmental quality standards for surface waters are given
by the EU 7137. These are listed in Appendix Table 5.
31
-------
Appendix Table 5 Environmental quality standards stated by EU /13/and Denmark. For Denmark values in
normal writing are effective /14/, while values in italic are planned to come in force within
2010/15/.
Group
Heavy metals
Organic
pesticides
Compound
Cr(VI)
Cu
Zn
Flutriafol
EU
Inland
surface
water
ljg/L
Other
surface
water
M9/L
Denmark
Fresh water
ljg/L
4.9 (dissolved)
1 (dissolved)
max 12
7.8
(dissolved)
Soft water: (H<24 mg
CaCOs/L)
3. 1 (dissolved)
31
Marine water
ljg/L
3.4 (dissolved)
1 (dissolved)
max 2. 9
7.8 (dissolved)
3.1
2.2 Application based needs
A validity check is required according to ISO 11348-3. The validity check involves
analysis of three reference standards which should cause 20 to 80% inhibition after 30
minutes of contact time. The results from the validity check are shown in Appendix Table
6, as reported for the LUMIStox by vendor.
Appendix Table 6 Vendor quality data for LUMIStox according to ISO 11348-3 /5/.
Standard concentration
No. of data set
Range of inhibition
Mean inhibition
RSD
3,5 dichlorophenol
3.4 mg/L
70
22.46-63.53%
44.1%
27%
Zn2+
2.2 mg/l
(zinc sulphate
heptahydrate)
60
20.82-49.19%
30.6%
23%
Cr2072-
18.7 mg/L (potassium
dichromate)
70
47.90-78.77%
63.4%
11%
In ISO 11348-3, results from an interlaboratory trial with the three reference standards are
listed for information. The results are shown in Appendix Table 7.
Appendix Table 7 Interlaboratory trial, Annex C, ISO 11348-3.
No. of
laboratories
Average cone.
RSD
3,5 dichlorophenol
EC20
14
2.32 mg/L
18.6%
EC50
13
3.36 mg/L
9.6%
Zn2+
EC20
15
1.08 mg/L
43.6%
EC50
14
2. 17 mg/L
33.6%
Cr2O72'
EC20
15
3.60 mg/L
52.4%
EC50
14
18.71 mg/L
32.9%
32
-------
State of the art performance
Other similar luminometers exist on the market. Some selected luminometers are listed in
Appendix Table 8. Information as to whether they have been verified is included.
Appendix Table 8 Luminometers and verification of these.
Name
Verification
Reference
Portable
BioFix Lumi-10
Triathler
ToxScreen-ll
Deltatox
None known
None known
U.S. EPA ETV
U.S. EPA ETV
no/
mi
/8/
in
Laboratory
Microtox
U.S. EPA ETV
/6/
Field installation
TOXcontrol BioMonitor
TESTNET
/9/
The three U.S. EPA ETV verifications have all been performed using drinking water with
a focus on chemical compounds toxic to humans. One compound, cyanide, is also relevant
with regards to wastewater. Performance on cyanide measurements for the three products
is listed in Appendix Table 9. The toxicity threshold is the lowest concentration of the
tested dilutions where toxic effects were significant. For ToxScreen-II a special set-up was
used and ECso could therefore not be retrieved.
Appendix Table 9 Results from U.S.EPA ETV verification on cyanide.
Luminometer
Cyanide EC50 at 5 minutes
Cyanide EC50 at 15 minutes
Repeatability. Range of relative standard
deviation
Toxicity threshold
Microtox
8 mg/L
4 mg/L
0-4%
0.25 mg/L
Deltatox
7.6 mg/L
Not measured
1-4%
0.25 mg/L
ToxScreen-II
Not measured
Not measured
0-29%
0.25 mg/L
For the TOXcontrol BioMonitor the LoD, RSD, repeatability etc. were tested and reported
for several test set-ups. The compounds used were Zn
the results are summarized in Appendix Table 10.
2+
and 3,5 dichlorophenol. Some of
Appendix Table 10 Results from TESTNET verification of TOXcontrol BioMonitor.
Lowest detectable change
RSD
Repeatability
Day-to-day repeatability
Memory effect
Interference (Tropaeolin-color)
Range
7.2-17.4% inhibition
5.7-39.3%
2.4-5.8% inhibition
2.5-31.2% inhibition
Not relevant
Not relevant
Comment
Calculated based on solution of
approximately 20%, 50% and 80%
inhibition
Calculated based on solution of
approximately 20% and 80%
inhibition
No significant effect
Increased inhibition was significant at
concentrations from 0.25 mg/L
33
-------
Vendors of Vibrio fischeri test the bacteria lots and state an interval for ECso for selected
standard parameters. They also test each lot before shipment. An example of such a test
from an anonymous vendor including user laboratory reference testing is shown in
Appendix Table 11.
Appendix Table 11 ECso performance of Vibrio fischeri on standard parameters stated by vendor and tested
by vendor and user laboratory.
Standard parameter
Phenol
Zinc sulfate
Zinc2* (ion)
Specification from vendor
EC50 interval at
specification
Test time
1 3.0-26.0 mg/L
5 minutes
3.0-1 0.0 mg/L
15 minutes
0.6-2.2 mg/L
15 minutes
Vendor test result
No. of LOTs
Mean
RSD
9
18.0 mg/L
19%
9
4.88 mg/L
27%
9
1 .04 mg/L
25%
User laboratory test result
No. of LOTS
No. of tests
Mean
RSD
9
14
18. 3 mg/L
10%
9
15
5.52 mg/l
20%
Not tested
-
-
-
4 Performance parameter definitions
Based on the above-mentioned performance requirements, a set of relevant ranges of
performance parameters for activated sludge tanks (and treated wastewater) have been set
up and are listed in Appendix Table 12.
Appendix Table 12 Relevant ranges of performance parameters in effluent wastewater.
LUMIStox
ECLOX
Criterion of
detection
% inhibition
<10
<10
Range of
application
L/L
>1/2-< 1/32
>1/2-< 1/32
Precision (RSD)
%
Repeatability
<20
<20
Reproduce-
ability
<30
<30
Agreement
with
accepted
values
%
100 ±50
100 ±50
Robustness
%
100±50
100±50
The limit of quantification is set to 10% because this is equal to the vendor claim for linear
range and because ECio-values often are used for reporting ecotoxicological results.
The range of application for a chemical analysis is usually the range of analyte
concentration from the limit of detection to the highest concentration with linear response.
This concept is not meaningful for a toxicity test of a water sample, because the test does
not measure a concentration but an inhibitory effect as a function of the dilution of the
sample. The range of application for determining ECso therefore has to be considered in
terms of dilution. According to the HACH-LANGE manual estimation of an ECso of a
water sample requires a minimum of three measurements where the inhibition is between
34
-------
10% and 90%. In addition one of the three measurements must be above 50%. If the
standard dilution row is considered as described in the LUMIStox 300 Operation manual
and in Annex B of the ISO 11248-3:2007 with 9 dilutions (2, 3, 4, 6, 8, 12, 16, 24, 32
times dilution in the test suspension) then ECso should be in the range of dilutions > 2 and
< 32 times dilution assuming three measurements with inhibition between 10 and 90%.
Based on test results will be given ranges of concentrations of the compounds tested in this
study, which will give a inhibition within the range of application.
Repeatability in Appendix Table 9 and Appendix Table 10 is less than 6% in all cases,
except for the ToxScreen-II, where a repeatability of 0-29% is seen. The vendor claims a
precision for the products of < 20%, see Appendix Table 4. A repeatability of less than
20% is chosen, since the vendor claims to fulfill this.
The day-to-day repeatability for TOXcontrol BioMonitor, as shown in Appendix Table 10,
lists RSD values up to 31.2%. The vendor states, as mentioned, a test precision of < 20%,
while the quality check of LUMIStox in Appendix Table 6 shows a reproducibility of up
to 27%. Here a reproducibility of 30% is chosen.
The agreement with accepted values is evaluated by looking at the ECso-values specified
by a vendor of Vibrio fischeri LOTs in Appendix Table 11. The largest relative interval is
given for zinc +, the "mean" here is 1.4 mg/L with an acceptable range of ± 57%. The ISO
standard 11348-3 requires inhibition of 20-80% of specified concentrations. These
numbers cover both reproducibility and repeatability. The agreement with accepted values
is set to ± 50%.
Robustness has been tested directly for the TOXcontrol BioMonitor, where the dye
chemical tropaeolin was added. The results showed a significant interference at 0.25 mg
tropaeolin/L, where an increased inhibition was seen. Color correction is part of the
LUMIStox product, see section 1.3 Target(s). The robustness can be interfered by other
parameters. The general robustness is set to the level seen without color correction, here
values of 148% of true value were seen.
35
-------
DANETV
BalteJie
Canada T r D"™"1" *'
LUMIStox 300 Bench Top Luminometer
ECLOX Handheld Luminometer
Joint test plan
Luminescent bacteria test for use in wastewater
Handheld ECLOX
January 2010
Approved
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LUMIStox 300 Bench Top Luminometer
ECLOX Handheld Luminometer
Joint test plan
Agern Alle 5
DK-2970 H0rsholm
Denmark
Tel: +4545169200
Fax: +4545169292
clj@dhigroup.com
www.dhigroup.com
Vendor
HACH-LANGE GmbH
Vendors representative
Dr. Elmar Grabert
Project
DANETV
Authors
Claus J0rgensen, Mette Tjener Andersson
2
1
0
Revision
Approved Test plan
Test plan for approval
Test plan for review
Description
Key words
Acute toxicity; EC50; ECLOX; ISO 11348-3;
Luminescent bacteria; LUMIStox; Wastewater
Project No
11800378
Date
January 2010
Approved
by
Sten Lindberg
CLJ/MTA
CLJ/MTA
CLJ/MTA
By
MWN S
MWN S
MWN S
Checked Appr
L 13/01-10
L 11/12-09
L 16/11-09
oved Date
Classification
E3 Open
n Internal
n Proprietary
Distribution
DHI
HACH-LANGE
Battelle
ETV Canada
CLJ, MTA, MWN
Dr. Elmar Grabert
Mary Schrock
Mona EI-Hallak
No of copies
File distribution
only
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1 TABLE OF CONTENTS
1 TABLE OF CONTENTS
2 INTRODUCTION 1
2.1 Verification protocol reference 1
2.2 Name and contact of vendor 1
2.3 Name of center/test responsible 1
2.4 Expert group 2
3 TEST DESIGN 2
3.1 Test sites 6
3.1.1 Types 6
3.1.2 Addresses 6
3.2 Tests 6
3.2.1 Test methods 7
3.2.2 Test staff 8
3.2.3 Test schedule 8
3.2.4 Test equipment 9
3.2.5 Type and number of samples 9
3.2.6 Operation conditions 10
3.2.7 Operation measurements 10
3.2.8 Product maintenance 11
3.2.9 Health, safety and wastes 12
4 REFERENCE TESTS AND ANALYSIS 12
4.1 Analytical laboratory 13
4.2 Analytical parameters 13
4.3 Methods of test and analysis 13
4.4 Analytical performance requirements 14
4.5 Preservation and storage of reference samples 15
5 DATA MANAGEMENT 15
5.1 Data storage, transfer and control 15
6 QUALITY ASSURANCE 16
6.1 Test plan review 16
6.2 Performance control - reference test and analysis 16
6.3 Test system control 18
6.4 Data integrity check procedures 19
6.5 Test system audits 19
6.6 Test report review 20
7 TEST REPORT 20
7.1 Test site report 20
7.2 Test data report 20
7.3 Amendment report 20
7.4 Deviations report 21
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APPENDIX 1 22
Terms and definitions used in the test plan 22
APPENDIX 2 26
References 26
APPENDIX 3 28
Reference methods 28
APPENDIX 4 30
In-house test methods 30
APPENDIX 5 50
I n-house analytical methods 50
APPENDIX 6 52
Data reporting forms 52
APPENDIX? 67
Data management 67
APPENDIX 8 70
Deviations and amendments 70
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2 INTRODUCTION
Environmental technology verification (ETV) is an independent (third party) assess-
ment of the performance of a technology or a product for a specified application, under
defined conditions and quality assurance.
This verification is a joint verification between DANETV, the U.S. EPA ETV Ad-
vanced Monitoring Systems (AMS) Center and the ETV Canada. The objective of the
verification is to evaluate the performance of a wastewater rapid toxicity technology
that could be used to monitor industrial or domestic wastewater.
This verification and test plan includes two products from one vendor.
2.1 Verification protocol reference
This test plan is prepared in response to the test design established in the LUMIStox
and ECLOX, verification protocol, for luminescent bacteria test for use in wastewater,
2009 III.
2.2 Name and contact of vendor
HACH-LANGE GmbH, Willstatterstrasse 11, 40549 Dusseldorf, Germany, phone +49
211 52880.
Contact: Dr. Elmar Grabert, email: elmar.grabert@hach-lange.de, phone +49 211 5288
241.
Web site: www.hach-lange.de
2.3 Name of center/test responsible
The Danish Centre for Verification of Climate and Environmental Technologies,
DANETV), DHI DANETV Water Centre, DHI, Agern Alle 5, DK-2970 H0rsholm,
Denmark.
Test responsible: Claus J0rgensen, email clj@dhigroup.com, phone +45 16 95 62.
U.S. EPA ETV Advanced Monitoring Systems Center (Battelle), Battelle Memorial
Institute, 505 King Avenue, Columbus, Ohio 43201-2693, U.S.A.
Test responsible: Mary E. Schrock, email schrock@battelle.org, phone +1 614 424
4976.
ETV Canada, 2070 Hadwen Road Suite 201 A, Mississauga, Ontario L5K 2C9, Cana-
da.
Test responsible: Mona El-Hallak, email melhallak@etvcanada.ca, phone +1 905 822
4133 extension 239.
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2.4 Expert group
The expert group assigned to this test and responsible for review of the test plan and
test report includes:
Dr. Joel Allen, email: allen.joel@epa.gov, phone +1 513 487 2806. U.S.EPA, Office of
Research and Development/National Risk Management Research Laboratory/Water
Supply and Water Resources Division/Water Quality Management Branch.
Associate Professor Kresten Ole Kusk, email: kok@env.dtu.dk, phone +45 4525 1569.
Technical University of Denmark, Department of Environmental Engineering.
Dr. Ali Amiri, email: aamiri@oceta.on.ca, phone +1 905 822 41 33 ext 222. Ontario
Center for Environmental Technology Advancement (OCETA).
TEST DESIGN
Test compounds are selected as described in the joint verification protocol Appendix 3
III, and are summarized in Table 3.1. The pesticide flutriafol has been chosen together
with the Danish company Cheminova. Flutriafol is a pesticide included in their effluent
wastewater analyses.
Table 3.1 Selected test compounds.
Group
Heavy metals
Organic pesticides
Organic pollutants
Industrial pollutants
Surfactants
Compound
Cu^+ as CuSO4
Zn2+ as ZnSO4+7H2O
Cr2O72" as K2Cr2O7
Flutriafol
Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether)
Cyanide (CM") as KCN
SDS (sodium lauryl sulphate)
CTAB (cetyl trimethyl ammonium bromide)
Nonylphenol ethoxylate
Compounds which are easy to handle in the laboratory and are toxic to Vibrio fischeri
will be used. For the specific test set-up, use of a non-colored compound in the test of
color robustness or use of a chemical not having a pKa value in the range of 6.0-8.5 for
the pH robust test will be considered.
The test design outlined in the test protocol III is summarized in Table 3.2. Acronyms
are explained in Appendix 1.
Test results will be EC20 and ECso values, representing the concentration causing re-
spectively 20% and 50% inhibition of luminescence of the Vibrio fischeri population.
For tests where the luminometer is not connected to a PC with LUMISsoft4 software,
results will be as % inhibition. This will be the case for a few tests performed with the
ECLOX.
All test will be performed with use of plastic cuvettes except for test L were LUMIStox
robustness towards cuvettes (glass or plastic) will be tested.
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Table 3.2 Test design.
Test
No.
A
B
C
D
E
Test of dilution series (9 dilutions) for all compounds. 3 test
replicates (includes 2 measurement replicates each). Optim-
al concentrations will result in inhibitions of 10-90%.
Test of series of 9 blanks (incl. bacteria suspension, but no
sample).
Test of 2 dilution series (9 dilutions) for 1 compound. Max
concentrations in dilution EC30, and EC60, respectively. 3
tests replicates (includes 2 measurement replicates each).
Test of dilution series for 1 compound. Dilution as used in
test A. 3 test replicates (includes 2 measurement replicates
each). Repeated on 4 different days with 4 different bacteria
batches (test A is equal to first test day).
3 concentrations ~ EC20, EC50, EC80 for 2 compounds (metal
and organic). 3 test replicates (no further replicates). Per-
formed at 3 possible outdoor temperatures: room tempera-
ture, 15°C climate room, and outdoor temperature in Den-
mark at wintertime (~0-5°C). Measurement only after 15
minutes of inhibition.
Equipment to be tested
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Test
No.
F
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tures of 14°C, 15°C and 16°C. 3 test replicates (includes 2
measurement replicates each). If test of thermostat shows
greater variation than from 14-16°C, these temperatures will
be used instead.
Just for concentration ~ EC20 for 1 compound. For pH 6.0,
7.0 and 8.5, 3 test replicates (includes 2 measurement repli-
cates each).
Just for concentration ~ EC20 for 1 compound. Addition of
color in three concentrations and 1 with no color. 3 test repli-
cates (includes 2 measurement replicates each). Include
blind with color and no sample. LUMIStox with color correc-
tion, ECLOX with correction cuvettes according to ISO
11348-3.
Just for concentration ~ EC20 for 1 compound. Addition of
turbid reagent/material in three concentrations and 1 with no
material. The third being visibly turbid. 3 test replicates (in-
cludes 2 measurement replicates each). Include blind with
turbid reagent/material and no sample.
Spiked non- inhibiting domestic and industrial wastewater.
Just for concentration ~ EC20 for 5 compounds. 3 test repli-
cates (includes 2 measurement replicates each). If needed
wastewater is diluted to be non-inhibitory.
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Test
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Test of dilution series for undiluted and unspiked industrial
and domestic wastewater. 3 test replicates (includes 2 mea-
surement replicates each).
Just for concentration ~ EC20 for 2 compounds. 3 replicates.
Test of use of glass and plastic cuvettes.
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3.1 Test sites
Both the laboratory tests and outdoor dependent test will be conducted at DHI, H0rs-
holm, Denmark.
3.1.1 Types
Domestic wastewater samples for laboratory testing are planned to be obtained from
Lundtofte wastewater treatment plants. Industrial wastewater will be retrieved from an
industrial wastewater treatment plant at Cheminova, a producer of pesticides in north-
western Jutland, Denmark.
The wastewaters will be collected by the personnel at the treatment plant. The sample
from Cheminova is sent cold to DHI. The sample from Lundtofte wastewater treat-
ment plant is stored cold at the plant and will picked up by DHI personnel. Both
wastewater samples will be stored at DHI at 5 °C until use.
MilliQ water from the DHI laboratory, added NaCl to a concentration of 2%, will be
used for standard dilution series.
3.1.2 Addresses
Addresses of all sites are listed:
Laboratory and outdoor test: DHI, Agern Alle 5, DK-2970 H0rsholm.
Domestic wastewater: Renseanlaeg Lundtofte, Hjortekaersbakken 12, DK-2800
Lyrigby.
Industrial wastewater: Cheminova, Thyboranvej 78, DK-7373 Harbo0re.
3.2 Tests
The test program is designed to comply with ISO 11348-3. Water quality - Determina-
tion of the inhibitory effect of water samples on the light emission of Vibrio fischeri
(Luminescent bacteria test). Ill and to retrieve information needed to determine per-
formance parameters, as described in ISO/TR 13530 guide to analytical quality control
for water analysis /12/, ISO 15839, Water Quality - On-line sensors/analysing equip-
ment for water - Specifications and performance tests. /3/, ICH Harmonised Tripartite
Guideline for validation of analytical procedure /I I/ as well as previous verifications
of similar equipment for drinking water performed by U.S. EPA ETV and described in
a public testplan /4/.
The test design, as described in Table 3.2, includes three test set-ups:
• LUMIStox 300 bench top lumiometer with LUMIStherm thermostat and LU-
MISsoft4 PC software.
• ECLOX with LUMIStherm thermostat and LUMISsoft4 PC software.
• ECLOX with use of firmware.
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The main focus is on the laboratory set-up of LUMIStox 300 bench top and ECLOX
in connection with LUMIStherm thermostat and LUMISsoft4 PC software, while
ECLOX using firmware is tested to a less extent.
The test will be performed mainly in the laboratory, while one test on the ECLOX will
be performed outdoors at DHL
3.2.1 Test methods
Luminescence tests with Vibrio fischeri are described in a three-part standard ISO me-
thod 111. Part 1 requires use of freshly prepared bacteria, part 2 uses liquid-dried bacte-
ria, while part 3 uses freeze-dried bacteria. The LUMIStox and ECLOX use freeze-
dried bacteria. Therefore the following applies:
ISO 11348-3 Water quality - Determination of the inhibitory effect of water
samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -
Part 3: Method using freeze dried bacteria.
This standard is incorporated in the manuals for LUMIStox and ECLOX.
Dilution series of 9 dilutions will be performed for all selected compounds in test A.
The dilution series will be prepared according to Annex B in ISO 11348-3. Each dilu-
tion will be prepared and analyzed twice and the average will be used as result. This
will be repeated 3 times. All other tests will be performed on selected compounds of
the 9 target compounds at various dilutions described in Table 3.2 except criterion of
detection (Test B), which will be performed on NaCl dilution. Robustness for tempera-
ture at lab use (Test F), pH (Test G), color (Test H), turbidity (Test I) and type of cu-
vettes (Test L) will be performed at one concentration (EC2o), while robustness to-
wards start concentration will be performed at two concentrations (ECso and ECeo).
Robustness towards wastewater matrix with undiluted and unspiked industrial and
domestic wastewater (Test K) will be performed on dilutions series with 9 dilutions.
Stock solutions will be prepared in 2% NaCl MilliQ water. Solid NaCl will be added
to wastewater to obtain the salt concentration required for testing with the saltwater
bacteria, Vibrio fischeri. Dilution series will be prepared with dilution saltwater (2%
NaCl) provided from HACH-LANGE.
The LUMISsoft4 PC software calculates ECso-values as an overall result of the testing
of a dilution series. When a certain test concentration gives 0% or 100% inhibition, the
result cannot be used in the determination of ECso- Usually only results between 10%
and 90% inhibition are used in the calculation of ECso. For compounds not previously
tested it will therefore be necessary to perform a range finding test to determine con-
centrations within 10-90% inhibition. The software requires a minimum of three val-
ues between 10 and 90% to perform the calculation of ECso.
The principle of the dilution series in the thermostat is shown in Figure 3-1. In the
rows C and B are the two measurement replicates included in all tests where the ther-
mostat is used. When performing the test, bacteria suspension is added doubling the
volume. The final dilution series is therefore 32, 24, 16, 12, 8, 6, 4, 3, 2.
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dilution: 1 :
B
1 23456789 10
e In/on
cal.
Temperatur
+ 15°C -
000
Figure 3-1 LUMIStherm thermostat and dilution series.
Tests will be performed on effluent industrial and domestic wastewater. The toxicity
(as indicated by inhibition) of these samples will be tested prior to being used in the
spiking test (Test series J and K). If the wastewater is toxic, it will be diluted with 2%
NaCl MilliQ water to a non-toxic concentration, and then spiked with the selected
compounds.
For field portability (Test E), the ECLOX without the LUMISsoft4 software is used
(i.e., no PC is taken along). The firmware only shows readings of % inhibition. The
goal of this test is to assess how stable individual inhibition measurements are under
three different temperatures, a controlled 15 °C, ambient room temperature, and out-
door temperature (ambient and outdoor temperatures to be recorded at time of testing).
Solutions generating three inhibitions (20%, 50% and 80%) will be measured at each
temperature to give a sense of variability over a range of inhibitions in temperatures
that might be encountered in real-world field testing.
3.2.2 Test staff
The test responsible is Claus J0rgensen, and test technicians are Jane Bergstram and
Connie Seier0.
3.2.3 Test schedule
The test schedule is given in Table 3.3, see Table 3.2 for identification of experiment
labels.
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Table 3.3
Test schedule.
Task
Test plan review
Set-up of equipment
Test preparation
Testing
Test report draft
Test report QA
Test report
Test report review
Week no. 2009
51
X
X
X
52
53
Vacation
Week no. 2010
1
X
2
X
3
X
4
X
5
X
6
7
8
X
9
X
10
X
11
X
12
13
X
14
15
X
3.2.4 Test equipment
The test equipment and manuals include:
• LUMIStox 300 bench top luminometer and LUMIStherm thermostat. De-
scribed in:
o LUMIStox 300. Manual. HACH-LANGE. Version 3.02 and above.
BDA 356. January 2008.
o Luminescent bacteria test with freeze-dried bacteria according to
EN/ISO 11348-3. Luminescent bacteria test LCK 491. DR LANGE.
• ECLOX handheld luminometer with LUMIStherm thermostat and LUMIS-
soft4 PC software or with firmware. Described in:
o Luminescent bacteria test using the ECLOX™ instrument. User Ma-
nual. Hach Company. Edition Beta 2. September 2009.
• LUMISsoft4 PC software. Described in:
o Dr. Lange LUMISsoft 4. Manual. Version 1.001. LZV 093. 2000.
The DR. LANGE manual LCK 491 for LUMIStox 300 specifies use of glass cuvettes
(LZP 187) for the testing. HACH-LANGE informs that both glass cuvettes and plastic
test tubes (LZP 1480) can be used with LUMIStox 300. For ECLOX, HACH-LANGE
specifies that plastic test tubes (LZP 1480) must be used. /13/
General laboratory equipment procedures including cleaning and calibration are those
described and ISO 17025 accredited 151 for the DHI laboratories under the laboratory
services manual of the DHI Quality Management System 161.
3.2.5 Type and number of samples
The types and number of samples are summarized in Table 3.4.
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Table 3.4 Summary of analyses of samples and blanks.
Test
No.
A
B
C
D
E
F
G
H
1
J
K
L
Performance parameters
Range
Repeatability
Agreement with accepted values
Criterion of detection
Robustness, start concentration
Reproducibility
Repeatability, field
Robustness, sample temperature
Robustness, pH
Robustness, color
Robustness, turbidity
Robustness, matrix
Robustness, matrix
Robustness, cuvettes
Total
Analyses of sam-
ples
LUMIStox
486
108
162
18
18
30
30
60
108
24
1044
ECLO
X
486
108
162
54
18
18
30
30
60
108
1074
Analyses of blanks
LUMIStox
54
9
12
18
18
18
30
30
60
12
24
286
ECLOX
54
9
12
18
18
18
18
30
30
60
12
280
Except for test B, the number of blanks may be changed slightly during the tests if
some blanks can be used for several tests.
3.2.6 Operation conditions
The operation conditions applied during the verification of the product are generally as
required in ISO 11348-3. EC-values will be determined for 15 and 30 minutes.
When using the ECLOX in the field, it is possible to fulfill the requirements in the ISO
11348-3 on adjustment of pH and salinity, and settling of turbid samples; however, it
is not possible to adjust the temperature of the testing samples. Therefore a test on var-
iation in sampling temperatures (indoor and outdoor in Danish winter time) is in-
cluded.
3.2.7 Operation measurements
During operation, the following conditions are recorded, when relevant, see Appendix
6 for data recording and reporting forms:
• Conductivity/salinity of stock solutions.
• pH of stock solutions.
• Temperature in the thermostat, will be done on a daily basis.
Salinity and pH will be adjusted if required according to ISO 11348-3.
The vendor has experienced that cyanide is difficult to work with in the laboratory.
Therefore, a regular determination of the concentration of cyanide in the dilutions will
be performed with a test kit. This is expected to be done by adding a dilution with
cyanide to a beaker before starting luminescence testing. The cyanide concentration of
this solution will be determined with the test kit and after the end of luminescence test-
ing, the cyanide concentration in the beaker will be determined again. The test kit,
10
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LCK 315 from HACH-LANGE, uses a babituric acid-pyridine method and has a range
from0.01-0.6mg/L.
If the loss of cyanide exceeds 20% the results of the cyanide test will not be included
in the verification.
3.2.8 Product maintenance
The following storage information for the bacteria is provided by the vendor:
• The test reagents must be stored at -18°C until the date of expiry. Reactivated bac-
teria should be used within 4 hours if possible. Undiluted, reactivated bacteria
should be placed only temporarily in a refrigerator. The sensitivity spectrum of
reactivated bacteria may shift as time elapses. Tubes containing thawed but not
reactivated freeze-dried luminescent bacteria can be refrozen.
For the LUMIStox the vendor provides the following information on product mainten-
ance and optimal performance:
• The system diskette must be inserted into the drive before the instrument is
switched on! Whenever the instrument is moved, the diskette must be removed
from the drive first.
• The LUMIStox 300 measuring instrument should not be operated in an ambient
temperature below 16°C or above 29°C, otherwise problems may occur with the
cooling of the measuring shaft. Do not operate the instrument in direct sunlight!
• Soiling impairs the functioning of the cuvette lowering system. For this reason, do
not pipette reagents into measuring cuvettes in the measuring shaft. The measuring
shaft should also be closed in the mode when the measurements have been
completed. When the LUMIStox 300 is in use, the measuring shaft is automatical-
ly closed after a 10-minute idle period. It can be opened again by pressing any key.
• Before any measurements the LUMIStox 300 must have been switched on for at
least 30 minutes so that the photomultiplier and the cooled components are ready
for operation.
For the ECLOX the vendor provides the following information on product mainten-
ance and optimal performance:
• All cleaning and maintenance of the ECLOX™ Water Test Kit is to be performed
in a suitable clean, dry area. Make sure the kit is clean before removing any access
or battery covers. Do not permit foreign material to enter the kits as this can cause
equipment damage.
• The ECLOX Water Test Kit is designed for field use. No routine maintenance is
required, provided all cleaning, test, and calibration procedures are followed.
• The luminometer must be kept clean at all times. If the surface is dirty, wipe it
down with a clean damp cloth. Do not let water get into the luminometer cell. If
water gets into the cell, remove the cell insert and wipe out the moisture with a
clean, dry cloth. Replace the cell insert.
11
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• When replacing the battery a special procedure described in the manual must be
followed.
3.2.9 Health, safety and wastes
The use of the product does not imply special health, safety and waste issues.
Laboratory work during testing will be done according to the DHI Safety Rules that
are compliant with the Danish rules for safe occupational health and the European
regulations of work with chemicals. The test substances will be handled carefully in
accordance with material datasheets of the test substances. Wastewater will be hand-
led according to DHI's safety rules.
Outdoor work will be done according to DHI's rules for safe field work included in
DHI's safety rules.
Chemicals and test solutions are discarded according to Danish regulations for chemi-
cal waste by collection and destruction, in cam by collection and shipment to con-
trolled destruction when required.
4 REFERENCE TESTS AND ANALYSIS
The true value of a toxicity test cannot be determined, since no bacteria vials are fully
identical, and are prepared slightly differently from vendor to vendor. Therefore, the
test results will react slightly differently in every test. According to ISO 11348-3, the
true or accepted ECso-value of a substance is obtained, as long as the criteria in the
ISO are met. The reference tests will not be used as true values as seen in other verifi-
cations, but will give an indication of the sensitivity of the test organisms and will help
to identify false negative tests performed with the LUMIStox or ECLOX equipment.
As a result the test of reference samples will be limited. Reference tests are performed
on selected samples:
• Test A: samples with start concentration for all 9 target compounds (expect
cyanide). 3 replicates will be performed for one compound.
• Test K: samples of one spiked, non-inhibiting domestic wastewater and one
spiked, non-inhibiting industrial wastewater. 3 replicates will be performed for
one of the wastewater samples.
Reference samples will be sent to an independent laboratory for analysis under ISO
17025 accreditation 151 using the ISO 11348-3 Luminescent bacteria test method. The
equipment from Microtox® will be used. Microtox test will not be performed for cya-
nide. The volatility of cyanide is supposed to impact the quality of the results. Since
cyanide is toxic there will also be difficulties by sending the sample and the reference
laboratory will have to take special precautions working with a toxic compound. Due
to the difficulties of working with a toxic sample and lack of confidence in the results,
cyanide will be excluded from the reference testing.
Samples of stock solutions for confirmation of test concentration will be sent to an in-
dependent laboratory to be analysed under ISO 17025 accreditation 151 with appropri-
ate methods. When performing luminescent bacteria tests, solid NaCl salt is added to
12
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the samples. Therefore this salt will be added to the stock solutions before shipment to
ensure that measured concentrations are similar to the concentrations in the lumines-
cent bacteria test. Stock solutions are not stored over longer periods (weeks). Analyses
of stock solutions are done at start of toxicity testing, except for cyanide where a test
kit will be used to monitor concentration changes in open vials.
4.1 Analytical laboratory
Reference test of toxicity will be performed by ALcontrol AB, Olaus Magnus vag 27,
S-583 30 Linkoping, Sweden. SWEDAC accreditation registry number 1006.
Contact Britt Aurell, email: britt.aurell@alcontrol.se, phone: +46 13-254987
Chemical analyses on stock solution will be done by Eurofins Danmark A/S, Lade-
lundvej 85, 6600 Vejen, Denmark. DANAK accreditation registry number 168.
Contact Vivi Handberg, email: vivihandberg@eurofms.dk, +45 70 22 42 66.
4.2 Analytical parameters
Samples are tested with Microtox®, results are EC20- and ECso-values.
All stock solutions will be analysed for the concentration of the added compound.
The wastewater samples will be analysed for general wastewater parameters as listed
in Table 4.1.
Table 4.1 Analytical parameters for wastewater.
Analytical parameters
Turbidity
TOC
Conductivity
Alkalinity
PH
COD
Suspended solids (SS)
Nitrogen (total)
Phosphorus (total)
BOD5
4.3 Methods of test and analysis
The reference test method will be ISO 11348-3 Luminescent bacteria test method. The
equipment from Microtox® will be used. The ISO 11348-3 method includes tests of
reference substances, to ensure validity of the test. ALcontrol performs regular tests
for zinc sulphate heptahydrate and 3,5-dichlorophenol. These test results will be avail-
able for review. ALcontrol has also participated in one interlaboratory trial, results
from this will be available for review.
Stock solutions are analyzed according to the methods listed in Table 4.2, while waste-
water parameters are analyzed according to methods listed in Table 4.3.
13
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Table 4.2 Analytical methods and performance expectations from the contracted laboratory.
Group
Heavy
metals
Organic
pesticides
Organic
pollutants
Industrial
pollutants
Surfactants
Compound
Cr
Cu
Zn
Flutriafol
Triclosan^
Cyanide (CN~)
Nonylphenol ethoxylate
SDS (sodium lauryl
sulphate)
CTAB (cetyl trimethyl
ammonium bromide)4
Method
ISO 1 7294m - ICP-MS
ISO 1 7294m - ICP-MS
ISO 17294m -ICP-MS
GC/MSJ
-
DS/EN ISO14403
MK0250-GC/MS
MK8230-LC-MSJ
VKI
Limit of
detection
ug/l
1.0
3.0
0.5
0.1
-
1
0.1
5.0
100
Uncertainty
%
151
151
102
10
-
10
15
15
20
Table 4.3 Method for analytical parameters analysed in waste water.
Parameters
Turbidity
TOC
Conductivity
Alkalinity
PH
Method
DS290
DS/EN 1484
DS/EN 27888
DS/EN I 9963
DS287
Parameter
COD
Suspended solids (SS)
Nitrogen (total)
Phosphorus (total)
BOD5
Method
ISO 15705
DS/EN 872
DSENI 11 905 Auto
DS/EN ISO 6878
DS/EN 01 899-1
For analyses performed under accreditation, internal and external quality control data
will be available from Eurofins.
4.4 Analytical performance requirements
The analytical performance requirement for the reference test performed with Micro-
tox® should be equal to performance parameters as derived for LUMIStox and
ECLOX, summarised in Table 4.4. The available quality control data from ALcontrol,
as described in Section 4.3, is not expected to give information on all these parame-
ters.
1 Eurofins states that salt content in samples can give higher RSD.
2 The inclusion of triclosan in wastewater analyses is relatively new. Triclosan will be set up by Eurofins in Decem-
ber 2009. This method is therefore not included under Eurofins accreditation.
3 Method is not included under Eurofins accreditation.
4 CTAB will be analysed with a general method for cationic detergents. The method is calibrated with benzyl di-
methyl tetradecyl ammonium chloride-dihydrate. The concentration of CTAB will be calculated based on the mole
weight of the two compounds.
14
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Table 4.4 Required analytical performance.
LUMIStox
ECLOX
Criterion of de-
tection
% inhibition
<10
<10
Precision
(RSD)%
<30
<30
Agreement with
accepted values
%
100 ±50
100 ±50
Robustness
%
100±50
100±50
4.5 Preservation and storage of reference samples
Samples for Microtox® testing will be frozen before shipment according to instruc-
tions from the reference laboratory.
Stock solutions for chemical analyses will be preserved according to instructions of
the reference laboratory. Wastewater samples will be stored cold at 5°C. The samples
will be shipped on ice or according to the instructions of the lab.
5.1
DATA MANAGEMENT
Data filing and archiving will follow the procedures of the DHI Quality Management
System. In here is stated that, the entire material is filed after the project has been
completed. The project material comprises all documents, calculations, analyses, re-
sults etc. that will enable another DHI employee to scrutinise the work carried out. Af-
ter 10 years, the project files should still be sufficiently complete to make possible a
reconstruction of the work.
Data storage, transfer and control
The data to be compiled and stored are summarized in Table 5.1.
Analytical raw data will be filed and archived according to the specifications of the la-
boratory, Eurofins, quality management systems under their ISO 17025 accreditation.
Table 5.1 Data compilation and storage summary.
Data type
Test plan and
report
Test details in
laboratory and
field
Calculations
Analytical re-
ports
Data media
Protected PDF
files
Log book and
pre-prepared
forms
Excel files
Paper
Data recorder
Test responsi-
ble, DHI
Technician, DHI
Test responsi-
ble, DHI
Test responsi-
ble, DHI
Data recording
timing
When approved
During collec-
tion
During calcula-
tions
When received
Data storage
Files and arc-
hives at DHI
Files and arc-
hives at DHI
Files and arc-
hives at DHI
Files and arc-
hives at DHI
Forms for data recording are given in Appendix 6. Record will be made for tests at 15
and 30 minutes.
15
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6 QUALITY ASSURANCE
The tests are performed under the quality management system of DHI which is ISO
9001 compliant 111, but not certified. The DHI laboratories have ISO 17025 accredita-
tions 151 and OECD GLP approvals /8/ for a range of tests and ISO 17025 for sampling
of drinking water. As part of the ISO 17025 and GLP inspections, the procedures for
general laboratory processes, quality assurance and documentation/archiving are as-
sessed.
6.1 Test plan review
The test plan will be subject to internal review by the verification responsible from
DHI DANETV Water Centre: head of innovation Margrethe Winther-Nielsen. The test
plan will also be subject to review by the Battelle Advanced Monitoring Systems Cen-
ter Verification Test Coordinator and Quality Manager (Mary Schrock and Zachary
Willenberg, respectively), as well as by the US EPA ETV AMS project officer and
quality manager (John McKernan and Michelle Henderson, respectively). Further-
more, the test plan will be subject to review by ETV Canada by Director Technology
Assessment and Quality Assurance Services Mona El-Hallak.
External review of the test plan will be done by the expert group assigned to this veri-
fication.
6.2 Performance control - reference test and analysis
Generally, our control of reference test and analysis is based on reference laboratories
performing analyses under ISO 17025 accreditation 151. Information on the laboratory
quality assurance will be gathered. Physical inspection (audit) will only be performed
if disagreements are suspected. Further for this verification the reference test and ana-
lyses are of minor extent and verification of the products will only to a minor extent be
based on these results.
Performance control of ALontrol Microtox® tests will be performed by test of 3 repli-
cates of a spiked water sample and a wastewater samples. 2 blank (pure MilliQ water)
samples will be tested for control. Information of the laboratory quality assurance, me-
thod validation etc. will be gathered.
Performance control of Eurofms analysis will be performed by sending 2 blanks (2% NaCl MilliQ
water) to analyses for each of the target compounds. Information of the laboratory
quality assurance, proficiency test etc. will be gathered. Eurofms is including standard
reference samples when they analyze. Details on their acceptance range and action if
standard is out acceptance range are given in Table 6.1.
16
-------
Table 6.1 Eurofins reference standards and acceptance criteria.
Group
Heavy metals
Organic pesti-
cides
Organic pollu-
tants
Industrial pollu-
tants
Surfactants
Compound
Cr
Cu
Zn
Flutriafol
Triclosan
Cyanide (CN~)
Nonylphenol
ethoxylate
Method
Use of NIST stan-
dard 1643d
Quality control is
performed by spik-
ing a sample and
calculate retrieval.
Use of standards
prepared from pure
chemicals from dif-
ference batches and
suppliers. A stan-
dard concentration
near LoD is included
as well as a high
standard concentra-
tion
Include standards of
NaCN: 5 ug/l and 50
ug/l
And K3(Fe(CN)6):
10 ug/l and 100 ug/l
Replicate on every
20. Samples and
minimum per series.
As fortriclosan.
As standards are
used 4-nonylphenol
of the following
compounds:
NP (technical mix-
ture 4-nonylphenol)
NPE1 (isomere mix-
ture 4-nonylphenol)
NPE2 (isomere
mixture 4-
nonylphenol).
Acceptance crite-
ria
1 .79-2.42 ug/L
1.89-2. 55 ug/L
6.5-8.8 ug/L
70-120%
The result for stan-
dard near LoD has
to be convincing.
Result of the sam-
ples has to be below
concentration in the
high standard.
For NaCN:
4,45-5,55 ug/l and
44,5-55,5 ug/l
For K3(Fe(CN)6):
> 9,0 ug/l and
>90 ug/l
Accepted difference
< 1 8 %
Action
If control is not with-
in acceptance crite-
ria the series will be
reanalyzed.
If retrieval is not
with acceptance
criteria sample and
spiked sample is
reanalyzed.
Performance on the
apparatus will be
improved and the
samples reana-
lyzed.
Either reextraction
with less sample
material in use or
the first extract will
be diluted.
If controls are not
within acceptance
criteria the series
will be reanalyzed.
17
-------
Table 6.2 Eurofins reference standards and acceptance criteria (cont.).
Group
Surfactants
(cont.)
Compound
SDS (sodium
lauryl sul-
phate)
CTAB (cetyl
trimethyl am-
monium bro-
mide)
Method
As fortriclosan.
As standards is
used LAS-mix cas
no. 69669-44-9
(Dodecyl benzene
sulfonicacid, so-
dium salt).
Further is also in-
cluded quality con-
trol performed by
spiking a sample
and calculate re-
trieval.
Include standards of
Benzyl-dimethyltetra
ammoniumchlorid
dihydrat: 0,3 mg/l
and 1 ,5 mg/l
Replicate on every
20. Samples and
minimum per series.
Acceptance crite-
ria
The result for stan-
dard near LoD has
to be convincing.
Result of the sam-
ples has to be below
concentration in the
high standard.
70-120%
0,1 1-0,49 mg/l and
0,9-2, 10mg/L
Accepted difference
< 1 8 %
Action
Performance on the
apparatus will be
improved and the
samples reana-
lyzed.
Either reextraction
with less sample
material in use or
the first extract will
be diluted
If retrieval is not
with acceptance
criteria sample and
spiked sample is
reanalyzed.
If controls are not
within acceptance
criteria the series
will be reanalyzed.
At the moment Eurofins has not provided information included standard and accep-
tance criteria for cyanide, SDS and CTAB. The principles for these compounds are
expected to be as for the compounds listed in Table 6.1.
Triclosan which is under implementation and Eurofins, and if one of the compounds
cyanide, SDS and CTAB should lack acceptance criteria, performance evaluation (PE)
audits will be considered at time of testing, depending on implementation progress for
triclosan and the reaming quality control data from Eurofins.
6.3 Test system control
System control is planned to test the DHI DANETV Water Centre test system of the
LUMIStox and ECLOX.
All stock solutions will be analyzed in duplicate to control the concentration of target
compounds. Before testing with luminometers solid NaCl salt is added to the samples.
Therefore, the samples sent for reference analysis will be stock solutions with added
salt. This will give information about whether the salt addition causes precipitation of
added compounds.
Luminescent bacteria tests of 2 blank samples will be performed at the reference la-
boratory to ensure that no sources of contamination are present in MilliQ water used
for preparation of stock solutions. These blank samples are also used for control of the
18
-------
reference laboratory Microtox® test. If test results indicate results of toxic water sam-
ples, both the water quality in DHI DANETV Water Centre and the test set-up at the
reference laboratory will be inspected.
Luminescent bacteria test of blank samples (MilliQ water) are expected to produce
very little or no inhibition. If the inhibition is different from what the vendor suggests
for a blank sample, the analysis will be repeated once. For this re-analysis, a new
blank sample will be prepared and clean vials/containers and fresh reagents will be
used. If similar results persist, the vendor will be notified, but the verification test will
proceed.
According to ISO 11348-3 three reference substances shall be tested for each batch of
bacteria. These tests will be performed solely on the LUMIStox equipment at DHI.
The testing of the batches is related to the bacteria and not to the equipment, therefore
tests on one instrument are considered sufficient.
An overview of the reference performance control, described in Section 6.2, and the
DHI DANETV Water Centre test system, described in this section, is given in Table
6.3.
Table 6.3 Summary of reference performance control and test system control.
Information/control type
Blank samples
Reference test according to ISO 1 1348-3
Control, stock solutions
Wastewater
Quality control
Proficiency test
Reference
laboratory
Detection limit
-
Precision
Precision
Precision
Trueness
DHI
Test laboratory
Quality of MilliQ water
Test of bacteria batches
-
-
-
-
6.4 Data integrity check procedures
All transfer of data from printed media to digital form will be checked. Transfer be-
tween digital media is checked by spot check of not less than 5% of the data. If errors
are found in a spot check, all data from the transfer are checked.
6.5 Test system audits
An internal audit by DHI, following the GLP audit procedures by a trained auditor,
will be performed (see the verification protocol for details).
The Battelle Quality Manager, Zachary Willenberg, will perform a technical systems
audit (TSA) at least once during this verification and test. The purpose of this audit is
to ensure that the verification test is performed in accordance with the AMS Center
quality management plan 191, this test plan, published reference methods and any me-
thods used in the tests. In the TSA, the Battelle Quality Manager may review the ref-
erence methods used and compare actual test procedures to those specified or refe-
renced in this plan. In the TSA, the Battelle Quality Manager will observe testing in
progress, inspect documentation, and review technology-specific record books. He
will also check standard certifications and may confer with other Battelle staff. A TSA
report will be prepared, including a statement of findings and the corrective actions
19
-------
taken. The AMS Center Quality Manager, the U.S. EPA Quality Manger and the DHI
DANETV Water Centre Verification Responsible will receive a copy of Battelle's
TSA report. The TSA findings will be communicated to technical staff at the time of
the audit and documented in a TSA report.
The Battelle Quality Manager will perform an audit of data quality (ADQ). This will
be a review of data acquisition and handling procedures and an audit of at least 10% of
the data acquired in the test and verification. The Battelle Quality Manager will trace
the data from initial acquisition, through reduction and statistical comparisons, to final
reporting. All calculations performed on the data undergoing the audit will be checked.
ETV Canada is not planning for a physical audit of the test operation.
6.6 Test report review
The test report will be subject to internal review by the verification responsible from
DHI DANETV Water Centre: head of innovation Margrethe Winther-Nielsen.
U.S. EPA staff, John McKernan (JMK) and Michelle Henderson (MH), and Mona El-
Hallak (MEH) from ETV Canada will review the test report.
External review of the test report will be done by the expert group together with the
review of the verification report.
7 TEST REPORT
The test report will follow the template of the DHI DANETV verification center quali-
ty manual /10/. The test report will contain the test plan, except for this Chapter 7 on
test report format, with the data and records from the tests to be inserted as new Chap-
ter 7. For this joint verification, the principles (contents) of the U.S. EPA ETV and
ETV Canada format will be complied with as well.
One joint test report will be prepared for LUMIStox and ECLOX.
7.1 Test site report
No tests will be performed in the field. Outdoor testing will be performed at DHI la-
boratory location in H0rsholm.
7.2 Test data report
The test data will include all data recorded during the test and the data reported by the
analytical laboratories, see Appendix 6 for data forms.
7.3 Amendment report
The report section on deviations will compile all changes of this test plan occurring
before testing with justification of deviations and evaluation of any consequences for
the test data quality. A template for an amendment report is included in Appendix 8.
20
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7.4 Deviations report
The report section on deviations will compile all deviations from this test plan occur-
ring during testing with justification of deviations and evaluation of any consequences
for the test data quality. A template for the deviation report is included in Appendix 8.
21
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A P P E N D I X 1
Terms and definitions used in the test plan
22
-------
The abbreviations and definitions used in the verification test plan are summarized below.
Where discrepancies exist between DANETV and US EPA ETV terminology, definitions from
both schemes are given.
Word
ADQ
Agreement
with accepted
values
AMS Center
Analytical la-
boratory
Application
BOD5
CD
CTAB
DANAK
DANETV ETV
DS
Effect
EN
ETV
EU
Evaluation
Experts
GC
GLP
ICP
ISO
LC
LID
Limit of detec-
tion
LoD
LUMISsoft4
NOWATECH
Audit of data quality: An examination of a set
of data after is has been collected and 100%
verified by project personnel, consisting of
tracing at least 1 0% of the test data from
original recording through transferring, calcu-
lating, summarizing and reporting.
Here defined as the % agreement between
literature values and test results
Advanced Monitoring Systems Center at Bat-
telle
Independent analytical laboratory used to
analyze reference samples
The use of a product specified with respect
to matrix, target, effect and limitations
Five-day biological oxygen demand
Criterion of detection
Cetyl trimethyl ammonium bromide
The Danish Accreditation and Metrology
Fund
The Danish Centre for Verification of Climate
and Environmental Technologies
Danish Standard
The way the target is affected
European standard
Environmental technology verification (ETV)
is an independent (third party) assessment of
the performance of a technology or a product
for a specified application, under defined
conditions and adequate quality assurance.
European Union
Evaluation of test data for a technology
product for performance and data quality
Independent persons qualified on a technol-
ogy in verification or on verification as a
process
Gas chromatography
Good laboratory practice
Inductively coupled plasma
International Standardization Organization
Liquid chromatography
Lowest ineffective dilution. Often seen as the
dilution in a dilution series causing less than
20 % inhibition.
Calculated from the standard deviation of
replicate measurements at less than 5 times
the detection limit evaluated. Corresponding
to less than 5% risk of false blanks.
PC software from HACH-LANGE , produced
for LUMIStox
US ETV
EPA program that develops generic
verification protocols and verifies the
performance of innovative environ-
mental technologies that have the
potential to improve protection of hu-
man health and the environment
An examination of the efficiency of a
technology
Peer reviewers appointed for a verifi-
cation
23
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Word
LUMIStherm
LUMIStox
Matrix
Method
MS
OECD
PE
Performance
claim
Performance
parameters
Precision
(Environmen-
tal) product
QA
Range of ap-
plication
Reference
analyses
Reference test
Repeatability
Reproducibility
Robustness
RSD
SM
SS
Standard
NOWATECH
Thermostat from HACH-LANGE , produced
for LUMIStox
LUMIStox 300 bench top luminometerfrom
HACH-LANGE
The type of material that the product is in-
tended for
Generic document that provides rules, guide-
lines or characteristics for tests or analysis
Mass spectroscopy
Organisation for Economic Co-operation and
Development
Performance evaluation
The effects foreseen by the vendor on the
target (s) in the matrix of intended use
Parameters that can be documented quanti-
tatively in tests and that provide the relevant
information on the performance of an envi-
ronmental technology product
The relative standard deviation obtained
from replicate measurements, here meas-
ured under repeatability or reproducibility
conditions.
Ready to market or prototype stage product,
process, system or service based upon an
environmental technology
Quality assurance
Generally: the range from the LoD to the
highest concentration with linear response.
For this verification the range is based on
range of dilution of a test sample.
Analysis of content of compounds in stock
solutions by a specified reference methods in
an accredited (ISO 17025) laboratory.
Luminescence bacteria test performed ac-
cording to ISO 1 1348-3 by an accredited
(ISO 17025) laboratory.
The precision obtained under repeatability
conditions, that is with the same measure-
ment procedure, same operators, same
measuring system, same operating condi-
tions, and same location and system, and
replicate measurements on the same or simi-
lar objects over a short period of time.
The precision obtained under reproducibility
conditions, that is with measurements that
include different locations, operators, measur-
ing systems, and replicate measurements on
the same or similar objects
% variation in measurements resulting from
defined changes in matrix properties.
Relative standard deviation in %.
Standard method
Suspended solids
Generic document established by consensus
and approved by a recognized standardiza-
tion body that provides rules, guidelines or
USETV
(Environmental) technology
24
-------
Word
SWEDAC
Target
(Environmen-
tal) technology
Test/testing
TOC
Trueness
ISA
U.S. EPA
Vendor
Verification
Vibrio fischeri
VKI
NOWATECH
characteristics for tests or analysis
Swedish Board for Accreditation and Con-
formity Assessment
The measurable property that is affected by
the product.
The practical application of knowledge in the
environmental area
Determination of the performance of a prod-
uct by parameters defined for the application
Total organic carbon
The % recovery of true value obtained either
from knowledge on the preparation of test
solutions or from measurements with refer-
ence methods.
Technical system audit
United States Environmental Protection
Agency
The party delivering the product or service to
the customer
Evaluation of product performance parame-
ters for a specified application under defined
conditions and adequate quality assurance
Light producing bacteria used in luminescent
bacteria test
Former Danish Water Quality Institute, today
DHI
USETV
An all-inclusive term used to describe
pollution control devices and systems,
waste treatment processes and sto-
rage facilities, and site remediation
technologies and their components
that may be utilized to remove pollu-
tants or contaminants from, or to pre-
vent them from entering, the environ-
ment.
The technology developer, owner, or
licensee seeking verification
Establishing or proving the truth of the
performance of a technology under
specific, predetermined criteria, test
plans and adequate data QA proce-
dures
25
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APPENDIX 2
References
26
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1. DANETV. LUMIStox 300 Bench Top Luminometer, ECLOX Handheld Luminometer. Luminescent bac-
teria test for use in wastewater. Joint verification protocol. DHL 2009.
2. ISO 11348-3. Water quality - Determination of the inhibitory effect of water samples on the light emis-
sion of Vibrio fischeri (Luminescent bacteria test). 11348-3. 2007.
3. ISO. Water Quality - On-line sensors/analysing equipment for water - Specifications and performance
tests. ISO 15839. 2006.
4. Battelle. Environmental technology verification program. Advanced monitoring systems center. Test/QA
plan for verifications of rapid toxicity technologies. June 2003.
5. ISO. General requirements for the competence of testing and calibration laboratories. ISO 17025. 2005.
6. DHL DHI Quality Manual. 2008.
7. International Standardization Organisation. EN ISO 9001. Quality management systems - Requirements.
15-11-2008.
8. OECD. OECD Principles of Good Laboratory Practice. OECD GLP Document No. 1. 21-1-1998.
9. Battelle. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center. Version
7.0. Dated November 2008.
10. ETV Test Center and Test Organization. Center quality manual - Water technology. Version 2. October
2009.
11. ICH Harmonised Tripartite Guideline. Validation of analytical procedure: Text and methodology Q2(R1).
International conference on harmonization of technical requirements for registration of Pharmaceuticals
for human use. Current Step 4 version. November 2005.
12. ISO. Water quality - Guide to analytical quality control for water analysis. ISO/TR 13530. 1997.
13. Email correspondence between Mette Tjener Andersson, DHI and Dr. Elmar Grabert, HACH-LANGE.
Regarding: Glass and plastic vials. Dated 6. November 2009.
27
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APPENDIX 3
Reference methods
28
-------
Reference test and reference analyses are described in the test plan Section 4.3 Methods of test and
analysis.
Conductivity and salinity measurement methods are provided with the instrument.
A cyanide test kit will be used according to the method description included in the kit.
29
-------
APPENDIX 4
In-house test methods
30
-------
DHL
Laboratory protocol for verification of LUMIStox 300 Bench
Top Luminometer and ECLOX Handheld
Luminometer
1. Objective
The objective of this protocol is to describe in detail the work to be carried out for the verification
of LUMIStox 300 Bench Top Luminometer and ECLOX Handheld Luminometer in accordance
with the verification protocol III and test plan 111.
2. Identification
Project No.: 11800378-2
3. Vendor
HACH-LANGE GmbH,
Willstatterstrasse 11, 40549 Dusseldorf,
Germany,
phone+49 211 52880.
Contact
Dr. Elmar Grabert
email: elmar.grabert@hach-lange.de,
phone+49 211 5288241.
4. Test facility
DHI
Agern Alle 5
DK-2970 H0rsholm
Denmark
5. Personnel responsible for the test
Test responsible: Claus J0rgensen
Technicians: Connie Seier0
Jane Bergstram
31
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6.
Instruments to be tested
The test covers two instruments from the same vendor, both instruments determine acute toxici-
ty with luminescent bacteria. The instruments are LUMIStox 300 bench top luminometer and
ECLOX handheld luminometer. Both will be operated in connection with a LUMIStherm
thermostat and the PC software LUMISsoft4 ver 1.001 /8/ except for test series E where the
ECLOX will be operated with the firm ware.
7. Safety handling
The test compounds will be handled in accordance with the MSDSs which are available to the
technicians.
8. Test principle
To verify the instruments the following performance parameters will be analysed:
The criterion of detection (CD)
The range of application
Precision
- repeatability
- reproducibility
Agreement with accepted values
Robustness
The tests will be performed in a series of experiments according to the test plan 111:
Test series.
A
B
C
D
E
F
G
H
1
J+K
L
Performance parameters
Range, Repeatability, Agreement with accepted values
Criterion of detection
Robustness, effect of start cone, on repeatability
Reproducibility
Robustness, sample temperature at field use
Robustness, sample temperature at laboratory use
Robustness, pH
Robustness, color
Robustness, turbidity
Robustness, matrix
Robustness, cuvettes
Equipment
LUMIStox
X
X
X
X
X
X
X
X
X
X
ECLOX incl.
thermostat and
software
X
X
X
X
X
X
X
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32
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9. Procedure
9.1 Start up procedure for LUMIStox 300.
Follow the procedure in the LUMIStox 300 operation manual /3/ page 6.
Perform daily temperature control of the LUMIStherm heating block(s) (se section 0).
Adjust the measuring shaft temperature according to section 3.7 of the operating manual /3/.
9.2 Start up procedure for ECLOX
Follow the procedure in the ECLOX user manual /4/ page 7.
Ensure that the temperature of the LUMIStherm heating block(s) is set to 15 °C.
9.3 Temperature control of LUMIStherm
9.3.1 Initial temperature control
The three LUMIStherm thermo blocks will initially be tested for temperature variation at 15 °C
in all wells. A high quality traceable calibrated thermo sensor will be used with a precision of
0.1 °C.
1. Mark the three LUMIStherms A, B, and C respectively.
2. Switch on the LUMIStherms
3. Insert plastic vials (0) in all small wells (Al to CIO) and add 1 mL of sodium chloride
solution (0). Insert reaction vials (0) in the two large right hand side wells and add 5 mL
of sodium chloride solution (0). Wait 15 minutes to allow for temperature equilibration.
4. Temperature equilibrate the thermo sensor in one of the wells.
5. Measure the temperature in all wells and record in a spread sheet.
6. Determine the Taverage, Tmax, Tmin and Tmedian temperature of the small wells for each
LUMIStherm. The temperature will be accepted if all wells are within 15 °C ± 0.8 °C.
Determine the temperature interval between max temperature and 16.0 °C ^T max), and
the temperature interval between min temperature and 14 °C (ATm;n)
7. Identify the small well with the median temperature.
8. Determine the temperature variation in well 5B over a period of 1.5 hours. A variation
of ± 0.3 °C is acceptable.
9.3.2 Daily temperature control
Determine and record on each day of operation, the temperature in the median temperature
well. The temperature will be accepted if the temperature is within the range between Tmedian +
AI max and 1 median ~ AI min-
33
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9.4 Storage and preparation of suspensions of luminescent bacteria
(Vibrio flscheri NRRL-B-11177).
9.4.1 Storage
The freeze-dried bacteria can be stored at -18 °C until the date shown on the package.
Reactivated bacteria should be used within 4 hours when possible. However longer storage
time is acceptable as long as the validity criteria stated in clause 11 of EN/ISO 11348-3 161 are
met. Reactivated bacteria should only be placed in temporary storage under undiluted condi-
tion. Tubes containing thawed but not reactivated freeze-dried bacteria can be refrozen. 151.
9.4.2 Preparation of stock suspension
(According to EN/ISO 11348-3: 2007 /6/.)
Remove the vial of the freeze-dried culture from the freezer immediately before reconstitution
in water. For the reconstitution, cool 1.2 mL of reconstitution solution LCX 047 (0) in a glass
testtubeto4°C±3°C.
Pour this volume of cooled water all at once into the lyophilized bacteria in the vial, thereby
minimizing cell damage during the rehydration process.
It is important that the water be added quickly to allow the bacteria to come into contact with
the water at once, thus avoiding clumping and loss of activity. Therefore do not use a pipette.
The exact volume of water is not critical.
The reconstituted luminescent bacteria suspension serves as a stock suspension; store at 4 °C ±
3°C.
9.4.3 Preparation of test suspension
(According to EN/ISO 11348-3:2007, variant B 161.)
The test suspension will be prepared outside the test tubes in a conical flask (volume e.g. 250
mL).
Ad 1 volume of stock suspension (0) to 50 volumes of the solution for freeze-dried bacteria (0)
maintained at 4 °C ± 3 °C and mix the resultant suspension thoroughly.
9.4.4 Quality control of test bacteria
All batches of bacteria must be controlled according to clause 11 of EN/ISO 11348-3 161. The
tests will be carried out on the first day of use of the specific bacterial batch.
Each stock suspension will be controlled as described in clause 11 of EN/ISO 11348-3 161. The
reference substance will be selected on the basis of preliminary test results.
9.5 Sample preparation
Samples made by adding test chemicals to sodium chloride solution (0) are called "artificial
samples" in this protocol.
34
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Measure the oxygen concentration in all samples. A concentration > 3 mg/L will be accepted.
161. Aerate if the concentration is < 3 mg/L
Measure the pH of all samples. If necessary adjust the pH with either HC1 (0) or NaOH (0).
Record the volumes used for pH adjustment. Restrict the volume added to no more than 5 % of
the total volume 161.
All artificial samples will be adjusted to pH 7.0 ± 0.2.
Waste water samples will be adjusted to be between pH 6.0 ± 0.2 and pH 8.5 ± 0.2 in agree-
ment with EN/ISO 11348-3: 2007 161
The salt concentration of the sample will be increased to 2 % by adding solid NaCl. For exam-
ple 2 g pr 100 mL of sample. /3/ If the salt concentration in the sample exceeds 20 g/L (guide
value: conductivity of 35 mS/cm) do not add NaCl. The salt content should not exceed 50 g/L.
151
9.6 Preparation of sample dilution rows
Dilution rows will be used in test series A, C, D, and K.
A dilution row will be produced in accordance with the standard dilution row described in the
LUMIStox 300 Operation manual page 33 /3/. The principle is illustrated in figure 9.1.
1,5 1,5 1,5 1,5 1,5 1,5 1,5 1,5 1,0 0,0 ml
1.QQQQQQ
1,5 1.5 1,5 1.5 1.5 1,5 1,5
2. DD
/'^r~""~^/^!T^.x/i
3. ~ ~
4.
Figure 9.1: Principles of preparation of dilution rows from /3/.
35
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1. Insert 10 vials into row A and pipet LCK 481 sodium chloride solution (0) into the vials
according to figure 9.1, i.e. 1.5 mL in position Al to A8 of the LUMIStherm thermo
block and 1 mL in position A9.
2. Add 1.5 mL sample into the vial in position A10, 2 mL sample in the vial in position A9
and 1.5 mL sample in the vial in position A8.
3. Pipet 1.5 mL from the vial in position A9 to the vial in position A7 and mix thoroughly
drawing the mixture into the pipette 3 times. Continue by pipetting 1.5 mL into the vials
in the positions A5 and A3 as illustrated in figure 9.1.
4. Pipet 1.5 mL from the vial in position A8 to the vial in position A6 and mix thoroughly
drawing the mixture into the pipette 3 times. Continue by pipetting 1.5 mL into the vials
in the positions A4 and A2 as illustrated in figure 9.1.
Leave the dilutions in 15 minutes in the LUMIStherm thermo block to bring them to the correct
temperature.
9.7 Test procedure
9.7.1 Determining inhibition under lab conditions
Connect the LUMIStox 300 and the ECLOX to the computers. Switch on the computers.
Switch on the LUMIStox 300 and the ECLOX. Switch on the LUMIStherm thermo block(s).
Allow 30 minutes for equilibration.
Prepare the dilution row as described in (0) or samples as described in (0). Prepare the test sus-
pension as described in (0).
Use plastic measuring tubes (0) except in test series L, where both plastic tubes and glass tubes
0 will be used.
1. Insert the appropriate number of plastic measuring tubes (0) in rows B and C.
2. Pipette 0.5 mL bacteria test suspension (0) into the measuring tubes and leave 15 mi-
nutes to acquire the correct temperature.
3. Open the LUMISsoft software and enter information on the samples to be analysed ac-
cording to the LUMISsoft manual p. 16-27 /8/.
4. Measure the initial luminescence in the vial in position Bl first on LUMIStox 300 then
on ECLOX.
5. Measure the initial luminescence in the vial in position Cl first on LUMIStox 300 then
on ECLOX. During the measurement of vial Cl add 0.5 mL of diluted sample from po-
sition Al into the measuring vial in position Bl and mix 3 times with the pipette.
6. Measure the initial luminescence in the vial in position B2 first on LUMIStox 300 then
on ECLOX. During the measurement of vial B2 add 0.5 mL of diluted sample from po-
sition Al into measuring tube Cl and mix 3 times with the pipette. Continue until all
measuring tubes have been measured and added sample. There is no need for changing
pipette tips except for the control.
7. After 15 minutes calculated from the time of the first reading, determine the lumines-
cence in the measuring tube Blfirst on the LUMIStox 300 then on the ECLOX. Meas-
ure the luminescence in the measuring tube Cl after the selected time interval (
Continue to measure the luminescence in the remaining measuring tubes.
8. Repeat 7 after 30 minutes after the first reading.
36
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9.7.2 Determining inhibition under field conditions
Follow the instructions in the ECLOX user manual pages 19-21.
10. Reagents and test tubes
10.1 Sodium chloride solution.
Dissolve 20 g of sodium chloride (NaCl) in MQ-water and make up to 1 L with MQ-water.
Store at 4 °C to 8 °C.
10.2 Hach-Lange sodium chloride solution (LCK 481)
Sodium choride solution (2 %) delivered by Hach-Lange. no. 10159, exp. date 10.2010. Store
at 4 °C to 8 °C.
10.3 Hach-Lange reconstitution solution (LCX 047)
Reconstitution solution after EN/ISO 11348-3 delivered by Hach-Lange. No. 04179, exp. date
10.2010. Store at 4 °C to 8 °C.
10.4 Sodium hydroxide solution
NaOH in MQ-water, 1 mol/L or another suitable concentration.
10.5 Hydrochloric acid
HC1 in MQ-water, 1 mol/L or another suitable concentration.
10.6 Hach-Lange test suspension solution for freeze-dried bacteria
(LCX 048)
Diluent after EN/ISO 11348-3 delivered by Hach-Lange. No. 10309, exp. date 10.2010. Store at
4 °C to 8 °C.
10.7 Reference substances
Do not adjust pH of the reference substance solutions.
10.7.1 Zinc sulphate heptahydrate
19.34 mg/L ZnSO4- 7 H2O in 2 % sodium chloride solution (0) .
10.7.2 3,5 - dichlorophenol
6.8 mg/L 3,5 - dichlorophenol (Purity > 99%) in 2 % sodium chloride solution (0) .
Potasium dichromate
105.8 mg/L K2Cr2O7 in 2 % sodium chloride solution (0).
37
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10.8 Test tubes
10.8.1 Plastic test tubes
Sarstedt tubes 3.5 mL, 55 x 12 mm, PS. ref no. 55.485. Delivered by Hach Lange
10.8.2 Glas test tubes
LZP 187 Glaskuvetten fur LUMIStox AR-Klar. 50.0 X 12.0/0.60 mm. Delivered by Hach
Lange.
10.8.3 Reaction vials with cap.
LZP 065 Reaktionsglaser mit verschluss, delivered by Hach-Lange.
11. Test setup
Generally, tests will be run in triplicate, i.e. three rows of dilution will be prepared from the
same artificial sample and tested in separate test runs. Each test run will be performed in dupli-
cate (i.e. row B and C). All test runs will include a blank consisting of 0.5 mL of test suspen-
sion (0) and 0.5 mL of sodium chloride solution (0).
Readings will be done after 15 minutes and 30 minutes.
If there is a visible colour at the EC20 concentration, colour correction will be applied.
11.1
Test series A
11.1.1 Purpose
To analyse range, repeatability and agreement with accepted values of EC20 and
11.1.2 Tests to be performed
EC2o and ECso will be determined on artificial samples made in sodium chloride solution (0)
with the compounds shown in table 11.1.
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
Table 11.1: Compounds to be tested in test series A
CAS no.
7758-99-8
7778-50-9
7446-20-0
76674-21-0
3380-34-5
151-50-8
151-21-3
57-09-0
104-35-8
Compound
CuSO4,5H2O
K2Cr2O7
ZnSO4,7H2O
Flutriafol
Triclosan
KCN
SDS
CTAB
4-NPE
Expected ECso (mg/L)
7.1 asCu2+
18.7asCr+b
2.2 as Zn2+
unknown
0.28
4 as CIST
2.09
0.97
unknown
38
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For KCN, a pre-experiment will be performed to examine evaporation of HCN. An artificial
KCN sample will be carried through the test procedure where the test suspension will be ex-
changed with the solution for freeze-dried bacteria (0) and without performing measurements
of luminescence. Instead the CN" concentration will be measured using a Hach-Lange test
(LCK 315). Test row B will be analysed at time 0 and test row C will be analysed after time 30
minutes. In addition, the concentration of the artificial KCN sample will be analysed. If the de-
crease in the average CN" concentration from time 0 to time 30 is higher than 20%, then the
KCN test will be aborted.
11.1.3 Sampling for chemical analyses
Samples for chemical analysis of CuSO/i, K2Cr2O7, ZnSO/i, KCN, Flutriafol and Triclosan will
be taken in duplicate from the prepared artificial samples, and shipped to the analytical labora-
tory.
SDS, CTAB and 4-NPE are expected to adsorb to the measurement tubes. Therefore samples
for chemical analysis will be prepared by adding 1.5 mL of artificial sample and 1.5 mL of so-
lution for freeze-dried bacteria (0) in each of 10 plastic tubes (0) at 15 °C and mixed three times
with the pipette. The mixtures will then be poured to glass sample containers. Only one sample
will be analysed pr. compound.
Performance control of Eurofms analysis will be performed by sending 2 blanks (MilliQ water)
to analysis for each of the target compounds.
Samples will be transferred to sample containers delivered by Eurofms.
Sample labeling will be coded.
11.1.4 Sampling for toxicity analysis at AlControl
Artificial samples will be taken for all target compounds except KCN. One of the samples will
be analyzed three times. KCN is exempted to avoid complications related to shipment and han-
dling by AlControl.
Two samples of 2 % NaCl solution (0) will be taken to ensure non-toxicity.
The samples are frozen at - 20 °C ± 3 °C and send to Alcontrol after the last sample is taken.
Sample labeling will be coded.
11.2 Test series B
11.2.1 Purpose
To determine criterion of detection.
11.2.2 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
A number (> 9) of test mixtures of 0.5 mL of 2% NaCl (0) and 0.5 mL test suspension (0) will
be measured in duplicate.
39
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11.3
Test series C
11.3.1 Purpose
To determine robustness of determination of ECso and EC20 in relation to the concentration.
11.3.2 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
A test compound and the concentration ranges will be decided after completion of series A.
The first concentration range will have the highest test concentration at approximately ECeo-
The second concentration range will have the highest test concentration at approximately
11.4
Test series D
11.4.1 Purpose
To determine reproducibility.
11.4.2 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
One test compound and the concentration range will be selected after completion of series A.
The reproducibility parameters will be: different days, different technicians, and different
batches of test bacteria according to Table 7-1.
Table 7-1: Variation of reproducibility parameters
Day
1
2
3
*4
Bacterial batch
A
B
C
D
Technician
J
C
J
C
*Will only be performed if a bacterial fourth batch is made available.
11.5
Test series E
11.5.1 Purpose
To determine robustness of the ECLOX instrument at different temperatures.
11.5.2 Tests to be performed
Tests will be performed on the ECLOX instrument with firmware according to procedure de-
scribed in the ECLOX user manual /4/ pages 19 to 21.
Based on the results obtained in series A, two compounds will be selected for test: one metal
and one organic compound. Each compound will be tested in triplicate and at three different
40
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temperatures: One at room temperature, one in a climate room at approximately 15 °C and one
at 0 °C to 6 °C, preferable outdoor, otherwise in cooler room.
The test setup is illustrated in Table 7-2. The concentration of the test compound in the test
sample shall be twice the ECso.
Table 7-2: Test setup for series E
Tube
1
2
3
4
Test suspension (0)
(mL)
0.2
0.2
0.2
0.2
2% NaCl (0)
(mL)
0.8
0.6
0.3
none
Sample
(mL)
none
0.2
0.5
0.8
Each of the two test compounds will be tested in triplicate.
The room temperature will be recorded.
11.6 Test series F
11.6.1 Purpose
To determine robustness at different sample temperatures.
11.6.2 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
The test compound will be selected based on results obtained in previous tests.
Adjust two of LUMIStherm thermo blocks to approximately 14 °C and 16 °C, respectively, af-
ter the procedure described in section 0. Adjustment of the temperature is done by turning the
"Cal." screw. It may not be possible to reach 14 °C and 16 °C. In this case maximum and min-
imum temperature adjustments will be selected.
The tests will be run at 14 °C, 15 °C and 16 °C at EC2o in triplicate.
The test will be performed as the last test to avoid temperature variations over the test series.
11.7 Test series G
11.7.1 Purpose
To determine robustness at different pH.
11.7.2 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
The test compound will be selected based on results obtained in previous tests.
The test will be performed at EC20 in triplicate.
41
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A stock solution of the test compound at a concentration corresponding to twice the £€20 will
be prepared and separated into three separate artificial samples, which will be adjusted to pH
6.0 ± 0.2, 7.0 ± 0.2, or 8.5 ± 0.2 respectively with either HC1 (0) or NaOH (0) and tested.
11.8 Test series H
11.8.1 Purpose
To determine robustness in relation to colour.
11.8.2 Tests to be performed
11.8.2.1 Screening of toxicity of dyes to determine dye test concentrations.
An artificial sample will be made in sodium chloride solution (0) as a mixture of 20 mg/L of
Ponceau 4R (E124), 20 mg/L of Green S (E142) and 20 mg/L of Yellow no.5 (E102). Alterna-
tive concentrations may be used if appropriate.
on this sample will be determined on the LUMIStox 300 with the colour correction fea-
ture switched on. See page 23 of the LUMIStox user manual /3/ and pages 70 to 77 of the LU-
MlSsoft 4 manual /8/.
The test data will be analysed with and without colour correction. A range of concentrations
with colour correction and without toxicity will be determined and used to define the dye con-
centrations to be used in the subsequent test.
11.8.2.2 Colour robustness on LUMIStox 300.
1 1 .8.2.2. 1 Preparation of test samples
A stock solution in sodium chloride (0) with an appropriate concentration of the selected test
compound will be made. An appropriate volume of the stock solution will be added to each of
three 100 mL measuring flasks to achieve a concentration of the test compound corresponding
to twice the EC 20 in the final test sample. Varying volumes of sodium chloride solution (0) and
the dye solution described in section 0 will be added to achieve the dye concentrations deter-
mined in the screening test in section 0.
1 1 .8.2.2.2 Preparation of dye control samples
The dye control samples will be made as the test samples (0) except that the stock solution will
be left out and replaced by sodium chloride solution (0).
1 1 .8.2.2.3 Preparation of test compound control samples
The test compound control samples will be made as the test samples (0) except that the dye so-
lution will be left out and replaced by sodium chloride solution (0).
11.8.2.2.4 Test setup
The test will be performed on LUMIStox 300 incl. thermostat and software.
The test will be performed in triplicate each with a blank consisting of 0.5 mL of test suspen-
sion (0) and 0.5 mL of sodium chloride solution (0), a dye control (0) and a test compound con-
trol (0) . Each triplicate in will be analysed in duplicate.
42
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11.8.2.3 Colour robustness on ECLOX.
Tests will be performed on ECLOX incl. thermostat and software with colour correction ac-
cording to EN/ISO 11348-3.
The tests will be performed on the same samples as used for the LUMIStox 300 (0).
11.9 Test series I
11.9.1 Purpose
To determine robustness in relation to turbidity.
11.9.2 Screening of toxicity of BaSO4.
This screening test will be run to ensure that BaSCu is non-toxic.
A volume of 10 mL of a 0.2 g/L of BaSCu in sodium chloride solution (0) will be centrifuged
10 minutes at approx. 5000 g.
The inhibitory effect of the supernatant will be determined in 5 duplicate tests with 5 blanks run
in the same rows. Readings after 15 minutes and 30 minutes.
If the average inhibition is significant higher than the CD determined in section 0 an alternative
turbidity sample will be selected. If the alternative also shows inhibition, then the test for tur-
bidity robustness will not be carried out.
11.9.3 Tests to be performed
11.9.3.1 Turbidity robustness on LUMIStox 300.
11.9.3.1.1 Preparation of test samples
A stock solution in sodium chloride (0) with an appropriate concentration of the selected test
compound will be made. An appropriate volume of the stock solution will be added to each of
three 100 mL measuring flasks to achieve a concentration of the test compound corresponding
to twice the EC20 in the final test sample. Varying volumes of sodium chloride solution (0) and
a 1 g/L BaSC>4 in sodium chloride solution (0) will be added to achieve final BaSC>4 concentra-
tions of 0.2 mg/L, 0.1 mg/L and 0.05 mg/L.
11.9.3.1.2 Preparation of turbidity control samples
The turbidity control samples will be made as the test samples (0) except that the stock solution
will be left out and replaced by sodium chloride solution (0).
11.9.3.1.3 Preparation of test compound control samples
The test compound control samples will be made as the test samples (0) except that the BaSC>4
suspension will be left out and replaced by sodium chloride solution (0).
11.9.3.1.4 Test setup
The test will be performed on LUMIStox 300 incl. thermostat and software.
43
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The test will be performed in triplicate each with a blank consisting of 0.5 mL of test suspen-
sion (0) and 0.5 mL of sodium chloride solution (0), a turbidity control (0) and a test compound
control (0). Each triplicate in will be analysed in duplicate.
11.9.3.2 Turbidity robustness on ECLOX.
Tests will be performed on ECLOX incl. thermostat and software with colour correction ac-
cording to EN/ISO 11348-3.
The tests will be performed on the same samples as used for the LUMIStox 300 (0).
11.10 Test series J
This test series will be performed after series K.
11.10.1 Purpose
To determine robustness in relation to the matrix.
11.10.2 Waste water samples
See section 0.
11.10.3 Preparation of test samples
11.10.3.1 Preparation of waste water test samples
If the waste water samples are found to be toxic, they will be diluted to non-toxicity level and
otherwise handled as described in section 0.
Five test compounds will be selected based on results obtained in previous tests.
For each compound a stock solution in sodium chloride solution (0) with a concentration cor-
responding to 4 times the £€20 will be made. Waste water test samples will be made by mixing
1 part of waste water samples with 1 part of the 4 times £€20 solutions.
11.10.3.2 Preparation of test compound control samples
Test compound control samples will be made by mixing 1 part of sodium chloride solution (0)
with 1 part of the 4 times £€20 solutions.
11.10.3.3 Preparation of waste water control samples
The waste water control samples will be made by mixing 1 part of sodium chloride solution (0)
with 1 part of the waste water sample.
11.10.3.4 Sampling for toxicity analysis at AlControl
Samples of one spiked, non-inhibiting domestic wastewater and one spiked, non-inhibiting in-
dustrial wastewater. Three replicates will be performed for one of the wastewater samples.
The samples are frozen at - 20 °C ± 3 °C and send to Alcontrol after the last sample is taken.
Sample labeling will be coded.
44
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11.10.4 Tests to be performed
The test will be performed on LUMIStox 300 and ECLOX incl. thermostat and software.
The test will be performed in triplicate each with a blank consisting of 0.5 mL of test suspen-
sion (0) and 0.5 mL of sodium chloride solution (0), a test compound control (0) and a waste
water control (0). Each triplicate in will be analysed in duplicate.
11.11 Test series K
This test series will be performed before test series J.
11.11.1 Purpose
To determine robustness in relation to the matrix.
11.11.2 Waste water samples
Treated industrial waste water (2 times 5 L in pp-plastic containers) was received from Che-
mionova on December 7, 2009. The samples were cool upon arrival and stored at 4 °C ± 2 °C
in the cooling room. The samples were marked with 0T-nr.: 09-0834, A and B respectively.
Treated domestic waste water will be obtained from Lundtofte Waste Water Treatment Plant.
11.11.2.1 Sampling for chemical analyses
Single samples for chemical analysis of the waste water will be taken prior to the test in the
sample containers provided by Eurofms. The analytical parameters are shown in Table 4.1.
Table 7-3 Analytical parameters for waste water.
Turbidity
TOC
Conductivity
Alkalinity
PH
COD
Suspended solids (SS)
Nitrogen (total)
Phosphorus (total)
BOD5
11.11.3 Preparation of waste water samples
The samples will be handled as described in section 0.
11.11.4 Tests to be performed
The test will be performed on LUMIStox 300 incl. thermostat and software with the colour cor-
rection feature switched on.
Test will also be performed on ECLOX incl. thermostat and software. If a significant effect of
colour correction is observed on the LUMIStox 300, colour correction according to ISO 11348-
3 will be performed.
11.12 Test series L
11.12.1 Purpose
45
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To determine robustness in relation to use of different measuring cuvettes
11.12.2 Tests to be performed
The test will be performed on LUMIStox 300 incl. thermostat and software.
Two test compounds will be selected based on results obtained in previous tests. One com-
pound will be selected among the compounds expected to adsorb to the plastic cuvette (SDS,
CTAB or 4-NPE) and one compound will be selected among the compounds not expected to
adsorb to the plastic cuvette (Cu2+, C^O?2" or Zn2+)
The test will be performed at EC20, i.e. at a sample concentration corresponding to twice the
EC20- The test will be run in 3 glass test tubes (0) with samples and 3 corresponding blanks (0)
in glass test tubes and in 3 plastic test tubes (0) with samples and 3 corresponding blanks (0) in
plastic tubes. The test will be performed three times in duplicate.
12. Data to be recorded
All measurements of luminescence will be recorded electronically on the PCs connected to the
instruments. At the end of a test day a copy of the data will be placed on the DHI server at
\\Dkstor\l 1800378_DAN_ETV\DHI delcenter\Verifikationer\HachLange\DHI laborato-
ry\results in separate folders named by the date (YYYY-MM-DD). In the test series E, data will
be retrieved and stored electronically in a folder named "series E".
The format of hard copies of the raw data will be decided at a later stage.
Data from initial and daily temperature control including will be recorded.
For each toxicity test, the following information will be recorded when relevant:
Date and time,
Test series, samples including controls and concentrations of test compounds,
Initials of the performing technician,
Bacterial batch, date and time of reconstitution and related quality control data,
Pipettes used,
pH of sample, pH meter used, pH adjustment,
Salinity, conductivity meter used,
Oxygen saturation, oxygen electrode used,
Stock solutions used.
46
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13. Time schedule
Tests will be started 2010.01.13 and will go on for 4 weeks.
The planned sequence of the tests and expected days required is shown in Table 7-4. It is antic-
ipated that approximately half of the time requires two technicians.
Table 7-4: Planned sequence of testing and expected time required
Series L
Series B
Series A
Series G+H+I
Series D
Series K
Series D
Series J
Series D
Series C
Series E
Series F
1
0.1
3
1
1
1
1
1
1
1
1
1
13
14. Quality Assurance
The quality assurance will be performed in accordance with the joint verification protocol III
15. Reports
Reporting will be performed in accordance with the joint test plan 111
16. Archives
All data generated and all other records and information relevant to the quality and integrity of
the study will be retained. They will be filed in the archives of DHI after termination of the
study and retained for a period of 10 years after issue of the final report.
17. Deviations and protocol amendments
Deviations and amendments will be handled in accordance with the joint test plan 111
47
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18. References
III LUMIStox 300 Bench Top Luminometer ECLOX Handheld Luminometer. Joint verifi-
cation protocol. Luminescent bacteria test for use in wastewater. December 2009. Mette
Tjener Andersson. DHL Project no. 11800378
121 LUMIStox 300 Bench Top Luminometer ECLOX Handheld Luminometer Joint test
plan. Luminescent bacteria test for use in wastewater. Claus J0rgensen and Mette Tjener
Andersson. Project no. 11800378.
/3/ LUMIStox 300. Manual. Hach Lange. January 2008. Version 3.02 and above. BDA 356.
/4/ Luminiscent bacteria test using the ECLOX Instrument. User manual. September 2009,
Edition beta 2. Hach Company.
151 Luminiscent bacteria test with freeze-dried bacteria according to EN/ISO 11348-3. Dr.
Lange. Luminiscent bacteria test LCK 491.
161 EN/ISO 11348-3:2007(E). Water Quality - Determination of the inhibitory effect of wa-
ter samples on the light emission of Vibriofischri (Luminiscent bacteria test). Part 3: Me-
thod using freeze-dried bacteria.
Ill Luminiscent bacteria test with freeze-dried bacteria according to EN/ISO 11348-3. Dr.
Lange. Luminiscent bacteria test LCK 491.
/8/ Dr. Lange LUMISsoft 4 Manual LZV 093, ver 1.001.
48
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PROTOCOL APPROVAL
Issued by Date:
Claus J0rgensen
Test Responsible
Concurred by Date:
Bodil Mose Pedersen
Quality Assurance Unit
Protocol copy no. of 3
49
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A P P E N D I X 5
In-house analytical methods
50
-------
None
51
-------
A P P E N D I X 6
Data reporting forms
52
-------
Test A
LUMIStox
[Test time: | 15 / 30 minutes ~|
Compound
1
Cr(VI)
2
Cu
3
Zn
4
Pesticide
5
Triclosan
6
Cyanide
7
Nonylphenol ethoxylate
8
SDS
9
CTAB
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
53
-------
Test A
ECLOX
[Test time: | 15 / 30 minutes ~|
Compound
1
Cr(VI)
2
Cu
3
Zn
4
Pesticide
5
Triclosan
6
Cyanide
7
Nonylphenol
8
SDS
9
CTAB
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
54
-------
TestB
iTesttime: 15 / 30 minutes
Batch No.
LUMIStox
ECLOX
No.
1
2
3
4
5
6
7
8
9
Mesurement
% inhibition
No.
1
2
3
4
5
6
7
8
9
Mesurement
% inhibition
55
-------
TestC
LUMIStox
|lest time:
15 / 30 minutes
Start concentration
~EC 60
Actual start cone:
rriR/L
-EC30
Actual start cone:
rriR/L
Replicate
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
ECLOX
Start concentration
~EC 60
Actual start cone:
rriR/L
-EC30
Actual start cone:
rriR/L
Replicate
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
56
-------
Test D
LUMIStox
[Test time: 15 / 30 minutes |
Day No.
1
2
3
4
Date
Replicates
1
2
3
1
2
3
1
2
3
1
2
3
Batch no.
EC20 (mg/L)
EC50(mg/L) |
Results from test A
ECLOX
Day No.
1
2
3
4
Date
Replicates
1
2
3
1
2
3
1
2
3
1
2
3
Batch no.
EC20 (mg/L)
EC50(mg/L) |
Results from test A
57
-------
TestE
ECLOX
Test time 15 minutes
[Compound 1:
Batch No.:
1. replicate
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
2. replicate
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
3. replicate
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
[Compound 2:
1. replicate
Batch No.:
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
2. replicate
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
3. replicate
Tube
1
2
3
4
Bacteria susp. (ml)
0.2
0.2
0.2
0.2
2% NaCI (ml)
0.8
0.6
0.3
no
Sample (ml)
no
0.2
0.5
0.8
% inhibition
n.a.
Sample cone.
non-toxic ref.
App. sample cone.
n.a.
20%
50%
80%
58
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TestF
Test time:
Test compound:
15 / 30 minutes
|Conc~EC20
Actual start cone:
LUMIStox
Temp. (°C)
15
Replicate
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
ECLOX
Temp. (°C)
15
Replicate
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
59
-------
TestG
Test time: 15 / 30 minutes
Test compound:
|Conc~EC20
Actual start cone:
LUMIStox
PH
6.0
7.0
8.5
Replicate
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
ECLOX
PH
6.0
7.0
8.5
Replicate
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
60
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TestH
Test time: 15 / 30 minutes
Test compound:
|Conc~EC20
Actual start cone:
LUMIStox
Color cone.
No
Blind (no sample)
Medium color
Intensity (nm)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
ECLOX
Color cone.
No
Blind (no sample)
Medium color
Intensity (nm)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
61
-------
Test I
Test time: 15 / 30 minutes
Test compound:
|Conc~EC20
Actual start cone:
LUMIStox
Turbide cone.
No
Blind (no sample)
Medium turbidity
Turbidity
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
ECLOX
Turbide cone.
No
Blind (no sample)
Medium turbidity
Turbidity
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
62
-------
TestJ
LUMIStox
Domestic wastewater
Bacteria batches as in Test A
Cone~EC 20
[lest time: 15 / 30 minutes
Compound
Cone (mg/L)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
Industrial wastewater
Compound
Cone (mg/L)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
63
-------
TestJ
ECLOX
Domestic wastewater
Bacteria batches as in Test A
Cone~EC 20
[lest time: 15 / 30 minutes
Compound
Cone (mg/L)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
Industrial wastewater
Compound
Cone (mg/L)
Replicate
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
Batch No.
% inhibition
64
-------
TestK
LUMIStox
Bacteria batches as in Test J
|Mark test time: | 15 / 30 minutes
Wastewater
Domestic
Industrial
Replicate
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
ECLOX
Wastewater
Domestic
Industrial
Replicate
1
2
3
1
2
3
Batch No.
EC20 (mg/L)
EC50 (mg/L)
65
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TestL
Compound 1
Cone ~EC 20
Mark test time:
15 / 30 minutes
Test compound 1:
Actual start cone:
LUMIStox
Cuvette
Glass
Plastic
Replicate
1
2
3
1
2
3
Batch No.
% inhibition
Compound 2
Cone ~EC 20
Mark test time:
15 / 30 minutes
Test compound 2:
Actual start cone:
LUMIStox
Cuvette
Glass
Plastic
Replicate
1
2
3
1
2
3
Batch No.
% inhibition
66
-------
A P P E N D I X 7
Data management
67
-------
In general, the data filing and archiving procedures of the DHI Quality Management
System will be followed.
All data recording and reporting is done in English, communication with Danish ex-
ternal and internal can be in Danish.
Data storage, transfer and control
The data to be compiled and stored are summarized in Table 5.1, in Section 5.1 Data
storage, transfer and control.
Analytical raw data will be filed and archived according to the specifications of the la-
boratories' quality management systems under their ISO 17025 accreditation and are
thus not the concern of DHI staff.
Implementation
All e-mail communication is filed in the Outlook Exchange folders, see below struc-
ture.
The DHI person receiving an e-mail (to field, not cc field) will file the e-mail. The
DHI person sending an e-mail will use the "send and file" option and thereby ensure
prompt filing of all e-mails sent. There is generally no need to widespread cc when
sending e-mails, unless specific action or communication is required.
All paper communication is immediately filed in the binder established by Mette Tjen-
er Andersson (MTA) and available in her office. The title page of the binder will re-
semble the folder structure in Outlook Exchange, see below.
All recordings during testing in the laboratory or outdoors are done in water proof
writing in hardback log-books with all pages numbered page/total page number. The
log books are filed with the staff member using them until the testing is completed,
then with Claus J0rgensen (CLJ), and will be available at his office.
All data needed for the tests are recorded in the data sheets available from Appendix 6
of the Test Plan. The format can be Word tables, Excel worksheets or paper sheets as
decided by CLJ as test responsible. The outline and format are mandatory and can on-
ly be deviated from by recording a deviation with justification, see the Test Plan.
All calculations are done using Excel spreadsheets with names identifying the contents
and with headings and notes explaining the calculations.
All electronic files are stored at dkstor in the folder structure shown below. File names
are constructed to identify the contents. Subfolders can be established as found conve-
nient, while again constructing folder names that identify the contents. When working
away from a network connection (offline), copies of files can be used on personal PCs,
but the server version is updated and the offline version deleted immediately after re-
turning to the network connection.
68
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dk stor:
Network > dkstor > 11800378DANETV
DHI delcenter > Verifikationer > HachLange
Favorite
g M..
M,,
Folders
.1 D -
^Cl D
i
ft
Name
Background material
Contract HACH-LANGE
COP15
Diverse
Hach Lange input to testplan
obsolete
Plan and protecol
Process documnet Battelle
Project management
Quick Scan
Reference tests and analyses
US CAN DK contract
Test and verification reports
Date modified
30-10-200914:55
27-10-200910:00
27-10-2009 09:55
02-11-2009 10:46
27-10-200916:08
19-10-200909:24
02-11-2009 09:01
02-11-200907:58
27-10-200909:31
20-10-2009 14:29
02-11-200910:46
19-10-2009 09:24
02-11-2009 13:09
Outlook Exchange:
Folder List
All Folders
All Outlook Items
d 14 11800378 - GTS EU ETV initiative'
L^J 1 Administration SL Control
Cj 2 Client Communication
±1 L^j 3 Other External Communic:
L^ 4 Internal Communication
L^ 5 Proposal & Bid Material
Qj 6 Contract St Invoices
IB E^J 7 Meetings 6: Presentations
SI Ql 8 Miscellaneous & Other Ar
Q CJJ Verifications
Q L^ Hach Lange
5J Battelle and OCETA
Lj^ External expert
Q L^J External laboratory
L^J AlControl
Lj Eurofins
_.u[ Hach Lange
Q t^jl Initial contact
L^J Batelle & OCETA
^ Internal corresponda
Q Other correspondan<
Type
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
File Folder
69
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APPENDIX 8
Deviations and amendments
70
-------
Deviation reports
The test plan version approved must be followed. If (or rather when) deviations are needed during testing, the deviations are noted and
justified in the format:
Deviation
number
Experiment
label
Test Plan
Deviation
Cause
Impact as-
sessment
Corrective
action, if
any
Date
Signature
test or field
responsible
Date
Signature
verification
responsible
Date
Signature
Battelle
AMSQM
Date
Signature
ETV
Canada
The verification protocol version approved must be followed. If deviations are needed during testing, the deviations are noted and justi-
fied in the format:
Deviation
number
Verification
protocol
Chapter
Deviation
Cause
Impact as-
sessment
Corrective
action, if
any
Date
Signature
verification
responsible
Date
Signature
internal
auditor
Date
Signature
Battelle
AMSQM
Date
Signature
ETV
Canada
Deviation reports are continuously filled in and filed in the appropriate folder at dkstor, see Appendix 7.
71
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Amendment reports
All changes in the protocol and test plan done in advance of verification and testing
must be done by the document owner (protocol Mette Tjener Andersson (MTA), plan
Claus J0rgensen (CLJ)) and approved by the verification responsible and the internal
auditor. Amendments shall be made available for all involved.
The amendments will mostly have the form of a revised section or chapter of the pro-
tocol or plan, with the front page given below.
72
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AMENDMENT
TESTING DOCUMENT TITLE AND DATE:
AMENDMENT NUMBER: _
DATE OF REVISED PART: _
PART TO BE CHANGED/REVISED:
CHANGE/REVISION:
Reference to revised part
REASON FOR CHANGE:
ORIGINATED BY:
DHIDANETV Water Centre Verification or Test Responsible
DATE
APPROVED BY:
DHI DANETV Water Centre Internal Auditor
DATE
Battelle AMS Center Quality Manager
DATE
ETV Canada Quality Manager
DATE
73
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