Unclassified ENV/JM/MONO(2011)38

Organisation de Coopération et de Développement Économiques

Organisation for Economic Co-operation and Development 22-Aug-2011

___________________________________________________________________________________________

_____________ English - Or. English

ENVIRONMENT DIRECTORATE

JOINT MEETING OF THE CHEMICALS COMMITTEE AND

THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY

REPORT OF PROGRESS ON THE INTERLABORATORY VALIDATION OF THE OECD 

HARPACTICOID COPEPOD DEVELOPMENT AND REPRODUCTION TEST

Series on Testing and Assessment 

No. 158

JT03306012

Document complet disponible sur OLIS dans son format d'origine

Complete document available on OLIS in its original format Unclassified ENV/JM/MONO(2011)38 English - Or. English
ENV/JM/MONO(2011)38

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ENV/JM/MONO(2011)38

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OECD Environment, Health and Safety Publications

Series on Testing and Assessment

No. 158

REPORT OF PROGRESS ON THE INTERLABORATORY VALIDATION OF THE OECD 

HARPACTICOID COPEPOD DEVELOPMENT AND REPRODUCTION TEST

Environment Directorate

ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT

Paris 2011
ENV/JM/MONO(2011)38

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Also published in the Series on Testing and Assessment:

No. 1, Guidance Document for the Development of OECD 

Guidelines for Testing of Chemicals (1993; reformatted 1995, revised 

2006)

No. 2, Detailed Review Paper on Biodegradability Testing (1995)

No. 3, Guidance Document for Aquatic Effects Assessment (1995)

No. 4, Report of the OECD Workshop on Environmental 

Hazard/Risk Assessment (1995)

No. 5, Report of the SETAC/OECD Workshop on Avian Toxicity 

Testing (1996)

No. 6, Report of the Final Ring-test of the Daphnia magna 

Reproduction Test (1997)

No. 7, Guidance Document on Direct Phototransformation of 

Chemicals in Water (1997)

No. 8, Report of the OECD Workshop on Sharing Information 

about New Industrial Chemicals Assessment (1997)

No. 9, Guidance Document for the Conduct of Studies of 

Occupational Exposure to Pesticides during Agricultural Application 

(1997)

No. 10, Report of the OECD Workshop on Statistical Analysis of 

Aquatic Toxicity Data (1998)

No. 11, Detailed Review Paper on Aquatic Testing Methods for 

Pesticides and industrial Chemicals (1998)

No. 12, Detailed Review Document on Classification Systems for

Germ Cell Mutagenicity in OECD Member Countries (1998)

No. 13, Detailed Review Document on Classification Systems for 

Sensitising Substances in OECD Member Countries 1998)

No. 14, Detailed Review Document on Classification Systems for 

Eye Irritation/Corrosion in OECD Member Countries (1998)

No. 15, Detailed Review Document on Classification Systems for 

Reproductive Toxicity in OECD Member Countries (1998)

No. 16, Detailed Review Document on Classification Systems for 

Skin Irritation/Corrosion in OECD Member Countries (1998)
ENV/JM/MONO(2011)38

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No. 17, Environmental Exposure Assessment Strategies for Existing 

Industrial Chemicals in OECD Member Countries (1999)

No. 18, Report of the OECD Workshop on Improving the Use of 

Monitoring Data in the Exposure Assessment of Industrial Chemicals 

(2000)

No. 19, Guidance Document on the Recognition, Assessment and 

Use of Clinical Signs as Humane Endpoints for Experimental Animals 

used in Safety Evaluation (1999)

No. 20, Revised Draft Guidance Document for Neurotoxicity 

Testing (2004)

No. 21, Detailed Review Paper: Appraisal of Test Methods for Sex 

Hormone Disrupting Chemicals (2000)

No. 22, Guidance Document for the Performance of Out-door 

Monolith Lysimeter Studies (2000)

No. 23, Guidance Document on Aquatic Toxicity Testing of Difficult 

Substances and Mixtures (2000)

No. 24, Guidance Document on Acute Oral Toxicity Testing (2001)

No. 25, Detailed Review Document on Hazard Classification 

Systems for Specifics Target Organ Systemic Toxicity Repeated 

Exposure in OECD Member Countries (2001)

No. 26, Revised Analysis of Responses Received from Member 

Countries to the Questionnaire on Regulatory Acute Toxicity Data 

Needs (2001)

No 27, Guidance Document on the Use of the Harmonised System 

for the Classification of Chemicals which are Hazardous for the 

Aquatic Environment (2001)

No 28, Guidance Document for the Conduct of Skin Absorption 

Studies (2004)

No 29, Guidance Document on Transformation/Dissolution of 

Metals and Metal Compounds in Aqueous Media (2001)

No 30, Detailed Review Document on Hazard Classification 

Systems for Mixtures (2001)

No 31, Detailed Review Paper on Non-Genotoxic Carcinogens 

Detection: The Performance of In-Vitro Cell Transformation Assays 

(2007) 
ENV/JM/MONO(2011)38

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No. 32, Guidance Notes for Analysis and Evaluation of RepeatDose Toxicity Studies (2000)

No. 33, Harmonised Integrated Classification System for Human 

Health and Environmental Hazards of Chemical Substances and 

Mixtures (2001)

No. 34, Guidance Document on the Development, Validation and 

Regulatory Acceptance of New and Updated Internationally Acceptable 

Test Methods in Hazard Assessment (2005)

No. 35, Guidance notes for analysis and evaluation of chronic 

toxicity and carcinogenicity studies (2002)

No. 36, Report of the OECD/UNEP Workshop on the use of 

Multimedia Models for estimating overall Environmental Persistence 

and long range Transport in the context of PBTS/POPS Assessment 

(2002)

No. 37, Detailed Review Document on Classification Systems for 

Substances Which Pose an Aspiration Hazard (2002)

No. 38, Detailed Background Review of the Uterotrophic Assay 

Summary of the Available Literature in Support of the Project of the 

OECD Task Force on Endocrine Disrupters Testing and Assessment 

(EDTA) to Standardise and Validate the Uterotrophic Assay (2003)

No. 39, Guidance Document on Acute Inhalation Toxicity Testing 

(in preparation)

No. 40, Detailed Review Document on Classification in OECD 

Member Countries of Substances and Mixtures Which Cause 

Respiratory Tract Irritation and Corrosion (2003)

No. 41, Detailed Review Document on Classification in OECD 

Member Countries of Substances and Mixtures which in Contact with 

Water Release Toxic Gases (2003)

No. 42, Guidance Document on Reporting Summary Information on 

Environmental, Occupational and Consumer Exposure (2003)

No. 43, Guidance Document on Mammalian Reproductive Toxicity 

Testing and Assessment (2008)

No. 44, Description of Selected Key Generic Terms Used in 

Chemical Hazard/Risk Assessment (2003)

No. 45, Guidance Document on the Use of Multimedia Models for

Estimating Overall Environmental Persistence and Long-range 

Transport (2004)
ENV/JM/MONO(2011)38

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No. 46, Detailed Review Paper on Amphibian Metamorphosis 

Assay for the Detection of Thyroid Active Substances (2004)

No. 47, Detailed Review Paper on Fish Screening Assays for the 

Detection of Endocrine Active Substances (2004)

No. 48, New Chemical Assessment Comparisons and Implications 

for Work Sharing (2004)

No. 49, Report from the Expert Group on (Quantitative) StructureActivity Relationships [(Q)SARs] on the Principles for the Validation of 

(Q)SARs (2004) 

No. 50, Report of the OECD/IPCS Workshop on Toxicogenomics 

(2005) 

No. 51, Approaches to Exposure Assessment in OECD Member 

Countries: Report from the Policy Dialogue on Exposure Assessment in 

June 2005 (2006)

No. 52, Comparison of emission estimation methods used in 

Pollutant Release and Transfer Registers (PRTRs) and Emission 

Scenario Documents (ESDs): Case study of pulp and paper and textile 

sectors (2006)

No. 53, Guidance Document on Simulated Freshwater Lentic Field 

Tests (Outdoor Microcosms and Mesocosms) (2006)

No. 54, Current Approaches in the Statistical Analysis of 

Ecotoxicity Data: A Guidance to Application (2006)

No. 55, Detailed Review Paper on Aquatic Arthropods in Life Cycle 

Toxicity Tests with an Emphasis on Developmental, Reproductive and 

Endocrine Disruptive Effects (2006)

No. 56, Guidance Document on the Breakdown of Organic Matter 

in Litter Bags (2006)

No. 57, Detailed Review Paper on Thyroid Hormone Disruption 

Assays (2006)

No. 58, Report on the Regulatory Uses and Applications in OECD 

Member Countries of (Quantitative) Structure-Activity Relationship 

[(Q)SAR] Models in the Assessment of New and Existing Chemicals 

(2006) 

No. 59, Report of the Validation of the Updated Test Guideline 407: 

Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats (2006)
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No. 60, Report of the Initial Work Towards the Validation of the 21-

Day Fish Screening Assay for the Detection of Endocrine Active 

Substances (Phase 1A) (2006)

No. 61, Report of the Validation of the 21-Day Fish Screening 

Assay for the Detection of Endocrine Active Substances (Phase 1B) 

(2006)

No. 62, Final OECD Report of the Initial Work Towards the 

Validation of the Rat Hershberger Assay: Phase-1, Androgenic 

Response to Testosterone Propionate, and Anti-Androgenic Effects of 

Flutamide (2006)

No. 63, Guidance Document on the Definition of Residue (2006)

No. 64, Guidance Document on Overview of Residue Chemistry 

Studies (2006)

No. 65, OECD Report of the Initial Work Towards the Validation of 

the Rodent Uterotrophic Assay - Phase 1 (2006)

No. 66, OECD Report of the Validation of the Rodent Uterotrophic 

Bioassay: Phase 2. Testing of Potent and Weak Oestrogen Agonists by 

Multiple Laboratories (2006)

No. 67, Additional data supporting the Test Guideline on the 

Uterotrophic Bioassay in rodents (2007)

No. 68, Summary Report of the Uterotrophic Bioassay Peer Review 

Panel, including Agreement of the Working Group of the National 

Coordinators of the Test Guidelines Programme on the follow up of 

this report (2006)

No. 69, Guidance Document on the Validation of (Quantitative) 

Structure-Activity Relationship [(Q)SAR] Models (2007)

No. 70, Report on the Preparation of GHS Implementation by the 

OECD Countries (2007)

No. 71, Guidance Document on the Uterotrophic Bioassay -

Procedure to Test for Antioestrogenicity (2007)

No. 72, Guidance Document on Pesticide Residue Analytical 

Methods (2007)

No. 73, Report of the Validation of the Rat Hershberger Assay: 

Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists 

and Negative Reference Chemicals by Multiple Laboratories. Surgical 

Castrate Model Protocol (2007) 
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No. 74, Detailed Review Paper for Avian Two-generation Toxicity 

Testing (2007)

No. 75, Guidance Document on the Honey Bee (Apis Mellifera L.) 

Brood test Under Semi-field Conditions (2007)

No. 76, Final Report of the Validation of the Amphibian 

Metamorphosis Assay for the Detection of Thyroid Active Substances: 

Phase 1 - Optimisation of the Test Protocol (2007)

No. 77, Final Report of the Validation of the Amphibian 

Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study 

(2007)

No. 78, Final Report of the Validation of the 21-day Fish Screening 

Assay for the Detection of Endocrine Active Substances. Phase 2: 

Testing Negative Substances (2007)

No. 79, Validation Report of the Full Life-cycle Test with the 

Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis 

and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007)

No. 80, Guidance on Grouping of Chemicals (2007)

No. 81, Summary Report of the Validation Peer Review for the 

Updated Test Guideline 407, and Agreement of the Working Group of 

National Coordinators of the Test Guidelines Programme on the 

follow-up of this report (2007) 

No. 82, Guidance Document on Amphibian Thyroid Histology 

(2007)

No. 83, Summary Report of the Peer Review Panel on the Stably 

Transfected Transcriptional Activation Assay for Detecting Estrogenic 

Activity of Chemicals, and Agreement of the Working Group of the 

National Coordinators of the Test Guidelines Programme on the 

Follow-up of this Report (2007)

No. 84, Report on the Workshop on the Application of the GHS 

Classification Criteria to HPV Chemicals, 5-6 July Bern Switzerland 

(2007)

No. 85, Report of the Validation Peer Review for the Hershberger 

Bioassay, and Agreement of the Working Group of the National 

Coordinators of the Test Guidelines Programme on the Follow-up of 

this Report (2007)

No. 86, Report of the OECD Validation of the Rodent Hershberger 

Bioassay: Phase 2: Testing of Androgen Agonists, Androgen 

Antagonists and a 5 α-Reductase Inhibitor in Dose Response Studies by 

Multiple Laboratories (2008)
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No. 87, Report of the Ring Test and Statistical Analysis of 

Performance of the Guidance on Transformation/Dissolution of Metals 

and Metal Compounds in Aqueous Media (Transformation/ Dissolution 

Protocol) (2008)

No. 88, Workshop on Integrated Approaches to Testing and 

Assessment (2008)

No. 89, Retrospective Performance Assessment of the Test 

Guideline 426 on Developmental Neurotoxicity (2008)

No.90, Background Review Document on the Rodent Hershberger 

Bioassay (2008)

No. 91, Report of the Validation of the Amphibian Metamorphosis 

Assay (Phase 3) (2008)

No. 92, Report of the Validation Peer Review for the Amphibian 

Metamorphosis Assay and Agreement of the Working Group of the 

National Coordinators of the Test Guidelines Programme on the 

Follow-Up of this Report (2008)

No. 93, Report of the Validation of an Enhancement of OECD TG 

211: Daphnia Magna Reproduction Test (2008)

No. 94, Report of the Validation Peer Review for the 21-Day Fish 

Endocrine Screening Assay and Agreement of the Working Group of 

the National Coordinators of the Test Guidelines Programme on the 

Follow-up of this Report (2008)

No. 95, Detailed Review Paper on Fish Life-Cycle Tests (2008)

No.96, Guidance Document on Magnitude of Pesticide Residues in 

Processed Commodities (2008)

No.97, Detailed Review Paper on the use of Metabolising Systems 

for In Vitro Testing of Endocrine Disruptors (2008)

No. 98, Considerations Regarding Applicability of the Guidance on 

Transformation/Dissolution of Metals Compounds in Aqueous Media 

(Transformation/Dissolution Protocol) (2008)

No. 99, Comparison between OECD Test Guidelines and ISO 

Standards in the Areas of Ecotoxicology and Health Effects (2008)

No. 100, Report of the Second Survey on Available Omics Tools 

(2009)

No. 101, Report of the Workshop on Structural Alerts for the OECD 

(Q)SAR Application Toolbox, 15-16 May 2008, Utrecht, the 

Netherlands (2009)
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No. 102, Guidance Document for using the OECD (Q)SAR 

Application Toolbox to Develop Chemical Categories According to the 

OECD Guidance on Grouping of Chemicals (2009)

No. 103, Detailed Review Paper on Transgenic Rodent Mutation 

Assays (2009)

No. 104, Performance Assessment: Conparsion of 403 and CxT 

Protocols via Simulation and for Selected Real Data Sets (2009)

No. 105, Report on Biostatistical Performance Assessment of the 

draft TG 436 Acute Toxic Class Testing Method for Acute Inhalation 

Toxicity (2009)

No. 106, Guidance Document for Histologic Evaluation of Endocrine 

and Reproductive Test in Rodents (2009)

No. 107, Preservative treated wood to the environment for wood held 

in storage after treatment and for wooden commodities that are not 

cover and are not in contact with ground. (2009)

No. 108, Report of the validation of the Hershberger Bioassay 

(weanling model) (2009)

No. 109, Literature review on the 21-Day Fish Assay and the Fish 

Short-Term Reproduction Assay (2009)

No. 110, Report of the validation peer review for the weanling 

Hershberger Bioassay and agreement of the working of national 

coordinators of the test guidelines programme on the follow-up of this 

report (2009)

No. 111, Report of the Expert Consultation to Evaluate an Estrogen 

Receptor Binding Affinity Model for Hazard Identification (2009)

No. 112, The 2007 OECD List of High Production Volume Chemicals 

(2009)

No. 113, Report of The Focus Session on Current and Forthcoming 

Approaches for Chemical Safety and Animal Welfare (2010)

No. 114, Performance Assessment of Different Cytotoxic and 

Cytostatic Measures for the In Vitro Micronucleus Test (MNVIT): 

Summary of results in the collaborative trial (2010)

No. 115, Guidance Document on the Weanling Hershberger Bioassay 

in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties 

(2009)
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No. 116, Guidance Document on the Design and Conduct of Chronic 

Toxicity and Carcinogenicity Studies, Supporting TG 451, 452 and 453 

(2010)

No. 117, Guidance Document 117 on the Current Implementation of 

Internal Triggers in Test Guideline 443 for an Extended One 

Generation Reproductive Toxicity Study, in the United States and 

Canada (2011)

No. 118, Workshop Report on OECD Countries Activities Regarding 

Testing, Assessment and Management of Endocrine Disrupters Part I 

and Part II (2010)

No. 119, Classification and Labelling of chemicals according to the 

UN Globally Harmonized System: Outcome of the Analysis of 

Classification of Selected Chemicals listed in Annex III of the 

Rotterdam Convention (2010)

No. 120, Part 1: Report of the Expert Consultation on Scientific and 

Regulatory Evaluation of Organic Chemistry Mechanism-based 

Structural Alerts for the Identification of DNA Binding Chemicals

No. 12. Part 2: Report of the Expert Consultation on Scientific and 

Regulatory Evaluation of Organic Chemistry Mechanism-based 

Structural Alerts for the Identification of DNA Binding Chemicals

No. 121, Detailed review paper (DRP) on Molluscs life-cycle Toxicity 

Testing (2010)

No. 122, Guidance Document on the determination of the Toxicity of 

a Test Chemical to the Dung Beetle Aphodius Constans (2010) 

No. 123, Guidance Document on the Diagnosis of Endocrine-related 

Histopathology in Fish Gonads (2010)

No. 124, Guidance for the Derivation of an Acute Reference Dose 

(2010)

No. 125, Guidance Document on Histopathology for Inhalation 

Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity: 28-

Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010)

No. 126, Short Guidance on the Threshold approach for Acute Fish 

Toxicity (2010)

No. 127, Peer review report of the validation of the 21-day 

androgenised female stickleback screening assay (2010)

No. 128, Validation Report of the 21-day Androgenised Female 

Stickleback Screening Assay (2010)
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No. 129, Guidance Document on using Cytotoxicity Tests to 

Estimate Starting Doses for Acute Oral Systemic Toxicity Tests

No. 131, Report of the Test Method Validation of Avian Acute Oral 

Toxicity Test (OECD test guideline 223) (2010)

No. 132, Report of the Multi-Laboratory Validation of the H295R 

Steroidogenesis Assay to Identify Modulators (2010)

No.133, Peer Review Report for the H295R Cell-Based Assay for 

Steroidogenesis (2010)

No.134, Report of the Validation of a Soil Bioaccumulation Test with 

Terrestrial Oligochaetes by an International ring test (2010)

No.135, Detailed Review Paper on Environmental Endocrine 

Disruptor Screening: The use of Estrogen and Androgen Receptor 

Binding and Transactivation Assays in Fish (2010)

No. 136, Validation Report of The Chironomid Full Life-Cycle 

Toxicity Test (2010)

No. 137, Explanatory Background Document to the OECD Test 

Guideline On In Vitro Skin Irritation Testing (2010)

No. 138, Report of the Workshop on Using Mechanistic Information 

in Forming Chemical Categories (2011)

No. 139, Report of the Expert Consultation on Scientific and 

Regulatory Evaluation of Organic Chemistry Mechanism Based 

Structural Alerts for the Identification of Protein-binding Chemicals 

(2011)

No. 141, Report of the Phase 1 of the Validation of the Fish Sexual 

Development Test for the Detection of Endocrine Active Substances 

(2011)

No. 142, Report of the Phase 2 of the Validation of the Fish Sexual 

Development Test for the Detection of Endocrine Active Substances 

(2011)

No. 141, Report of the Phase 1 of the Validation of the Fish Sexual 

Development Test for the Detection of Endocrine Active Substances 

(2011)

No. 142, Report of the Phase 2 of the Validation of the Fish Sexual 

Development Test for the Detection of Endocrine Active Substances 

(2011)
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No. 143, Peer Review Report for the Validation of the Fish Sexual 

Development Test and Agreement of the Working Group of National 

Co-ordinators of the Test Guideline Programme on the Follow-up of 

the Peer Review (2011) 

No. 144, Validation Report for the Acute Chironomid Assay (2011) 

No. 148, Guidance Document on the Androngenised Female 

Stickleback Screen (2011)

No. 152, Case Study: Assessment of an Extended Chemical Category, 

the Short-chain Methacrylates, Targeted on Bioaccumulation (2011)

No. 153, Guidance Document for the Derivation of an Acute 

Reference Concentration (Arfc) (2011) 

No. 156, Guidance Notes on Dermal Absorption (2011)

No. 158, Report of Progress on the Interlaboratory Validation of the 

OECD Harpacticoid Copepod Development and Reproduction Test

(2011)

© OECD 2011

Applications for permission to reproduce or translate all or part of this 

material should be made to: Head of Publications Service, 

RIGHTS@oecd.org. OECD, 2 rue André-Pascal, 75775 Paris Cedex 

16, France
ENV/JM/MONO(2011)38

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ABOUT THE OECD

The Organisation for Economic Co-operation and Development (OECD) is an intergovernmental 

organisation in which representatives of 34 industrialised countries in North and South America, Europe 

and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise 

policies, discuss issues of mutual concern, and work together to respond to international problems. Most of 

the OECD‟s work is carried out by more than 200 specialised committees and working groups composed 

of member country delegates. Observers from several countries with special status at the OECD, and from 

interested international organisations, attend many of the OECD‟s workshops and other meetings. 

Committees and working groups are served by the OECD Secretariat, located in Paris, France, which is 

organised into directorates and divisions.

The Environment, Health and Safety Division publishes free-of-charge documents in ten different series: 

Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides and 

Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety of 

Novel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission 

Scenario Documents; and Safety of Manufactured Nanomaterials. More information about the 

Environment, Health and Safety Programme and EHS publications is available on the OECD‟s World 

Wide Web site (www.oecd.org/ehs/).

This publication was developed in the IOMC context. The contents do not necessarily reflect the views or 

stated policies of individual IOMC Participating Organisations.

The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was established in 

1995 following recommendations made by the 1992 UN Conference on Environment and Development to 

strengthen co-operation and increase international co-ordination in the field of chemical safety. The 

Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD. 

UNDP is an observer. The purpose of the IOMC is to promote co-ordination of the policies and activities 

pursued by the Participating Organisations, jointly or separately, to achieve the sound management of 

chemicals in relation to human health and the environment.
ENV/JM/MONO(2011)38

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This publication is available electronically, at no charge.

For this and many other Environment,

Health and Safety publications, consult the OECD’s

World Wide Web site (www.oecd.org/ehs/)

or contact:

OECD Environment Directorate,

Environment, Health and Safety Division

2 rue André-Pascal

75775 Paris Cedex 16

France

Fax: (33-1) 44 30 61 80 E-mail: ehscont@oecd.org

E-mail: ehscont@oecd.org
ENV/JM/MONO(2011)38

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FOREWORD

This document presents a second validation report of the harpacticoid copepod (Amphiascus tenuiremis) 

development and reproduction test.

In 2002, Sweden submitted a project proposal to the Working Group of the National Coordinators of the 

Test Guidelines Programme (WNT) to develop a new Test Guideline on the development and reproduction 

of copepods, an aquatic arthropod species. In 2007, A first validation report of the full life-cycle test with 

the harpacticoid copepods Nitocra Spinipes and Amphiascus Tenuiremis and the calanoid copepod Acartia 

tonsa (phase 1) was published in the Series on Testing and Assessment as No. 79. Further validation work 

was needed to ensure the relevance and reliability of the test method, and the species Amphiascus 

tenuiremis was selected for further experimental work. 

In June 2009, a meeting of the OECD invertebrate testing expert group reviewed the outcome of the 

validation, acknowledged the challenges in applying this test method and made a number of 

recommendations for further work on this test method. These recommendations are included in the present 

report. The report was endorsed by the WNT at its meeting held on 12-14 April 2011. The Joint Meeting of 

the Chemicals Committee and Working Party on Chemicals, Pesticides and Biotechnology agreed to its 

declassification on 22 June 2011.

This document is published under the responsibility of the Joint Meeting of the Chemicals Committee and 

the Working Party on Chemicals, Pesticides and Biotechnology of the OECD.
ENV/JM/MONO(2011)38

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ACKNOWLEDGEMENT

Participating Laboratories:

University of South Carolina, Columbia, SC

Stockholm University, Stockholm, Sweden

NOAA Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC

Swedish University of Agricultural Sciences, Uppsala, Sweden

Co-Directors of Validation Exercise:

Dr. G. Thomas Chandler, USC-Columbia 

Dr. Magnus Breitholz, Stockholm University 
ENV/JM/MONO(2011)38

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TABLE OF CONTENTS

REPORT OF PROGRESS ON THE INTERLABORATORY VALIDATION OF THE OECD 

HARPACTICOID COPEPOD DEVELOPMENT AND REPRODUCTION TEST......................................3

FOREWORD.................................................................................................................................................17

ACKNOWLEDGEMENT.............................................................................................................................18

INTRODUCTION.........................................................................................................................................20

RESULTS:....................................................................................................................................................24

ATRAZINE RESULTS.................................................................................................................................24

LINDANE RESULTS...................................................................................................................................29

SUMMARY PRELIMINARY CONCLUSIONS TO DATE (MAY 22, 2009): ..........................................41

CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER WORK...............................................44
ENV/JM/MONO(2011)38

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INTRODUCTION

1. This report covers progress as of May 2009 on the “second inter-laboratory calibration” of the 

draft OECD Copepod Test Guideline. The below chart shows the history of the Test Guideline‟s 

development, from its inception, as a co-project among Sweden, Denmark and the UK, up to the present 

time). The USA became involved in 2003 with the publishing of an American Society for Testing and 

Materials (ASTM) water quality standard test method for copepod full lifecycle testing (ASTM E2317-04). 

That method has been adapted here for this OECD copepod test guideline. In brief, the Test Guideline 

details how to culture, isolate and expose freshly hatched copepod larvae for full lifecycle exposures as 

individuals in microwells of 96-well microplates. It recommends the benthic harpacticoid copepod 

Amphiascus tenuiremis, but there are other copepod species that may be similarly suited for lifecycle 

testing using this approach (e.g., Robertsonia propinquum). There are now a dozen peer-reviewed journal 

articles using this method successfully to evaluate eight anthropogenic chemicals (see appended 

bibliography). 

2002 Standard Project Submission Form for New Test Guideline (Sweden).

2003 First draft written as a co-project between UK, Denmark and Sweden. The

harpacticoids Nitocra spinipes and Tisbe battagliai were included 

together with the calanoid Acartia tonsa.

2003 After circulation: Paris-meeting decided to include Amphiascus

tenuiremis and change the experimental design from cohort exposure to 

individual exposure in microwells.

2004 Revised drafts sent out for comments (harpacticoid and calanoid parts 

separated). Reproduction added as new endpoint to the calanoid draft!

2005 Pre-validation including labs from Sweden, UK, Denmark (x2) and USA. Report 

finished in September and discussed in Paris. It was decided to continue with 

both drafts and use 3,5-DCP as a single reference substance. Minor changes 

to the drafts.

2006 Ring test including labs from Sweden (x3), USA (x2), UK (x1), Denmark (x2). 

Report finished in December. 

2007 Decision to proceed with a second intercalibration with A. tenuiremis.

1. Background – Short history of the OECD copepod guideline

2. Figure 1 below is a flow chart of the essence of this copepod-based test guideline. A revised 

draft guideline is forthcoming which will incorporate suggested improvements and eliminate some 

inconsistencies and ambiguities identified in the validation process for the most recent 2007 version. We 

direct the reader of this report to that draft OECD Copepod Test Guideline if there are questions regarding 

methods and materials used to produce the test data discussed and compared below for each participating 

laboratory. Four laboratories participated in this inter-laboratory calibration of the guideline: The 

University of South Carolina (hereafter called USC), the NOAA Center for Coastal Environmental Health 

and Biomolecular Research (NOAA), Stockholm University (Stockholm), and the SLU, Uppsala, 
ENV/JM/MONO(2011)38

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(Uppsala). Two reference chemicals (atrazine and lindane; analytical standard quality, > 98% purity) were 

tested individually over a range of 5 serially diluted concentrations plus controls for a 36 day (or less) “full 

lifecycle through two broods” exposure period. Although not part of the OECD copepod test guideline, 

life-table data produced by this method were used to estimate population instantaneous population growth 

rates  and predict population growth and stage structure as a function of concentration as a 

demonstration for the reference chemical lindane (i.e., Table 18 and Figures 8 -16).

3. As of December 2008, all four labs (NOAA, USC, Stockholm and Uppsala) have completed 

lindane validation tests. NOAA, USC and Stockholm have also completed atrazine validation tests. 

Uppsala had planned to run a definitive atrazine test beginning March 9, 2009, but they encountered 

problems with algal culture contamination and paternity leave issues for their technician, and requested a 

further delay until June 2009. Fresh algal stocks were shipped to Uppsala by USC on May 15. After June 

2009, the Uppsala atrazine test could not be carried out due to the persistent difficulties mentioned above 

with stock culture of marine algae. Copepods cannot be cultured in good health or at all if algal stocks 

cannot be established and maintained in good health. Uppsala has abandoned further efforts to validate the 

copepod Test Guideline.

4. In February 2008, all supplies and chemicals (atrazine and lindane) for the validation were 

ordered and received. Reference chemical concentrated stocks were prepared at the USC Chandler 

laboratory in Columbia, SC, using technical-grade chemicals (Atrazine = 98% purity; Lindane = 99.6%) in 

pesticide-grade acetone. Prior to shipping, chemical stocks were analyzed by Dr. Lee Ferguson (USC 

Chemistry Dept.) to verify nominal concentrations. Atrazine was measured in concentrate stocks using 

HPLC-UV (214 ± 38 ug/mL), and lindane using GC-ECD (47.3 ± 2.2 ug/mL). Limit of detection (LOD) 

for atrazine was 0.001 ug/mL. LOD for lindane was 0.0005 ug/mL. Supplies and stock reference 

chemicals were driven by courier to the NOAA lab in Charleston, SC. Supplies, pesticide-grade acetone 

and stock reference chemicals were sent to the laboratory of Dr. Magnus Breitholz in Stockholm via FedEx 
ENV/JM/MONO(2011)38

22

International Priority. However, strict regulations on the importation and use of hazardous substances (i.e., 

the chemical stocks) in Sweden resulted in a significant delay in delivery (April 2008). Chemicals were 

returned to USC and had to be re-shipped to Sweden via a commercial shipping company that was able to 

gain Swedish Customs approval to deliver the concentrated pesticides in acetone. This initial obstacle set 

Sweden behind US lab test schedules by approximately two months. All participating laboratories used the 

same stocks of acetone and reference chemicals through all tests. All measured chemistries were 

performed by Dr. Lee Ferguson, Department of Chemistry, University of South Carolina.

5. Amphiascus tenuiremis for the validation study were cultured in a clean, sediment-based, flowthrough seawater system. Sediment was collected from a pristine site located on Bread and Butter Creek in 

the NOAA national estuarine research reserve of North Inlet, Georgetown, South Carolina, and 

washed/processed per ASTM Guidelines (E2317-04). The water used was sterile-filtered 30 g/L Instant 

Ocean synthetic seawater with a dissolved oxygen (DO) level greater than 90% and a pH of 8.0. 

Preliminary rangefinders were used to set exposures at or below the expected 96 h LC30 for A. tenuiremis. 

USC served as the intercalibration source laboratory for copepod-culture seed stocks, a common lot of 

measured concentrated stock reference chemicals in acetone, a source of algal phytoplankton food stocks, 

and a supplier of all microplates, Hamilton® syringes, Costar Netwell® brood chambers, and silanized 

pipettes for copepod picking (see guideline). USC also hosted one technician from Stockholm for one 

week in early 2008 to learn copepod culturing and testing. NOAA participated in the first pre-validation 

test with 3,5 DCP in 2005 and had experience with the method prior to this broader intercalibration 

exercise. Uppsala received training from Dr. Magnus Breitholz and his technician in spring/summer 2008. 

Dr. Lee Ferguson (USC Chemistry Dept.) provided chemical analysis of all water samples from all 

participating laboratories for both reference chemicals.

 

6. Beginning March 3rd, 2008, the NOAA laboratory began their atrazine validation. 

Subsequently, USC (April 4th), Stockholm (May 21st) and Uppsala (June 19th) started their atrazine 

validations. Bioassay experimental treatments (n=7 concentrations) consisted of a highest nominal 

concentration of 200 ug/L, a series of four dilutions decreasing by 60%, a solvent seawater control and a 

normal blank seawater control. Microplates were checked daily for temperature, mortality and 
ENV/JM/MONO(2011)38

23

developmental life-stage (e.g., nauplius, copepodite and adult copepod). Every third day, each microwell

was renewed (250 uL test media) and water quality criteria (temperature, salinity, pH and DO) was 

recorded. Every sixth day, test organisms were fed a 1:1:1 (by volume) algal mixture of a chrysophyte 

(Isochrysis galbana), a chlorophyte (Dunaliella tertiolecta) and a cryptophyte (Rhodomonas salina). This 

mixture provided each microwell with ~ 2 x 104 mixed cells / microwell per feeding. During testing, the 

microplates were placed in an opaque, covered humidity chamber and held in a temperature-regulated 

incubator at 25°C. A 12:12h light:dark cycle was also maintained throughout the duration of the test.

7. From March through December 2008, data and water samples were collected from each 

laboratory as they completed their atrazine and lindane bioassays. With the exception of Uppsala, all 

laboratories began testing lindane within a month of completing the atrazine bioassay. Testing periods for 

atrazine and lindane ranged from 30 – 36 days. The following are the results for lindane reference 

chemical exposures for all four participating laboratories. Atrazine results are reported for USC, NOAA, 

and Stockholm. Uppsala is conducting their atrazine validation in June 2009 with samples and data to 

follow. 

8. For both reference chemicals, some laboratories experienced difficulties outside of the draft Test 

Guideline regarding consistent execution of reference chemical spikes using the agreed-upon Hamilton 

syringe delivery method. Some were unable to achieve spikes at or near nominal targets (see Tables 1 and 

10 below), but generally all laboratories could achieve a serially ascending (descending) concentration 

range that was fairly evenly spaced. All results here are reported using nominal concentrations, but have 

identified in red those concentrations where mean measured concentrations exceeded nominal targets by 

more (or less) than 50%, and where toxicity responses are possibly affected. 

9. Tables and figures in this report provide summary statistics (means and standard deviations) 

derived from spreadsheets and SAS® programming using data provided by each participating laboratory.
ENV/JM/MONO(2011)38

24

RESULTS:

ATRAZINE RESULTS

10. Chemical analyses: (Table 1) Based on measured chemistry, atrazine nominal spike 

concentrations exceeded + 50% of nominal targets for NOAA time-zero seawater sample means at 26, 43 

and 72 ug/L. Stockholm exceeded nominal targets by 50% or more at 43 and 120 ug/L. Across all labs, 

atrazine concentrations in pooled microwell water removed at 72 hours showed very little loss relative to 

initial measured atrazine concentrations. Atrazine was highly stable over each 72 hour spike:renewal time 

period for the 34-36 day bioassays. Hydrogel coating of microplate walls appears to prevent passive 

binding of atrazine to the test vessel.

Table 1: Atrazine measured chemistry (μg/L at 0 and 72 hrs; mean + 1 SD)

LAB

Atrazine

[nominal]

USC

0 hours 

72 hours

NOAA

0 hours 

72 hours

Stockholm

0 hours 

72 hours

Uppsala

0 hours 

72 hours

Control (ND)

(ND)

(ND)

(ND)

(ND)

(ND)

--

--

Carrier (ND)

(ND)

(ND)

(ND)

(ND)

(ND)

--

--

26 μg/L 26.4 ± 4.0

31.1 + 4.1

45.5 ± 12.5 *

31.5 + 11.1

26.9 ± 6.6

36.1 + 6.1

--

--

43 μg/L 53.0 ± 11.4

48.8 + 5.0

70.7 ± 22.6 *

54.9 + 4.5

74.7 ± 38.9 *

63.9 + 21.5

--

--

72 μg/L 93.3 ± 35.1

94.1 + 10.0

109.8 ± 32.5 *

93.6 + 20.0

92.3 ± 26.9

88.6 + 5.3

--

--

120 μg/L 140.3 ± 30.1

159.5 + 34.2

163.6 ± 28.0

162.2 + 14.9

186.0 ± 82.3 *

144.9 + 33.4

--

--

200 μg/L 207.8 ± 25.2

208.5 + 38.2

268.4 ± 48.1

210.4 + 14.9

290.9 ± 88.6

221.0 + 55.9

--

--

Note: (0 hour values in red are > 50% above or below nominal targets)
ENV/JM/MONO(2011)38

25

Bioassay results: 

Table 2: Percent total mortality (over 36d; mean + 1 SD) 

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 11.1% ± 3.1

(ND)

13.3% ± 5.8

(ND)

33.4% ± 9.7# *

(ND)

--

Carrier 7.3% ± 3.9

(ND)

18.3% ± 5.8 

(ND)

11.9% ± 6.1

(ND) --

26 μg/L 17.5% ±4.4

26.4 ± 4.0

10.0% ± 5.0

45.6 ± 12.5

10.3% ± 5.4

26.9 ± 6.6 --

43 μg/L 23.8% ± 3.3

53.0 ± 11.5

13.3% ± 2.9

70.7 ± 22.6

23.6% ± 7.9

74.7 ± 38.9 --

72 μg/L 23.3% ± 8.9

96.83 ± 29.1

6.7% ± 7.6

115.3 ± 22.9

32.9% ± 12.6 

92.3 ± 26.9

--

120 μg/L 23.9% ± 9.5

140.3 ± 30.1

10.0% ± 5.0

163.6 ± 28.0

21.6% ± 5.9 

186.00 ± 82.3

--

200 μg/L 14.8% ± 2.8

207.8 ± 25.3

5.2% ± 0.3

268.4 ± 48.1

28.7% ± 11.3

290.9 ± 88.6 --

Note: 

- # Control mortality for Stockholm not typical due to non-random selection of individuals at start, 

and too few animals in stock cultures;

- red bold* means statistical significance

Total atrazine mortality divided among the life-stages is available in Annex 1 of the report.
ENV/JM/MONO(2011)38

26

Table 3: Sex ratio under exposure to atrazine (F:M)

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 1.1 0.4

(ND)

2.1 0.4

(ND)

0.8 ± 0.2

(ND) --

Carrier 0.5 0.1

(ND)

1.4 1.3

(ND)

1.3 ± 0.4

(ND) --

26 μg/L 1.4 0.5

26.4 ± 4.0

2.5 0.9

45.6 ± 12.5

0.8 ± 0.4

26.9 ± 6.6 --

43 μg/L 1.4 0.7

53.0 ± 11.5

1.7 0.5

70.7 ± 22.6

1.1 ± 0.5

74.7 ± 38.9 --

72 μg/L 1.0 0.3

96.8 ± 29.1

1.3 0.4

115.3 ± 22.9

1.0 ± 0.5

92.3 ± 26.9 --

120 μg/L 0.5 0.1

140.3 ± 30.1

1.1 0.3

163.6 ± 28.0

1.1 ± 0.3

186.0 ± 82.3 --

200 μg/L 0.4 0.0

207.8 ± 25.3

1.6 1.2

268.4 ± 48.1

1.1 ± 0.6

290.9 ± 88.6 --

Table 4: Total Development Time -- Nauplius to Adult (80% pop.)

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 19.6

(ND)

15.7

(ND)

19.8

(ND) --

Carrier 21.3

(ND)

15.5

(ND)

18.8

(ND) --

26 μg/L 19.4

26.4 ± 4.0

15.5

45.6 ± 12.5

17.5

26.9 ± 6.6 --

43 μg/L 20.0

53.0 ± 11.5

15.5

70.71 ± 22.57

17.1

74.7 ± 38.9 --

72 μg/L 20.7

96.8 ± 29.1

15.8

115.1 ± 22.9

18.3

92.3 ± 26.9 --

120 μg/L 20.7

140.3 ± 30.1

15.3

163.6 ± 28.0

17.7

186.0 ± 82.3 --

200 μg/L 18.1

207.8 ± 25.3

15.9

268.4 ± 48.1

17.3

290.9 ± 88.6 --
ENV/JM/MONO(2011)38

27

Table 5: Percent mating success through two broods

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 82.1% ± 16.0

(ND)

72.6% ± 8.4

(ND)

--

(ND) --

Carrier 96.7% ± 5.6

(ND)

74.2% ± 3.9

(ND)

44.2% ± 38.8 *

(ND) --

26 μg/L 73.1% ± 8.0

26.4 ± 4.0

73.0% ± 12.4

45.6 ± 12.5

56.7% ± 35.1

26.9 ± 6.6 --

43 μg/L 68.5% ± 17.6

53.0 ± 11.5

58.6% ± 20.0

70.7 ± 22.6

34.2% ± 31.7

74.7 ± 38.9 --

72 μg/L 90.0% ± 14.9

96.8 ± 29.1

55.6% ± 31.4

115.3 ± 22.9

36.7% ± 32.2

92.3 ± 26.9 --

120 μg/L 65.9% ± 13.5

140.3 ± 30.1

53.6% ± 5.1

163.6 ± 28.0

36.7% ± 32.2

186.0 ± 82.3 --

200 μg/L 74.1% ± 22.9

207.8 ± 25.3

57.1% ± 20.2

268.4 ± 48.1

35.6% ± 33.6

290.9 ± 88.6 --

Note: red bold* means statistical significance

Table 6: Mean viable offspring in two broods

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 11.6 ± 4.9

(ND)

11.8 ± 9.3

(ND)

--

(ND) --

Carrier 12.6 ± 2.6

(ND)

12.7 ± 8.4

(ND)

9.9 ± 7.8

(ND) --

26 μg/L 9.1 ± 5.6

26.4 ± 4.0

12.4 ± 8.6

45.6 ± 12.5

11.2 ± 8.7

26.9 ± 6.6 --

43 μg/L 8.0 ± 5.2

53.0 ± 11.5

6.8 ± 6.4

70.7 ± 22.6

10.2 ± 8.5

74.7 ± 38.9 --

72 μg/L 8.0 ± 6.3

96.8 ± 29.1

7.1 ± 5.9

115.3 ± 22.9

13.1 ± 6.8

92.3 ± 26.9 --

120 μg/L 6.5 ± 4.8 *

140.3 ± 30.1

6.8 ± 6.9 *

163.6 ± 28.0

9.8 ± 7.9

186.0 ± 82.3 --

200 μg/L 5.5 ± 4.4 *

207.8 ± 25.3

6.1 ± 6.2 *

268.4 ± 48.1

10.9 ± 8.0

290.9 ± 88.6 --

Note: red bold* means statistical significance
ENV/JM/MONO(2011)38

28

USC

11. USC survival rates over the atrazine lifecycle test (36 days) were 89.0% and 92.7% for the 

solvent control (carrier) and water controls respectively (Table 2), and fell within the copepod guideline 

survival rate criterion of > 80%. USC found no concentration dependency or treatment-specific 

significant differences in mortality, development rate, sex ratios, or mating success at any test 

concentration or for any life-stage relative to controls (i.e., nauplius, copepodite, or adult; see Annex 1 at 

end). However, the number of viable offspring produced through two clutches or broods at 120 and 200 

ug/L atrazine were significantly depressed 52 to 57% relative to controls (see Table 6). 

NOAA

12. NOAA control survival rates over the atrazine lifecycle test (35 days) were 86.7% and 81.7% for 

the carrier and non-solvent controls respectively (Table 2), and both rates fell within the guideline control 

survival rate criterion of > 80%. NOAA found no concentration dependency or treatment-specific 

significant differences in mortality, development rate, sex ratios, or mating success at any atrazine test 

concentration for any life-stage (nauplius, copepodite, adult) relative to controls (Tables 3-5). However, 

the number of viable offspring produced through two clutches at 120 and 200 ug/L atrazine were both 

significantly depressed 46 to 52% relative to controls; and very similar to the USC response (Table 6).

STOCKHOLM

13. Stockholm control survival rates over the atrazine lifecycle test (34 days) were 66.6% and 88.1% 

for the carrier and non-solvent controls respectively (Table 2). The water-control survival rate was 

significantly below the OECD draft test guideline criterion of 80%, but the solvent control rate was similar 

to other laboratories and well within the guideline criterion. Stockholm attributed the normal-control result 

to non-random selection of control nauplii at experimental set up, and low stock culture densities prior to 

the beginning of the test. The Stockholm laboratory found no significant mortality differences relative to 

water control or solvent controls for any atrazine concentrations tested. No concentration dependency or 

significant differences were detected for development rate at any life-stage, nor did Stockholm see any 

treatment-related effects at any concentration for sex ratios, reproductive success or viable offspring 

production relative to controls. Solvent controls performed similarly to other laboratories for all endpoints 

except mating success. The guideline criterion for “percentage of control pairs able to produce two 

broods” is > 70%. All labs achieved >70% mating pair success in controls except for Stockholm which 

achieved 44.2% success. Stockholm normal control matings could not be completed because that set of 

microplates was accidentally dropped prior to clutch extrusion. Solvent control matings that were 

successful by Stockholm produced two-brood clutch sizes of ~10 offspring which was close to the mean 

response seen in all atrazine treatments, and within the guideline two-brood clutch-size criterion of > 8

viable offspring in controls (Table 6).
ENV/JM/MONO(2011)38

29

LINDANE RESULTS

Chemical analyses: (Table 7) 

14. Water control and solvent control seawater showed non detectable lindane levels in every sample 

for every lab (i.e., for time zero seawater and 72-h microplate pooled seawater). Lindane nominal spike 

mean concentrations exceeded 150% for every Uppsala time-zero seawater sample. However, beyond the 

2.5 and 5.0 ug/L nominal treatments, this laboratory did achieve a declining measured concentration 

spacing of 58-60% as requested in the validation plan. USC exceeded nominal concentrations by 50% or 

more for the 15 ug/L nominal time-zero seawater target. Stockholm spikes were all within + 50% of the 

nominal target concentrations. Across all labs, lindane concentrations in pooled microwell water removed 

at 72 hours usually showed higher loss rates than seen for atrazine, but concentrations were still reasonably 

stable for each 72 hour spike:renewal time period (Table 7; loss rates from 5 to 50%). Hydrogel coating of 

microplate walls appears to minimize passive binding of hydrophobic lindane to the test vessel. 

Table 7: Lindane measured chemistry (μg/L at 0 and 72 hrs; mean + 1 SD)

LAB

Lindane

[nominal]

USC

0 hours 

72 hours

NOAA

0 hours 

72 hours

Stockholm

0 hours 

72 hours

Uppsala

0 hours 

72 hours

Control (ND)

(ND)

(ND)

(ND)

(ND)

(ND)

(ND)

(ND)

Carrier (ND)

(ND)

(ND)

(ND)

(ND)

(ND)

(ND)

(ND)

2.5 μg/L 3.9 ± 1.4

3.7 + 1.2

5.1 ± 2.9 *

3.1 + 1.2

2.2 ± 0.8

1.5 + 0.39

7.0 + 1.6 *

16.3 + 14.8

5 μg/L 7.6 ± 3.3

5.2 + 0.70

7.5 ± 4.6

4.3 + 2.2

4.7 ± 2.0

2.6 + 0.50

8.9 + 4.2 *

8.6 + 0.9

10 μg/L 12.7 ± 4.2

25.3 + 10.4 *

15.9 ± 7.7 *

7.3 + 4.7

7.6 ± 3.4

4.3 + 3.0

16.7 + 6.7 *

53.8 + 71.8

15 μg/L 23.4 ± 7.6 *

22.8 + 6.8

28.3 ± 15.1 *

31.3 + 28.3

11.6 ± 3.8

10.1 + 2.3

29.3 + 9.4 *

25.6 + 6.2

20 μg/L 23.2 ± 5.1

22.4 + 6.3

36.7 ± 20.9 *

24.5 + 17.6

15.7 ± 7.3

11.5 + 2.7

53.2 + 9.1 *

51.1 + 21.7

Note: (0 hour values in red are > 50% above or below nominal targets) 
ENV/JM/MONO(2011)38

30

Bioassay results

Survival

Table 8: Total mortality (over 36 d; mean + 1 SD)

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 5.2% ± 2.6

(ND)

2.2% ± 1.9

(ND)

11.7% ± 12.6

(ND)

12.7% ± 6.2

(ND)

Carrier 10.2% ± 2.3

(ND)

6.7% ± 5.8

(ND)

13.9% ± 11.1

(ND)

2.0% ± 3.4

(ND)

2.5 μg/L 6.9% ± 5.3

3.9 ± 1.4

30.0% ± 8.8

5.1 ± 2.9

5.0% ± 5.0

2.2 ± 0.8

41.7% ± 36.3

7.0 + 1.6

5 μg/L 10.3% ± 4.5

7.6 ± 3.3

31.1% ± 16.8 *

7.5 ± 4.6

10.4% ± 5.0

4.7 ± 2.0

10.9% ± 5.8

8.9 + 4.2

10 μg/L 33.4% ± 1.7 *

12.7 ± 4.2

81.1% ± 11.7 *

15.9 ± 7.7

22.2% ± 14.9

7.6 ± 3.4

44.9% ± 18.9 *

16.7 + 6.7

15 μg/L 19.2% ± 13.1

23.4 ± 7.6

78.9% ± 3.9 *

28.3 ± 15.1

8.5% ± 10.3

11.6 ± 3.8

15.5% ± 12.5

29.3 + 9.4

20 μg/L 34.4% ± 7.8 *

23.2 ± 5.1

57.8% ± 12.6 *

36.7 ± 20.9

21.9% ± 11.3 *

15.7 ± 7.3

14.8% ± 7.4

53.2 + 9.1

15. USC total control survival rates over the lindane lifecycle test were 94.8% and 89.8% 

respectively for the solvent control and water control (Table 8), and similar to the atrazine test control 

rates. These rates are within the copepod guideline survival rate criterion of > 80%. USC saw strong 

concentration dependency and treatment-specific significant differences in mortality at 10, 15 and 20 ug/L. 

The naupliar larval stage was strongly sensitive to lindane at each of these concentrations (Annex 2, Table 

1), but the copepodite stage (i.e., survivors from the preceding naupliar exposure window) was insensitive 

to lindane until 20 ug/L (Annex 2, Table 2). Adult surviving copepods were practically insensitive to all 

exposures over all laboratories (Annex 2, Table 3) for the mortality endpoint, except for 15 ug/L at 

NOAA. No USC mean mortalities exceeded 35% at any treatment level which is the guideline 

recommendation for treatment-related maximum mortalities.

16. NOAA total control survival rates over the lindane lifecycle test were 97.8% and 93.3% 

respectively for the water control and solvent control (Table 8), and significantly higher than the atrazine 

test control rates. All of these rates are within the copepod guideline survival rate criterion of > 80%. 

NOAA saw strong and significant mortalities at all test concentrations which generally increased with 

concentration. Similar to USC, Stockholm and Uppsala, most mortality was manifest at the naupliar larval 

stage (Annex 2, Table 1). The 10, 15 and 20 ug/L treatments exceeded guideline recommendations of < 

35% total mean mortality at any test concentration. The copepodite stage survivors were insensitive across 

all exposures (Annex 2, Table 2) for the mortality endpoint, but adults showed significant additional 

sensitivity at 5 and 15 ug/L which was not seen for other laboratories (Annex 2, Table 3). 

Stockholm total control survival rates over the lindane lifecycle test were 88.3% and 86.1% respectively 

for the solvent control and water control (Table 8), and much improved over the atrazine-test normal 
ENV/JM/MONO(2011)38

31

control rates. These rates are within the copepod guideline survival rate criterion of > 80%. Stockholm 

mortality rates were variable across microplates within treatment, and showed no significant differences 

for any treatment. Mean mortality maximum was 17 nauplii at the highest 20 ug/L concentration (Annex 

2, Table 1). Copepodites and adults were generally insensitive for the mortality endpoint -- similar to 

responses seen for the other laboratories (Annex 2, Tables 1-3). No Stockholm mean mortalities 

exceeded 35% which is the test guideline recommendation for any treatment maximum mortality. 

Overall, Stockholm measured lindane concentrations were closer to the nominal targets than any other 

participating laboratory, but they were consistently lower by ~ 12 to 45%.

17. Uppsala total control survival rates over the lindane lifecycle test were 87.3% and 98.0% 

respectively for the water and solvent controls (Table 8). These rates are within the copepod test guideline 

survival rate criterion of > 80%. Uppsala saw strong and significant mortalities at the 2.5 and 10 ug/L test 

concentrations, but concentration response was inconsistent for the mortality endpoint at Uppsala. Like 

USC, NOAA and Stockholm, observed mortality was almost completely manifest at the naupliar larval 

stage (Annex 2, Table 1) with almost zero copepodite and adult mortalities at any concentration (Annex 2,

Tables 1-3). The 2.5 and 10 ug/L treatments exceeded the guideline recommendation of < 35% total mean 

mortality at any test concentration.

Development

Figure 1: Mean No. of Days from Nauplius to Copepodite (80% pop) 

7.8

0

3

6

9

12

15

18

21

24

27

30

33

36

STOCKHOLM USC NOAA UPPSALA

Mean No. of Days

Laboratory

Control Carrier 2.5 ug/L 5.0 ug/L 10 ug/L 15 ug/L 20 ug/L

Figure 2: Mean No. of Days from Nauplius to Adult (80% pop) 
ENV/JM/MONO(2011)38

32

0

3

6

9

12

15

18

21

24

27

30

33

36

STOCKHOLM USC NOAA UPPSALA

Mean No. of Days

Laboratory

Control Carrier 2.5 ug/L 5.0 ug/L 10 ug/L 15 ug/L 20 ug/L

USC. Unlike atrazine, the pesticide lindane had a strong concentration dependent affect on USC, NOAA 

and Stockholm copepod naupliar larval development time to adult (Figure 2). For USC, the normal and 

carrier-control development times for the nauplius-to-copepodite progression were almost identical at 7.8 

and 8.0 days respectively (Figure 1). The USC copepodite-to-adult progression were similarly close at 

14.8 and 16.2 days respectively. Lindane significantly delayed naupliar development at 10, 15 and 20 ug/L 

by 3 to 4.5 days relative to controls. Additional significant delays for the less acutely-sensitive copepodite 

stage were not seen by USC as all treatments and controls fell within the normal 5 to 7 day copepodite-toadult time window. 

18. NOAA control and carrier-control development rates for nauplii and copepodites were similar at 

10 and 11 days, or 6.7 and 6.8 days, respectively. NOAA produced the most consistently high measured 

concentration of lindane among the four laboratories at the high end of the nominal range; and they saw 

correspondingly the strongest delays with concentration across the naupliar-to-copepodite time 

progressions (Figure 2). Significant naupliar delays were seen at 5, 15 and 20 ug/L that ranged from 7 to 

16 days longer than that required for controls. 10 ug/L also elicited a strong mean delay of 8 additional 

days, but the response was highly variable across microplates possibly due to low sample sizes from high 

treatment mortalities. Additional significant copepodite delays were not seen by NOAA as all treatments 

and controls fell within the normal 5 to 7 day copepodite-to-adult time window. 

19. Stockholm water control and solvent control development rates for nauplii and copepodites were 

similar to NOAA at 9.5 or 11.2 days (to copepodite), and 15.7 or 17.0 days (to adult), respectively. 

Stockholm produced the most consistently even spread of lindane concentrations of the four laboratories 

(Table 10). Correspondingly, they saw the least variable concentration-dependent increase in mean 

developmental delays across the naupliar-to-adult time window (Figure 2). Significant naupliar-tocopepodite delays of 2.7 to 4.1 days were seen at 5, 15 and 20 ug/L relative to controls. Additional 

significant copepodite delays were not seen as all treatments and controls fell within the normal 5 to 7 day 

copepodite-to-adult time window. 

20. Uppsala water and solvent control development rates for nauplii and copepodites were the most 

rapid of all labs (Figure 2) at 7.0 or 6.3 days (to copepodite), and 13.3 or 12.3 days (to adult), respectively. 
ENV/JM/MONO(2011)38

33

Uppsala saw almost zero differences in development time for the naupliar-to-copepodite progression with 

concentration (6.3 to 7.7 days total; < 1.5 day delay with concentration), but saw strong concentration

dependency in copepodite-to-adult development rates at 15 and 20 ug/L (i.e., 3 to 3.3 days). 

Sex ratios

Table 9: Sex ratios under Lindane exposure (F:M)

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 1.1 ± 0.3

(ND)

1.3± 0.7

(ND)

0.8 ± 0.3

(ND)

1.1 ± 0.6

(ND)

Carrier 0.9 ± 0.1

(ND)

1.4 ± 0.3

(ND)

1.0 ± 0.2

(ND)

1.7 ± 0.7

(ND)

2.5 μg/L 1.1 ± 0.6

3.9 ± 1.4

2.5 ± 1.6

5.1 ± 2.9

1.1 ± 0.5

2.2 ± 0.8

0.8 ± 0.5

7.0 + 1.6

5 μg/L 1.1 ± 0.1

7.6 ± 3.3

3.7 ± 2.3 *

7.5 ± 4.6

0.9 ± 0.1

4.7 ± 2.0

1.7 ± 0.5

8.9 + 4.2

10 μg/L 0.7 ± 0.3

12.7 ± 4.2

5.5 ± 4.9 *

15.9 ± 7.7

1.0 ± 0.3

7.6 ± 3.4

1.1 ± 0.8

16.7 + 6.7

15 μg/L 0.7 ± 0.6

23.4 ± 7.6

15.0 ± 2.0 *

28.3 ± 15.1

1.1 ± 0.5

11.6 ± 3.8

0.9 ± 0.5

29.3 + 9.4

20 μg/L 0.5 ± 0.2

23.2 ± 5.1

4.8 ± 5.6 *

36.7 ± 20.9

0.9 ± 0.5

15.7 ± 7.3

1.0 ± 0.4

53.2 + 9.1

21. USC water control and solvent control female-to-male ratios at adulthood were 1.1 and 0.9 

respectively in the lindane test (Table 9). The guideline criterion for female-to-male adult ratio in controls 

is 0.6 to 1.8. One significant sex ratio shift to 0.5 + 0.2 was observed by USC at 20 ug/L.

22. NOAA control and carrier-control female-to-male ratios at adulthood were 1.3 and 1.4 

respectively in the lindane test (Table 9). The guideline criterion for female-to-male adult ratio in controls 

is 0.6 to 1.8. Strong and significant sex ratio shifts toward females were seen in the test populations at 10 

through 20 ug/L. These shifts may have been caused by lindane acting on sex determination processes in 

larval and juvenile copepods, or possibly (likely) caused by differential male mortality from lindane since 

treatment mortality rates were high (81 to 58% at 10, 15 and 20 ug/L respectively) for NOAA. No other 

labs saw such strong sex ratio shifts with increasing concentration.

23. Stockholm water control and solvent control female-to-male ratios at adulthood were 0.8 and 1.0 

respectively in the lindane test (Table 9). The guideline criterion for female-to-male adult ratio in controls 

is 0.6 to 1.8. No sex ratio shifts were seen by Stockholm at any test concentrations (range across 

treatments = 0.9 to 1.1; Table 15). 

Uppsala. Similar to Stockholm and USC, Uppsala saw no significant sex ratio differences relative to 

normal (SR = 1.1) and carrier (SR = 1.7) controls (Table 9). 

Mating success
ENV/JM/MONO(2011)38

34

Table 10: Mating pairs able to produce two broods

Lab

Lindane

USC NOAA Stockholm Uppsala

(all matings in 

one microplate)

Control 84.6% ± 15.4

(ND)

100% ± 0.0

(ND)

77.3% ± 19.8

(ND)

66.7

(ND)

Carrier 50.0% ± 7.1 *

(ND)

96.9% ± 5.8

(ND)

85.0% ± 6.6 

(ND)

50.0 *

(ND)

2.5 μg/L 57.6% ± 19.5

3.9 ± 1.4

79.0% ± 12.5

5.1 ± 2.9

92.6% ± 7.3

2.2 ± 0.8

84.6

7.0 + 1.6

5 μg/L 72.4% ± 11.7

7.6 ± 3.3

71.4% ± 14.4

7.5 ± 4.6

90.0% ± 6.4

4.7 ± 2.0

61.9

8.9 + 4.2

10 μg/L 50.0% ± 38.2

12.7 ± 4.2

25.0% ± 28.9 *

15.9 ± 7.7

45.5% ± 32.8 *

7.6 ± 3.4

91.7

16.7 + 6.7

15 μg/L 10.0% ± 9.6 *

23.4 ± 7.6

0% (n=1) *

28.3 ± 15.1

69.6% ± 23.2

11.6 ± 3.8

75.0

29.3 + 9.4

20 μg/L 37.5% ± 50 *

23.2 ± 5.1

14.3% ± 19.2

36.0 ± 20.9

27.3% ± 22.3 *

15.7 ± 7.3

66.7

53.2 + 9.1

24. USC rates of control and carrier-control copepod mating pair success (i.e., ability of individually 

paired male:female couples to produce two viable broods of nauplii) were 84.6% and 50.0% respectively 

(Table 10). The test guideline criterion for minimal mating success is 70% . USC easily passed this 

criterion for water controls, but failed this criterion for the solvent control. Success rates were depressed at 

10, 15 and 20 ug/L lindane, but were also variable across microplates within treatments. A significant and 

sharply depressed mating success rate of only 10% was seen at 15 ug/L. 

25. NOAA rates of control and carrier-control copepod mating pair successe were 100% and 96.9% 

respectively (Table 10), and were the highest rates among all labs. The test guideline criterion for minimal 

mating success is 70% . NOAA success rates were sharply depressed at 5 through 20 ug/L lindane (Table

16), but were also variable across microplates within treatments. A significant and sharply depressed 

mating success rate of 0% was seen at 15 ug/L.

26. Stockholm rates of water control and solvent control copepod mating-pair success were 77.3% 

and 85.0% respectively (Table 10). The test guideline criterion for minimal mating success is 70%. 

Success rates were depressed at 10 and 20 ug/L lindane. A significant and sharply depressed mating 

success rate of only 27% occurred at 20 ug/L.

27. Uppsala rates of control and carrier-control copepod mating-pair success were 66.7% and 50.0% 

respectively (Table 10). The test guideline criterion for mating success of > 70% in controls was not met 

by Uppsala. Success rates were not significantly different from controls for any lindane treatment in the 

Uppsala dataset.

Viable offspring production

Table 11: Mean viable offspring in two broods
ENV/JM/MONO(2011)38

35

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 12.0 ± 3.3

(ND)

20.4 ± 4.6

(ND)

12.6 ± 7.3

(ND)

16.8±10.6

(ND)

Carrier 12.4 ± 3.9

(ND)

20.0 ± 6.0

(ND)

19.2 ± 6.2

(ND)

10.3 ± 7.3

(ND)

2.5 μg/L 11.2 ± 3.9

3.9 ± 1.4

15.5 ±7.7

5.1 ± 2.9

15.9 ± 7.0

2.2 ± 0.8

11.8 ± 8.0

7.0 + 1.6

5 μg/L 13.0 ± 2.3

7.6 ± 3.3

11.7 ± 9.2

7.5 ± 4.6

15.4 ± 6.2

4.7 ± 2.0

9.1 ± 7.4 *

8.9 + 4.2

10 μg/L 12.0 ± 2.9

12.7 ± 4.2

4.0 + 5.7 *

15.9 ± 7.7

6.7 ± 6.8

7.6 ± 3.4

13.8 ± 6.2

16.7 + 6.7

15 μg/L 0.0 ± 0.0 *

23.4 ± 7.6

0 (n=1) *

28.3 ± 15.1

8.8 ± 7.2

11.6 ± 3.8

13.6 ± 7.7

29.3 + 9.4

20 μg/L 0.0 ± 0.0 *

23.2 ± 5.1

4.3 + 7.5 *

36.0 ± 20.9

3.7 ± 6.4 *

15.7 ± 7.3

10.2 ± 7.4

53.2 + 9.1

28. USC control and solvent control mean viable offspring production (i.e., number of live hatchlings 

through two broods) were almost identical at 12.0 or 12.3 offspring per mating pair respectively (Table 

11). Offspring production remained stable for 2.5, 5 and 10 ug/L treatments, but was strongly depressed at 

15 and 20 ug/L with zero offspring production. The test guideline criterion of > 8 offspring per mating 

pair on average in controls was met by USC. Figure 3 is a graph of the USC production response.

29. NOAA water control and solvent control mean viable offspring productions (i.e., number of live 

hatchlings through two broods) were also highest among all labs at 20.4 or 20.0 offspring per mating pair 

respectively (Table 11). Offspring production declined sharply at 5 ug/L to only 11.7 offspring, and then 

declined significantly to < 4.0 offspring per mating pair at 10 to 20 ug/L. Only one mating pair could be 

constructed at 15 ug/L (offspring = 0) because of high lindane-induced mortality in earlier lifestages, and a 

dramatically skewed sex ratio that limited the number of mating pairs that could be constructed. The test 

guideline criterion of > 8 offspring per mating pair on average in controls was met by NOAA. Figure 4 is 

a graph of the NOAA production response.

30. Stockholm water control and solvent control mean viable offspring production was 12.6 or 19.2 

nauplii respectively, and sharply depressed at 10, 15 and 20 ug/L (Table 11). Offspring production 

declined to only 3.7 offspring per pair on average at 20 ug/L which was statistically significant. The test 

guideline criterion of > 8 offspring per mating pair on average in controls was met by Stockholm. Figure 

5 is a graph of the Stockholm production response.

31. Uppsala water control and solvent control mean viable offspring production was 16.8 or 10.3 

nauplii per mating pair respectively (Table 11). No significant mating pair production rates were seen at 

any lindane concentration by Uppsala. The test guideline criterion of > 8 offspring per mating pair on 

average in controls was met by Uppsala. Figure 6 is a graph of the Uppsala production response.

Figure 3: Lindane -- Mean viable offspring in 2 broods - University of South Carolina, Columbia, SC, 

USA*
ENV/JM/MONO(2011)38

36

0

5

10

15

20

25

30

Control Carrier 2.5 (3.9) 5 (7.6) 10 (12.7) 15 (23.4) 20 (23.2) Mean Viable

Concentration (mg/L)

* number in ( ) is measured concentration
ENV/JM/MONO(2011)38

37

Figure 4: Lindane: Mean viable offspring in 2 Broods - NOAA, Charleston, SC, USA* 

0

5

10

15

20

25

30

Control Carrier 2.5 (5.1) 5 (7.5) 10 (15.9) 15 (28.3) 20 (36.7) Mean Viable

Concentration (mg/L)

* number in ( ) is measured concentration

Figure 5: Lindane: Mean viable offspring in 2 Broods - Stockholm University, Stockholm, Sweden* 

0

5

10

15

20

25

30

Control Carrier 2.5 (2.2) 5 (4.7) 10 (7.6) 15 (11.6) 20 (15.7) Mean Viable

Concentration (mg/L)

* number in ( ) is measured concentration
ENV/JM/MONO(2011)38

38

Figure 6: Lindane: Mean viable offspring in 2 Broods - SLU, Uppsala, Sweden*

0

5

10

15

20

25

30

Control Carrier 2.5 (7.0) 5 (8.9) 10 (16.7) 15 (29.3) 20 (53.2) Mean Viable

Concentration (mg/L)

* number in ( ) is measured concentration

*

Microplate Life-table Modeling: population growth rate

Table 12: Instantaneous Population Growth Rates λ

*

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 1.43 1.57 1.23 1.21

Carrier 1.33 1.57 1.37 1.13*

2.5 μg/L 1.26 1.51 1.35 1.09

10 μg/L 1.19 * 1.01 * 1.09 * 1.14

20 μg/L 0.96 * 1.08 * 0.98 * 1.11

*Normal control λ > 1.2 

32. USC measured lifetable parameters from the parental (F0) microplate populations were modeled, 

by lindane treatment, using a matriarchal 5-stage Leslie matrix to produce estimates of  (instantaneous 

rate of population increase; Table 12), projected life-stage abundances at the first filial generation (F1), and 

projected total population sizes at the second filial generation (F2) as a function of lindane exposure. The 
ENV/JM/MONO(2011)38

39

five stages of the lifetable matrix are embryo:nauplius:copepodite: barren-female:gravid-female. All 

abundance estimates by the model exclude embryos and adult males from population counts in order to be 

able to capture sex ratio comparative effects on population growth. Parameters of the model were initial 

abundance of 90 naupliar-stage individuals, ceiling-type density dependence (with carrying capacity K = 

20000 individuals), zero competition, and invoked stochasticity using microplate-measured standard 

deviations for each concentrations‟ stage-to-stage transition rates and for fecundity in the Leslie matrix. 

The recommended criterion for control copepod populations in microplate culture is  > 1.2 . USC water 

and solvent controls  „s were 1.43 and 1.33 respectively, which implies these populations would be 

expected to experience a 43% or 33% increase in abundance at each successive generation. Lindane at 10 

and 20 ug/L sharply reduced  to 1.19 and 0.96 respectively (Table 12). A  value of 1.0 means the 

population is exactly replacing itself over time; a  value less than one means the population is in decline 

over time. 

33. NOAA control and carrier-control  „s (Table 12) were both high and identical at 1.57. This 

implies these populations would be expected to experience a large 57% increase in abundance at each 

successive generation. Recall that production rates for NOAA (i.e., fecundity) were high at 20.0 to 20.4 

offspring through two broods, and  is very sensitive to low (or high) mortalities and/or high fecundity, 

and/or high female production relative to males. NOAA lindane  at 2.5 ug/L was close to control values 

at 1.51. However, „s at 10 and 20 ug/L were dramatically lower at 1.01 and 1.08, implying these 

populations would be close to “replacement” values over time. 

34. NOAA predicted total population abundance (Figure 7 below) at the F2 (second filial, or third 

total) generation of exposure for each laboratory for the solvent control (SOLV CON) was high at 3578 

individuals. Interestingly, at 2.5 ug/L the total predicted population size was only 46% of the control 

despite the identical  value estimated from the Leslie matrix. Decreased fecundity and naupliar mortality 

rates produced a lower production estimate at 2.5 ug/L, but some of that negative effect was overcome by 

the higher proportion of females transitioning into the 2.5 ug/L population (see Table 9) relative to 

controls. More dramatically, at 10 and 20 ug/L the predicted population is either declining (only 46 

individuals at 10 ug/L), or effectively just replacing itself at 20 ug/L just as  alone would have predicted. 

Annex 2, Figure 2 shows NOAA predicted life stage percentages in the population during the first filial 

generation (i.e., at the 6th through 10th time steps in the 3 generation model (15 time steps; 60 days). Stage 

structure is healthy and almost identical at 0, 2.5 and 10 ug/L -- similar to USC and Stockholm (Annex 2, 

Figures 1-3) -- but numbers are strongly depressed overall at 10 ug/L, and stage structure is predominated 

by barren females at 20 ug/L. High mortalities and low fecundities were the predominant drivers of low 

population predictions at these concentrations.
ENV/JM/MONO(2011)38

40

Figure 7: Leslie matrix projected population sizes (+ 1 SD) at the F2 

544

7794

1362

78

199

3578

1224

39

131

46 63

79

29

101

26

68

1

10

100

1000

10000

USC NOAA STOCKHOLM UPPSALA

SOLV CON 2.5 mg/L 10 mg/L 20mg/L

Figure 7 shows Leslie matrix predicted total population abundance at the F2 (second filial, or third total) 

generation of exposure for each laboratory for the solvent control (SOLV CON), and the 2.5, 10 and 20 

ug/L microplate datasets. Error bars represent one standard deviation of the mean. USC lifetable data 

produced a smooth concentration responsive decline in population size from 544 individuals in controls to 

only 29 individuals at 20 ug/L. Annex 2, Figure 1 shows USC predicted life stage percentages in the 

population during the first filial generation (i.e., at the 6th through 10th time steps in the 3 generation (15 

time step; 60 day) of the model (note: white numbers are predicted stage abundances at the F1. Stage 

structure is almost identical at 0 and 2.5 ug/L, with typical within population distributions of highly 

abundant nauplii, copepodites, and normally fewer but sufficient barren and gravid females to support the 

population; but at 10 ug/L, copepodite and naupliar abundances decline sharply, and at 20 ug/L the 

population is composed completely of copepodites and barren females with zero gravids. 

NOTE: Annex 2 Figures 5-8 show predicted F1 abundances by stage for each laboratory. This 

information is included as complementary to Annex 2, Figures 1-4 without discussion, because 

abundance differences and trends are discussed for the F2 in Figure 7.
ENV/JM/MONO(2011)38

41

35. Stockholm control and carrier-control  „s (Table 12) were normal at 1.23 or 1.37 respectively. 

This implies that these populations would be expected to experience a 23 or 37% increase in abundance at 

each successive generation. Lindane  at 2.5 ug/L was close to control values at 1.35. However,  „s at 10 

and 20 ug/L were similar to the NOAA lindane responses and were dramatically lower at 1.09 and 0.98. 

alone would predict that these populations would be close to “replacement” values over time, but that was 

not what the more integrative full model actually predicted: 

36. Model predicted total population abundances (Figure 7 above) at the F2 (second filial, or third 

total) generation for the solvent control (SOLV CON) and 2.5 ug/L treatment were high at 1362 or 1224 

individuals respectively. Decreased fecundity and increased mortality at 10 and 20 ug/L produced sharply 

lower abundance estimates of only 63 or 26 individuals respectively at the F2. Annex 2, Figure 3 shows 

the predicted life stage percentages (structure) in the Stockholm population during the first filial generation 

(i.e., at the 6th through 10th time steps) in the 3 generation model (i.e., 15 time steps total; 60 days). Stage 

structure is healthy and almost identical at 0, 2.5 and 10 ug/L -- similar to USC and NOAA (Figs. 9 and 

10) -- but stage structure at 20 ug/L has a much higher proportion of barren females and almost zero 

gravids to support the population through time. 

37. Uppsala‟s measured lifetable parameters from the parental (F0) microplate populations produced 

estimates of  for water controls and solvent controls of 1.21 and 1.13 respectively. The carrier fell below 

the recommended > 1.2 criterion for  when using this test guideline method. Lindane  „s at 2.5, 10 and 

20 ug/L were all close to the solvent control value of 1.13 (Table 12). Fecundity for Uppsala showed no 

concentration response, and, with the exception of 5 ug/L, fecundity was relatively flat at 10.2 to 13.8 

offspring per mating pair across treatments and in the solvent control (Table 11). Uppsala mortalities were 

high at the 2.5 and 10 ug/L concentrations (42-45%), but much lower at 20 ug/L (only 15%). Mating pair 

success rates were sharply impaired at 20 ug/L and in the solvent control (only 50% and 67% respectively). 

Unlike the 2.5 and 10 ug/L treatments, the solvent control experienced only a 2% mortality rate; but with 

only a 50% mating success rate, and average fecundity, these microplate data yielded a model total 

abundance prediction for the control that was not significantly different from the lindane treatments. 

Therefore, not surprisingly and quite elegantly, the population model did what it should have done and 

predicted similar population abundances with high precision across 0, 2.5, 10 and 20 ug/L lindane (Figure 

7). Correspondingly, population stage structures (Annex 2, Figure 4) were almost identical across 

treatments and control and uniformly showed sharp depressions in production of gravid females to support 

these populations. 

SUMMARY PRELIMINARY CONCLUSIONS TO DATE (MAY 22, 2009): 

 All participating laboratories experienced varying degrees of difficulty in producing consistently 

accurate and precise measured concentrations of atrazine and lindane. Measured chemistries of 

initial spike renewal seawater for some labs often exceeded 150%. Microplate pooled seawater 

was often much closer to the nominal targets at 72 h due to modest loss, bioaccumulation or 

degradation.
ENV/JM/MONO(2011)38

42

 All labs except for USC (the inventor of the method) experienced more frequent and more severe 

difficulties executing the guideline on the first validation run with atrazine than the second or 

later trials with lindane. 

 Most labs were able to maintain physical parameters within guideline recommendations and 

requirements. Physical and copepod response data were not always recorded and reported 

consistently across labs.

 Atrazine mortality response was consistently low across labs, with toxicity manifest primarily in 

mating pair success rates and fecundity (offspring production) at exposures above 100 ug/L. 

Uppsala has not tested atrazine yet (planned for June 2009), so the atrazine dataset is not 

complete. 

 For three of four participating laboratories, lindane produced consistent concentration-dependent 

delays in development rates, consistent naupliar-specific mortality, consistent mating-success 

depressions, and consistent reduction of fecundity (offspring production through 2 broods).

 Interlab comparisons of lindane lifetable data from the microplate exposures using the Leslie 

matrix model produced consistent patterns of response that could be explained integratively from 

the mortality, sex ratio, and fecundity measurements (and variance estimates) collected by each 

laboratory. One lab failed mating success criteria for controls, and therefore we could not 

discriminate a lindane effect on population growth using their data in the model. 

 values alone were often predictable of Leslie matrix integrative projections of population 

abundance, but not always. Sex ratio shifts and mating success differences are often important 

indicators of reproductive and endocrine-based toxicity but they are not captured by 

alone. The OECD expert testing group should discuss the value of including population 

modeling in this guideline approach. The method provides all of the information one needs to 

produce an individualized lifetable for each test population at each test concentration, which can 

then be easily modeled with off-the-shelf software (e.g., RAMAS Stage® or Metapop® 

programs).

 All laboratories demonstrated solid ability most of the time to produce control survival rates well 

within guideline criteria (80% or higher), control mating success above 70%, control clutch sizes 

above ten offspring through 2 broods, mean sex ratios for non solvent and solvent controls 

between 0.4 to 2 taking variability into account, and control lifetime development rates of 20 

days or less at 25C. Uppsala conducted their lindane test at 21-22C rather than the guideline 

25C, but still produced acceptable and the most rapid development rates to adult. 

 All laboratories were able to culture Amphiascus tenuiremis in static bulk culture using seed 

stocks, sterile processed sediments, algal feed stocks, and 30S seawater shipped from USC, 

Columbia. All labs were able to harvest gravid copepods from culture, and get them to hatch 

healthy larval nauplii into Netwell® 12-well plates for harvest and delivery to the microplate test.

 Algal ration control seemed adequate for most labs given the relatively consistent control clutch 

sizes achieved across labs; but in some cases rations may have been set too high (e.g., the NOAA 

mean fecundity values of 20 offspring in controls is unusual and possibly tied to overfeeding.). 

The OECD expert testing committee should discuss whether a minimum and maximum two 

brood range should be included in the guideline. [We have tentatively suggested a criterion of 

>

ration per microwell every 6th
ENV/JM/MONO(2011)38

43

35% increase in total clutch size in controls. Good laboratory practice requires labs to maintain 

calibrated micropipettors, and a poorly performing micropipettor can deliver too much or too 

little food. As an example of expected reproductive output, consistency and food ration effects, 

below is a table of Amphiascus tenuiremis average offspring production through two broods for 

approximately 630 mating pairs scored by four USC technicians for 10 bioassays conducted over 

the past 5 years. The bottom row is data from four bioassays by two technicians that used a 3

tabulated below): 

CONTROL TYPE AND 

RATION

Mean Two Brood 

Size

STD. DEV COEFFICIENT

OF 

VARIATION

2 mL DIET

NORMAL CONTROL 

(n=450 mating pairs)

11.13 3.14 28.2%

SOLVENT CONTROL 

(n=180 mating pairs)

ALL CONTROLS

(n=630 mating pairs)

11.63

11.27

3.78

3.32

32.5%

29.5%

3 mL DIET

NORMAL CONTROL

(n=180 mating pairs)

17.65 3.62 20.5%

 Most Frequently Reported Difficulties from Participants (not ranked):

 Lack of precision and accuracy in spiking pesticide: seawater solutions for each 72-h static 

renewal. 
ENV/JM/MONO(2011)38

44

 Complaints about time and effort required on some weekends to score plates and/or perform 

water changes and feedings when the third or sixth static-renewal day falls on a Saturday/Sunday. 

(Note: fewer complaints with experience and facility with the method).

 Accidental aspiration of larval/juvenile copepods during water changes.

 Dessication of copepods during water changes (either by open exposure to atmosphere for 

extended periods or leaving copepods too long under warm incandescent illumination).

 Difficulty with male: female sex determinations for proper construction of mating pairs. (Note: 

Seemed to be an issue for labs in their first attempt at running the microplate test. Abilities 

improved with experience).

 Insufficient or inferior microscope optics for microplate reading, sex determination, and clutch 

size counting. Two labs reported difficulty seeing/counting the second clutch of new nauplii 

after the first clutch had hatched. (Note: Research quality dissection stereomicroscope with 

darkfield lighting and fiber optics, and a Hoffman DIC inverted compound microscope (4X, 10X 

and 20X) are highly recommended and make the assay much easier to perform. Optics are 

everything when working with harpacticoid copepods.)

 Lack of electronic particle counter for easy algal cell density determinations of food ration (i.e., 

recommended ~20,000 cells per microwell feeding). 

CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER WORK

38. In June 2009, the Invertebrate Testing Expert Group met and reviewed the results and status of 

the validation of the copepod development and reproduction tests presented in this report. Several issues 

were addressed in the discussion: 1) the issue of proficiency and training in the application of the test 

method, 2) the absence of effects on most of the endpoints reported in the atrazine study, 3) the possible 

issue of sensitivity of the population of Amphiascus tenuiremis used in the study, 4) the influence of 

mortality (males more sensitive than females) on the sex ratio measured, 5) the expression of test results 

(NOEC/LOEC) based on nominal concentrations rather than measured concentrations, and finally 6) the 

manpower required in the study (50% time of a technician). Despite a recommendation from the group to 

collect the remaining data, laboratories with incomplete experiments were not able to finalize the 

recommended work.

39. The meeting encouraged the lead countries (Sweden and the United States) to further investigate 

the endpoints reliability through further experimental work. The following endpoints: mortality, fecundity 

(number of viable offspring in 2 broods), and development rate from nauplius to copepodite were 

considered to be important and reliable endpoints of the test method. The meeting also recommended 

performing the test method for controls alone to reassure that the test performance and the validity criteria 

set out in the revised draft TG can be met by the laboratories with no problem.
ENV/JM/MONO(2011)38

45

40. An effort led by the United States to carry out additional validation work is currently underway. 

The issues raised in this report in relation to consistent guideline execution, relative sensitivity and best 

selection of endpoints, and consistency of endpoint measurement across laboratories will hopefully be 

addressed in the validation study coordinated by the United States.

41. Additional recommendations for further work:

 There is a need for more information on the basic biology of this test species. It would be very 

useful to have more specific information on how different feeding levels affect the life history 

progression of this copepod.

Bibliography of Publications using the Copepod Microplate Test Method:

Templeton, R.C., P.L. Ferguson, K.M. Washburn, W.A. Scrivens and G.T. Chandler. 2006. Lifecycle 

effects of single-wall carbon nanotubes (SWNTs) on an estuarine meiobenthic copepod. Environmental 

Science and Technology, Special Issue on Emerging Contaminants, 40:7387-7393.

Bejarano, A.C., G.T. Chandler, L.He, T.L. Cary and J.L. Ferry. 2006. Risk assessment of the NIST 

petroleum crude oil standard water accommodated fraction (WAF): Further application of a copepodbased full life-cycle bioassay. Environmental Toxicology and Chemistry 25:1953-1960.

Bejarano, A.C., G.T. Chandler, L. He, and B.C. Coull. 2006. Individual to population effects of South 

Louisiana crude-oil water hydrocarbon accommodated fraction (WAF) on a marine meiobenthic 

copepod. Journal of Experimental Marine Biology and Ecology 332:49-59.

Bejarano, A.C., G. T. Chandler and A.W. Decho. 2005. Influence of natural dissolved organic matter 

(DOM) on acute and chronic toxicity of the pesticides chlorothalonil, chlorpyrifos and fipronil on the 

meiobentic estuarine copepod Amphiascus tenuiremis. J. Experimental Marine Biology and Ecology 

321:43-57.

Chandler, G.T., T.L. Cary, A.C. Bejarano, J.Pender and J.L. Ferry. 2004. Population consequences of 

fipronil and degradates to copepods at field concentrations: an integration of lifecycle testing with 

Leslie-matrix population modeling. Environmental Science and Technology 38:6407-6414.

Chandler, G.T. 2004. Standard Guide for Conducting Renewal Microplate-Based Life-Cycle Toxicity 

Tests with a Marine Meiobenthic Copepod. In: (S.J. Bailey et al., eds.) ASTM E 2317-04, American 

Society for Testing and Materials, W. Conshohocken, PA, USA, 15 pp.

Chandler, G.T., T.L. Cary, D.C. Volz, S.S. Walse, J.L. Ferry, and S.L. Klosterhaus. 2004. Fipronil effects 

on copepod development, fertility, and reproduction: A rapid life-cycle assay in 96-well microplate 

format. Environmental Toxicology and Chemistry 23:117-124. 

Bejarano, A.C., K.A. Maruya and G.T. Chandler. 2004. Toxicity assessment of sediments associated with 

various land uses in coastal South Carolina, USA, using a meiobenthic copepod bioassay. Marine 

Pollution Bulletin 49:23-32.

Cary, T.L., G.T. Chandler, D.C. Volz, S.S. Walse, and J.L. Ferry. 2004 Fipronil induces male infertility in 

the meiobenthic harpacticoid copepod, Amphiascus tenuiremis. Environmental Science and 

Technology 39:522-528.
ENV/JM/MONO(2011)38

46

Bejarano, A.C. and G.T. Chandler. 2003. Reproductive and developmental effects of atrazine on the 

estuarine meiobenthic copepod, Amphiascus tenuiremis. Environmental Toxicology and Chemistry 

22:3009-3016.

Bejarano A.C., A.Widenfalk, A.W. Decho and G.T. Chandler. 2003. Bioavailability of the 

organophosphorous insecticide chlorpyrifos to the suspension-feeding bivalve, Mercenaria 

mercenaria, following exposure to dissolved and particulate matter. Environmental Toxicology and 

Chemistry 22:2100-2105.

Block, D.S., A.C. Bejarano and G.T. Chandler. 2003. Ecdysteroid distribution through various life-stages 

of the meiobenthic harpacticoid copepod, Amphiascus tenuiremis, and the benthic estuarine amphipod, 

Leptocheirus plumulosus. General and Comparative Endocrinology 132:151-160.
ENV/JM/MONO(2011)38

47

ANNEX 1

ADDITIONAL ATRAZINE RESULTS

Table 1: Percent Naupliar Mortality

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 6.8% ± 3.9

(ND)

8.3% 10.4

(ND)

33.4% ± 9.7

(ND) --

Carrier 6.3% ± 3.7

(ND)

18.3% 5.8

(ND)

11.9% ± 6.1

(ND) --

26 μg/L 9.2% ± 5.8

26.4 ± 4.0

10.0% 5.0

45.6 ± 12.5

10.3% ± 5.4

26.9 ± 6.6 --

43 μg/L 18.2% ± 6.2

53.0 ± 11.5

13.3% ± 2.9

70.7 ± 22.6

21.7% ± 8.6

74.7 ± 38.9 --

72 μg/L 15.3% ± 11.0

96.8 ± 29.1

3.3% ± 5.8

115.3 ± 22.9

29.4% ± 16.2

92.3 ± 26.9 --

120 μg/L 23.0% ± 11.0

140.3 ± 30.1

6.7% 5.8

163.6 ± 28.0

18.1% ± 4.7

186.0 ± 82.3 --

200 μg/L 9.0% ± 8.1

207.8 ± 25.3

5.2% 0.32

268.4 ± 48.1

25.1% ± 13.4

290.9 ± 88.6 --

Table 2: Copepodite mortality

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 2.8% ± 2.8

(ND)

5.1% 5.0

(ND)

0.0% ± 0.0

(ND) --

Carrier 0.0% ± 0.0

(ND)

0.0% 0.0

(ND)

0.0% ± 0.0

(ND) --

26 μg/L 6.4% ± 3.6

26.4 ± 4.0

0.0% 0.0

45.6 ± 12.5

0.0% ± 0.0

26.9 ± 6.6 --

43 μg/L 3.5% ± 0.1

53.0 ± 11.5

0.0% ± 0.0

70.7 ± 22.6

0.0% ± 0.0

74.7 ± 38.9 --

72 μg/L 7.3% ± 0.8

96.8 ± 29.1

1.9% ± 3.2

115.3 ± 22.9

0.0% ± 0.0

92.3 ± 26.9 --

120 μg/L 1.1% ± 1.9

140.3 ± 30.1

1.9% 3.2

163.6 ± 28.0

4.2% ± 7.2

186.0 ± 82.3 --

200 μg/L 3.4% ± 3.3

207.8 ± 25.3

0.0% 0.0

268.4 ± 48.1

2.6% ± 4.4

290.9 ± 88.6 --
ENV/JM/MONO(2011)38

48

Table 3: Adult stage mortality

Lab

Atrazine

USC NOAA Stockholm Uppsala

Control 2.8% ± 2.8

(ND)

5.1% 5.0

(ND)

0.0% ± 0.0

(ND) --

Carrier 0.0% ± 0.0

(ND)

0.0% 0.0

(ND)

0.0% ± 0.0

(ND) --

26 μg/L 6.4% ± 3.6

26.4 ± 4.0

0.0% 0.0

45.6 ± 12.5

0.0% ± 0.0

26.9 ± 6.6 --

43 μg/L 3.5% ± 0.1

53.0 ± 11.5

0.0% ± 0.0

70.7 ± 22.6

0.0% ± 0.0

74.7 ± 38.9 --

72 μg/L 7.3% ± 0.8

96.8 ± 29.1

1.9% ± 3.2

115.3 ± 22.9

0.0% ± 0.0

92.3 ± 26.9 --

120 μg/L 1.1% ± 1.9

140.3 ± 30.1

1.9% 3.2

163.6 ± 28.0

4.2% ± 7.2

186.0 ± 82.3 --

200 μg/L 3.4% ± 3.3

207.8 ± 25.3

0.0% 0.0

268.4 ± 48.1

2.6% ± 4.4

290.9 ± 88.6 --
ENV/JM/MONO(2011)38

49

ANNEX 2

ADDITIONAL LINDANE RESULTS

Table 1. Naupliar mortality

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 2.6% ± 0.4

(ND)

2.2% ± 1.9

(ND)

5.0% ± 5.0

(ND)

12.7% ± 6.2

(ND)

Carrier 3.4% ± 2.9

(ND)

3.3% ± 3.3

(ND)

8.8% ± 11.0

(ND)

2.0% ± 3.4

(ND)

2.5 μg/L 1.7% ± 2.9

3.9 ± 1.4

23.3% ± 8.8

5.1 ± 2.9

1.7% ± 2.9

2.2 ± 0.8

41.7% ± 36.3

7.0 + 1.6

5 μg/L 5.1% ± 4.3

7.6 ± 3.3

31.1% ± 15.0

7.5 ± 4.6

8.5% ± 5.6

4.7 ± 2.0

10.9% ± 5.8

8.9 + 4.2

10 μg/L 28.8% ± 4.1 *

12.7 ± 4.2

78.9% ±15.0 *

15.9 ± 7.7

13.8% ± 13.2

7.6 ± 3.4

44.9% ± 18.9 *

16.7 + 6.7

15 μg/L 12.8% ± 10.1 *

23.4 ± 7.6

74.4% ± 1.9 *

28.3 ± 15.1

8.5% ± 10.3

11.6 ± 3.8

15.5% ± 12.5

29.3 + 9.4

20 μg/L 26.9% ± 5.1 *

23.2 ± 5.1

54.4% ± 10.2 *

36.7 ± 20.9

16.7% ± 17.6 *

15.7 ± 7.3

10.8% ± 9.4

53.2 + 9.1

Table 2. Copepodite mortality

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 0.9% ± 1.5

(ND)

0.0% ± 0.0

(ND)

1.7% ± 3.0

(ND)

0.0% ± 0.0

(ND)

Carrier 0.0% ± 0.0

(ND)

0.0% ± 0.0

(ND)

1.9% ± 3.2

(ND)

0.0% ± 0.0

(ND)

2.5 μg/L 2.7% ± 2.7

3.9 ± 1.4

0.0% ± 0.0

5.1 ± 2.9

1.8% ± 3.0

2.2 ± 0.8

0.0% ± 0.0

7.0 + 1.6

5 μg/L 3.6% ± 4.1

7.6 ± 3.3

1.6% ± 2.7

7.5 ± 4.6

2.1% ± 3.6

4.7 ± 2.0

0.0% ± 0.0

8.9 + 4.2

10 μg/L 5.2% ± 1.7

12.7 ± 4.2

0.0% ± 0.0

15.9 ± 7.7

0.0% ± 0.0

7.6 ± 3.4

0.0% ± 0.0

16.7 + 6.7

15 μg/L 6.6% ± 5.8

23.4 ± 7.6

0.0% ± 0.0

28.3 ± 15.1

0.0% ± 0.0

11.6 ± 3.8

0.0% ± 0.0

29.3 + 9.4

20 μg/L 14.4% ± 5.5 *

23.2 ± 5.1

2.6% ± 4.4

36.7 ± 20.9

0.0% ± 0.0

15.7 ± 7.3

2.2% ± 3.9

53.2 + 9.1
ENV/JM/MONO(2011)38

50

Table 3. Adult-stage mortality

Lab

Lindane

USC NOAA Stockholm Uppsala

Control 1.8% ± 1.6

(ND)

0.0% ± 0.0

(ND)

5.6% ± 9.6

(ND)

0.0% ± 0.0

(ND)

Carrier 7.5% ± 1.8

(ND)

3.5% ± 3.4

(ND)

4.1% ± 3.7

(ND)

0.0% ± 0.0

(ND)

2.5 μg/L 3.0% ± 0.3

3.9 ± 1.4

8.7% ± 4.5

5.1 ± 2.9

1.7% ± 2.9

2.2 ± 0.8

0.0% ± 0.0

7.0 + 1.6

5 μg/L 2.1% ± 1.9

7.6 ± 3.3

13.9% ± 5.5

7.5 ± 4.6

0.0% ± 0.0

4.7 ± 2.0

0.0% ± 0.0

8.9 + 4.2

10 μg/L 1.3% ± 2.3

12.7 ± 4.2

6.7% ±11.6

15.9 ± 7.7

10.3% ± 7.4

7.6 ± 3.4

0.0% ± 0.0

16.7 + 6.7

15 μg/L 1.9% ± 3.2

23.4 ± 7.6

28.1% ± 19.2 *

28.3 ± 15.1

0.0% ± 0.0

11.6 ± 3.8

0.0% ± 0.0

29.3 + 9.4

20 μg/L 0.0% ± 0.0

23.2 ± 5.1

6.1% ± 10.5

36.7 ± 20.9

5.3% ± 9.1

15.7 ± 7.3

2.1% ± 3.6

53.2 + 9.1

Figure 1. USC Leslie matrix F1 stage structure (stage abundance in white)

0% 50% 100%

SOLV CON

2.5 PPB

10 PPB

20 PPB

212

117

54

0

202

115

37

12

115

53

26

25

77

29

15

0

NAUPLII (DAY 18)

COPEDITES (DAY 24)

BARRENS (DAY 32)

GRAVIDS (DAY 34) 
ENV/JM/MONO(2011)38

51

Figure 2. NOAA Leslie matrix F1 stage structure (stage abundance in white) 

0% 50% 100%

SOLV CON

2.5 PPB

10 PPB

20 PPB

888

578

14

9.8

858

454

5.2

5

433

277

9.7

32

412

248

2.4

3

NAUPLII (DAY 18)

COPEDITES (DAY 24)

BARRENS (DAY 32)

GRAVIDS (DAY 34) 

Figure 3. STOCKHOLM Leslie matrix F1 stage structure (stage abundance in white)

0% 50% 100%

SOLV CON

2.5 PPB

10 PPB

20 PPB

345

355

55

14

328

350

51

12.6

83

91

14

8.2

75

84

7

1.5

NAUPLII (DAY 18)

COPEDITES (DAY 24)

VIRGINS (DAY 32)

GRAVIDS (DAY 34) 
ENV/JM/MONO(2011)38

52

Figure 4. UPPSALA Leslie matrix F1 stage structure (stage abundance in white)

0% 50% 100%

SOLV CON

2.5 PPB

10 PPB

20 PPB

49

13.5

56

68

48

10.3

41

63

22

4.5

10

18

1.5

0.9

2

3

NAUPLII (DAY 18)

COPEDITES (DAY 24)

VIRGINS (DAY 32)

GRAVIDS (DAY 34) 

Figure 5. USC Leslie matrix F1 stage abundances

1 10 100 1000

SOLV CON

2.5 PPB

10 PPB

20 PPB

212

117

54

1

202

115

37

12

115

53

26

25

77

29

15

1

GRAVID BARREN COPEPODITES NAUPLII
ENV/JM/MONO(2011)38

53

Figure 6. NOAA Leslie matrix F1 stage abundances

1 10 100 1000

SOLV CON

2.5 PPB

10 PPB

20 PPB

888

578

14

9.8

858

454

5.2

5

433

277

9.7

32

412

248

2.4

3

GRAVID BARREN COPEPODITES NAUPLII

Figure 7. STOCKHOLM Leslie matrix F1 stage abundance

1 10 100 1000

SOLV CON

2.5 PPB

10 PPB

20 PPB

345

355

55

14

328

350

51

12.6

83

91

14

8.2

75

84

7

1.5

GRAVID BARREN COPEPODITES NAUPLII

Figure 8. UPPSALA Leslie matrix F1 stage abundance 

1 10 100 1000

SOLV CON

2.5 PPB

10 PPB

20 PPB

49

13.5

56

68

48

10.3

41

63

22

4.5

10

18

1.5

0.94

2.3

3

GRAVID BARREN COPEPODITES NAUPLII