Unclassified ENV/JM/MONO(2007)26

Organisation de Coopération et de Développement Economiques 

Organisation for Economic Co-operation and Development 31-Aug-2007 

___________________________________________________________________________________________

English - Or. English 

ENVIRONMENT DIRECTORATE 

JOINT MEETING OF THE CHEMICALS COMMITTEE AND 

THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGY 

 

SERIES ON TESTING AND ASSESSMENT 

Number 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 

JT03231276 

Document complet disponible sur OLIS dans son format d'origine 

Complete document available on OLIS in its original format 

Unclassified ENV/JM/MONO(2007)26 English - Or. English 
ENV/JM/MONO(2007)26 

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 ENV/JM/MONO(2007)26 

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OECD Environment, Health and Safety Publications 

Series on Testing and Assessment 

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 

Environment Directorate 

Organisation for Economic Co-operation and Development 

2007 
ENV/JM/MONO(2007)26 

 4

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(2007)26 

 5

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(2007)26 

 6

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, Draft Guidance Document on Reproductive Toxicity 

Testing and Assessment (in preparation) 

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 Longrange Transport (2004) 
 ENV/JM/MONO(2007)26 

 7

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) 

Structure-Activity 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) 
ENV/JM/MONO(2007)26 

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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 fort he 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 One (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 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)

No. 74, Detailed Review Paper for Avian Two-generation 

Testing (2007)
 ENV/JM/MONO(2007)26 

 9

No. 75, Guidance Document on the Honeybee (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)

© OECD 2007 

Applications for permission to reproduce or translate all or part of 

this material should be made to: Head of Publications Service, 

OECD, 2 rue André-Pascal, 75775 Paris Cedex 16, France 
ENV/JM/MONO(2007)26 

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ABOUT THE OECD 

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

organisation in which representatives of 30 industrialised countries in North 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 the 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 

(http://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 Organizations. 

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 coordination in the field of chemical safety. The participating organisations are FAO, ILO, 

OECD, UNEP, UNIDO, UNITAR and WHO. The World Bank and UNDP are observers. 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(2007)26 

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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 
ENV/JM/MONO(2007)26 

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FOREWORD 

This document presents the report of the Phase 1 of the validation of the copepod development 

and reproduction test. 

A Standard Project Submission Form, proposing to develop a Test Guideline on the copepod 

development and reproduction test, was presented by Sweden at the 14th meeting of the Working Group of 

National Coordinators of the Test Guidelines Programme (WNT) in 2002. The project was included on the 

work plan of the Test Guidelines Programme. 

An OECD Invertebrate Expert Group was established in 2002 to follow the development and 

validation of test methods on invertebrate species. The group met for the first time in October 2003 and 

developed a proposal for the pre-validation of the test, using 3 species of harpacticoid copepods and one 

species of calanoid copepods, both groups of copepods being of ecological interest. Pre-validation work 

was led by Sweden and Denmark. The outcome of the pre-validation was presented to the Invertebrate 

Expert Group for discussion at its second meeting in November 2005. The protocol was streamlined for the 

validation exercise and a reference substance: 3,5-dichlorophenol was chosen by the Invertebrate Expert 

Group. 

The Phase 1 experimental work was undertaken on two species of harpacticoid copepods 

(Amphiascus tenuiremis and Nitocra spinipes) and on one species of calanoid copepods (Acartia tonsa). 

The outcome of the validation was presented to the Validation Management Group on Ecotoxicity testing 

(VMG-eco) in January 2007. The Task Force on Endocrine Disrupters Testing and Assessment and the 

WNT endorsed the report in March 2007. The validation work will continue in 2007-2008 to fully 

demonstrate the reproducibility of the test. 

This document is published on the responsibility of the Joint Meeting of the Chemicals 

Committee and Working Party on Chemicals, Pesticides and Biotechnology. 
 ENV/JM/MONO(2007)26 

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TABLE OF CONTENTS 

FOREWORD................................................................................................................................................ 12

ACKNOWLEDGEMENT............................................................................................................................ 14

ABSTRACT ................................................................................................................................................. 15

1. INTRODUCTION .................................................................................................................................... 17

1.1 Rationale for the Proposed Test Method Validation ........................................................................... 17

1.2 Experiences from the pre-validation using 3,5-Dichlorophenol ......................................................... 18

1.3 Scheduled work plan ........................................................................................................................... 20

1.4 Ring test .............................................................................................................................................. 20

2. MATERIALS AND METHOD................................................................................................................ 24

2.1 Full life cycle tests............................................................................................................................... 24

2.2 Chemical analysis................................................................................................................................ 24

2.3 Statistical analysis ............................................................................................................................... 25

3. RESULTS AND DISCUSSION............................................................................................................... 26

3.1 Harpacticoids....................................................................................................................................... 27

3.2 Calanoid .............................................................................................................................................. 39

3.3 Compilation of NOEC and LOEC for both groups of species ............................................................ 43

3.4 Chemical analysis of 3,5-DCP – potential reasons for declining levels.............................................. 44

3.5 Water quality within test vessels......................................................................................................... 48

4. SUMMARY AND CONCLUSIONS ....................................................................................................... 50

5. REFERENCES ......................................................................................................................................... 52

APPENDIX A............................................................................................................................................... 55

APPENDIX B: VALIDATION OF THE COPEPOD DEVELOPMENT AND REPRODUCTION TEST, 

PHASE 1 - DATA ANALYSIS.................................................................................................................... 62

APPENDIX C: INVITATION FOR PARTICIPATION IN RING TEST OF THE DRAFT OECD TEST 

GUIDELINE “HARPACTICOID AND CALANOID COPEPOD DEVELOPMENT AND 

REPRODUCTION TEST” ........................................................................................................................... 83

APPENDIX D: HARPACTICOID COPEPOD LIFE-CYCLE RING-TEST PROTOCOL ........................ 89

APPENDIX E: DETAILED CALANOID COPEPOD LIFE-CYCLE RING-TEST PROTOCOL............. 97
ENV/JM/MONO(2007)26 

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ACKNOWLEDGEMENT 

This report is the fruit of collaboration between laboratories in several countries: 

• Department of Applied Environmental Science (ITM), Stockholm University, Sweden 

• IVL Swedish Environmental Research Institute, Sweden 

• Toxicon AB, Sweden, 

• Block Environmental Inc., Pleasant Hill, California, USA, 

• NOAA Center for Coastal Environmental Health and Biomolecular Research (CCEHBR), 

Charleston, South Carolina, USA, 

• Institute of Environment & Resources, Technical University of Denmark, 

• DHI Water and Environment (DHI), Agern Allé 5, 2970 Hørsholm, Denmark, 

• Opus Plus Ltd, (OPUS), Flotta, Stromness, Orkney, KW16 3NP, United Kingdom 

• ECT Oekotoxikologie GmbH, Boettgerstr, Germany, 

and has been prepared by 1

M. Breitholtz, 1M. Linde 1J. Minten and 2J-O. Persson. 

1

Department of Applied Environmental Science (ITM), Stockholm University, S-106 91 Stockholm, Sweden 

2

Mathematical Statistics, Stockholm University S-106 91, Stockholm, Sweden 
 ENV/JM/MONO(2007)26 

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ABSTRACT 

With the purpose to develop an OECD Test Guideline (TG) for full-life cycle tests with sexually 

reproducing small crustaceans, ring test activities with the two harpacticoid copepods Nitocra spinipes and 

Amphiascus tenuiremis (OECD 2005b) and the calanoid copepod Acartia tonsa (OECD 2005a) were 

carried out during 2006 with participation of 10 laboratories from Denmark (2), Germany (1), UK (1), 

USA (3) and Sweden (3). Eight of these laboratories contributed with developmental data, whereas only 

six laboratories contributed with reproduction data. 3,5-dichlorophenol (DCP) was used as a single 

reference substance in the following concentrations: 6.6, 20, 60, 180 and 540 μg/L. 

The present ring test activity shows that most of the participating laboratories have succeeded to 

follow one of the two draft TGs and deliver both developmental and reproduction data, which have been 

treated and analysed at the Department of Applied Environmental Science (ITM) and Mathematical 

Statistics at Stockholm University, Sweden. However, the chemical analyses, which were also performed 

at ITM, showed that there was a significant decline of 3,5-DCP-levels in the test medium over time for all 

laboratories, which makes it difficult to draw thorough conclusions about exact concentration effect levels. 

Nevertheless, since there was a consistent decline evident for all laboratories, comparisons of LOECs were 

still possible based on the initial measured concentrations. 

The interpretational difficulties due to the exposure declines aside, the developmental data were 

generally consistent among all species and laboratories. For the harpacticoids, the LOEC for development 

ranged from 6.6 to 20 μg/L whereas for the calanoids the range was somewhat larger between the three 

participating laboratories; i.e. 6.6 to 180 μg/L. For the harpacticoids the reproduction data was also 

consistent, at least for two out of three “reproductive” endpoints (i.e. “time to first clutch” and “offspring 

production”). The reproductive data for the calanoids varied more, with LOECs ranging from 6.6 to 540 

μg/L. Overall, the LOECs for the developmental data were lower than the corresponding LOECs for 

reproduction. Whether this actually reflects the sensitivity of the species exposed to 3,5-DCP in the present 

ring test is difficult to say since the concentrations were not stable over time. On the other hand, the 

chemical analyses of the test medium samples for some laboratories showed that the levels of 3,5-DCP 

increased as the reproductive phase was reached, but the LOEC for development was still consistently 

lower for the developmental endpoints. 

The test time for the two laboratories that ran the harpacticoid full lifecycle test using N. spinipes 

was over 40 days, which indicates that this species should not be tested over the full life cycle. However, 

the test time for A. tenuiremis, for which the current experimental design has been more commonly used, 

the lifecycle test time was about 25 days. This indicates that A. tenuiremis is more appropriate to use for 

full life cycle testing in the sense that about 15 days reduced test time will significantly reduce the costs of 

testing. 

Taking into consideration the above mentioned findings, and if it is preferred that all the species 

used in the current ring tests are to be included in further validation work, then it is recommended that a 

partial life cycle test, which focuses on the larval and juvenile developmental phases, be used. However, if 

there is considered to be a greater regulatory need for a full life cycle test which includes reproduction 

endpoints, A. tenuiremis should be used as a single harpacticoid species, owing to its much shorter test 

time and more consistent reproductive output and success. For the calanoid A. tonsa, the large inter-
ENV/JM/MONO(2007)26 

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laboratory variability for reproduction illustrates that a partial life cycle test may be a better alternative. 

Other additional reference chemicals should be used for continuing validation work; at least until the 

reason(s) for the 3,5-DCP concentration decline using the current brackish and marine species has been 

thoroughly investigated. More laboratories should also participate in the ring test activities to avoid random 

errors and variability that may influence the overall judgement of the ring test results. Finally, in order to 

estimate more statistically robust EC50 values instead of LOEC/NOECs, non-linear models should be fitted 

to the harpacticoid and calanoid (partial) life cycle data. The Phase 2 validation work should consider the 

difficulties encountered in the Phase 1 studies in the planning of the test protocol and overall testing 

program. 
 ENV/JM/MONO(2007)26 

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1. INTRODUCTION 

1.1 Rationale for the Proposed Test Method Validation 

Copepods in general are often key species in freshwater as well as marine ecosystems. They 

constitute the food and energy link between primary producers and higher-level heterotrophic organisms 

such as macro crustaceans and fish larvae. Thus, they play an important role in the function of aquatic 

ecosystems. 

Harpacticoid copepods comprise more than 3.000 species, are mostly free-living benthic 

organisms, and are usually the second most abundant group of animals (after nematodes) in marine benthic 

communities (Huys et al., 1996). They almost always dominate the gut contents of bottom or phytal 

feeding larval and juvenile fish (Hicks and Coull, 1983). They generally have short generation times, 

which mean that they can be easily studied for the duration of a full life cycle, focusing on all crucial 

ontogenetic stages and life history traits, such as larval development, reproduction, etc.; this is essential for 

characterization of both lethal and sublethal effects of toxicants (Ingersoll et al., 1999; Preston and Snell, 

2001). As copepods in general, harpacticoid copepods have a sexual reproduction; the copulation almost 

always involves the attachment of a sperm sac - spermatophore - by the male to the copulatory pore of the 

female, immediately after the last moult (Wesenberg-Lund, 1937; Huys et al., 1996). Consequently, 

harpacticoid copepods have become one of the most studied metazoan groups in chronic ecotoxicity tests 

(e.g. Hutchinson et al., 1999a, b; Chandler et al. 2001; 2004a, b; Breitholtz and Bengtsson 2001; Breitholtz 

and Wollenberger 2003; Breitholtz et al. 2003). Also, an international standard method (ISO 1997) for 

acute toxicity testing with both harpacticoid and calanoid copepods exists since 1997, including the species 

Acartia tonsa, Tisbe battagliai and Nitocra spinipes. Within the regime of the ASTM (American Society of 

Testing and Materials) a full life cycle test using Amphiascus tenuiremis has also been published recently 

(ASTM 2004). 

At the moment there are no internationally harmonized (i.e. OECD) chronic toxicity test methods 

for marine invertebrates, although they are an ecologically important and large group of organisms that 

need to be protected. Therefore, the development of a test on reproduction and development of marine 

copepods has been added to the work plan of the OECD Test Guideline Program. The copepod life-cycle 

test is urgently needed in several multi-national regulatory systems. It will be of wide applicability and as 

such facilitate international harmonization of risk and hazard assessments. It addresses important endpoints 

and environmental compartments (marine/estuarine/brackish) that are poorly covered by existing methods. 

Two common and regularly used species in toxicity tests were originally proposed (SPSF, 2002) 

as model test organisms, namely the harpacticoid copepods Nitocra spinipes and Tisbe battagliai. Later 

one further harpacticoid species, Amphiascus tenuiremis, and the calanoid copepod Acartia tonsa were 

included. The new guidelines were intended for evaluation of adverse long-term effects (incl. endocrine 

disruptive effects) of various types of chemicals to aquatic invertebrates – e.g. industrial chemicals, 

pesticides and pharmaceuticals, as well as compounds used in off shore oil industry. 
ENV/JM/MONO(2007)26 

 18

1.2 Experiences from the pre-validation using 3,5-Dichlorophenol 

At the time of the copepod test pre-validation, the draft OECD guideline for life-cycle tests also 

included the harpacticoid copepod Tisbe battagliai. Due to difficulties in finding interested laboratories for 

the ring-test activities, this species was excluded after the pre-validation (OECD 2005c). 

Two types of copepod test methods were conducted by five laboratories using 3,5-dichlorophenol 

as a reference compound. Other candidate substances (e.g. fipronil, bisphenol-A, fenoxycarb and 

methoprene), which were believed to serve as possible reference compounds with a specific mode of 

action, were also tested using N. spinipes, but none of these candidate chemicals fulfilled the criteria for 

suitable reference substances (OECD 2005c). Fenarimol, which has been suspected of having ecdysteroid 

disruptive effects in freshwater daphniids (Mu et al 2000; Mu and LeBlanc 2002, 2004) was later also 

excluded from this list owing to “lack” of effects in A. tenuiremis (T. Chandler personal communication). 

1.2.1 Acute toxicity 

For both calanoids and harpacticoids (considering late juvenile or adult stage) the acute toxicity 

(96-hr LC50) of 3,5-DCP is in the range of 1 mg l-1 In A. tenuiremis, the most sensitive life stage was the 

nauplius stage, which resulted in a 96-hr LC50 of 0.38 mg l-1 (95%-CI= 0.16-0.54 mg l-1). This difference in 

juvenile and adult sensitivity to 3,5-DCP was considered when the concentration range for the full life 

cycle tests was set (see 1.2.2 - 1.2.4. below). 

1.2.2 Development 

To compare the sensitivity related to development between A. tonsa and the harpacticoid species, 

we have to make clear that larval/juvenile development ratio, which is often a sensitive test variable in 

toxicity testing using copepods, is measured in two different ways in these groups of species. This is due to 

differences in the experimental set up; A. tonsa is exposed in cohorts and the morphological distinction 

between the nauplius and copepodite stages (metamorphosis occurs between these two life stages) is used 

to measure effects on development (termed larval development ratio). The larval development ratio gives a 

picture of the mean developmental status among a large group of animals, but may not necessarily 

represent the mean development rate since only the ratio between the nauplius and copepodite stages at an 

arbitrarily chosen point in time is calculated. In the harpacticoid tests exposed and control copepods are 

held and tested individually. This means that the exact number of days for an individual to develop into the 

copepodite stages or the adult stage, respectively, is observed and this represents a rate. The larval 

development ratio may be presented using such data, but since the copepods are kept individually, one 

must calculate the ratio of copepodite- to naupliar-stage individuals in each replicate microplate at some 

fixed time interval (e.g., at day 6 for N. spinipes and at day 7 for A. tenuiremis) averaged across 

microplates. Consequently, such data collected at the fundamental level of replication (i.e. the microplate 

or the beaker for calanoids) may be used to do further statistical analysis. The number of microplate or 

beaker replicates used, the number of copepods tested, and the statistical sampling error (variance) will 

determine statistical power to reject the null hypothesis of no toxicant concentration effect relative to the 

toxicant-free control. 

If stage-specific mortality from a test compound is the same for calanoids and harpacticoids, then 

these two test endpoint variables indicate the same types of effects on development (a non-mortality based 

endpoint). It is therefore possible to compare the lowest observed effect concentrations (LOECs) for larval 

development ratio (in F0-calanoids or harpacticoids) and/or mean development rates (in F0-harpacticoids). 

The results from the tests with 3,5-DCP show that there are species differences but these differences cannot 

be generalized to indicate different sensitivity in the two taxonomic groups (i.e. calanoids vs 

harpacticoids). In A. tonsa the two (DTU vs DHI) LOECs for inhibition of larval development ratio was 
 ENV/JM/MONO(2007)26 

 19

100 and 220 μg/L, respectively. Among the harpacticoids, the LOEC for the test with A. tenuiremis was 

18.4 μg/L, which caused a significant delay in the development from the first nauplius stage to the first 

copepodite stage. Mean larval and juvenile developmental rate was also suppressed in N. spinipes but only 

in the highest concentration tested (i.e. 200 μg/L). In T. battagliai none of the two test rounds showed 

significant responses on delayed larval development up to and including 560 μg/L. This means that related 

to delay of mean larval/juvenile development, A. tenuiremis was the most sensitive species, followed by A. 

tonsa and N. spinipes. T. battagliai was the least sensitive species in these tests. 

1.2.3 Reproduction 

Since the test variables related to reproduction differ widely between the two protocols, it is 

difficult to make comparisons. In the harpacticoid protocol, the main test variables related to reproduction 

are fertilization success (i.e. number of mating pairs able to produce two successive clutches), total viable 

offspring production per mating pair (i.e. offspring through two clutches averaged over the number of 

mating pairs created and tested), time to production of first clutch and time interval between successive 

clutches. In A. tonsa, it is mainly the egg production rate that is used as a measure of reproductive effects. 

However, the use of this test variable with A. tonsa caused some concerns because of the low number of 

females present in each group making the statistics quite uncertain due to large confidence intervals. Thus, 

it was not possible from these experiments to evaluate if this was a proper test variable to examine with A. 

tonsa. As a consequence the method for measuring egg production is changed in the next draft guideline. 

As for reproduction in the harpacticoid copepods, none of the < 200 μg/L 3,5-DCP 

concentrations had significant effects on fertilization success or total viable offspring production per 

female in A. tenuiremis. However, the reproductive success (and thereby offspring production) of A. 

tenuiremis was significantly lowered (75%) at 200 μg l-1 with only one of four mating pairs able to produce 

two clutches. Likewise, for N. spinipes, total offspring production was significantly lowered when 

considering that in 200 μg l-1 no mating pairs were able to produce offspring. Otherwise the time to hatch 

and number of offspring produced per hatch at lower concentrations were not significantly different from 

the control. In T. battagliai significant reductions in offspring production at nominal 18, 180 and 560 μg l-1

test concentrations were observed compared with the control. However, no effects were observed at 56 μg 

l

-1. Irrespective of the deviation from dose response at 56 μg l-1, T. battagliai was the least sensitive 

species with regard to fertility (i.e., highest ability to produce clutches under 3,5-DCP exposure), but the 

most sensitive species with regard to offspring production by those females able to reproduce. 

1.2.4 Conclusions 

The comparison of results between the different harpacticoid and calanoid species should be 

treated with caution as such comparisons are based on a very small data set which was analyzed using 

different statistical techniques that makes direct comparison difficult. The sensitivity to 3,5-DCP between 

the four species differed depending on specific biological (e.g. physiology, metabolism, morphology) and 

ecological (e.g. life strategies) features of the respective species, and the endpoints chosen for analysis. 

Still, the results were within the same order of magnitude and the same concentration range was chosen for 

the ring test, i.e. 6.6, 20, 60, 180 and 540 μg/L 3,5-DCP.

To be able to find laboratories that were interested in participating (i.e. to reduce the work load) it 

was after the pre-validation at the OECD Invertebrate expert meeting in Paris 2005 decided that we should 

only proceed with 3,5-DCP as a reference substance. 
ENV/JM/MONO(2007)26 

 20

1.3 Scheduled work plan 

In June 2003 a Nordic Expert group under NordUtte prepared a proposal for a new guideline 

“OECD draft Guideline for Testing of Chemicals – Copepod Development and Reproduction Test”. This 

guideline describes testing of effects on development and reproduction of marine harpacticoid and calanoid 

copepods in full life-cycle tests. The method is intended also to cover effects of potential endocrine 

disrupters. 

This draft guideline was discussed at the first OECD meeting of the ad hoc expert group on 

invertebrate testing in November 2003 in Paris (OECD 2003). At this meeting it was decided to separate 

the first draft into two – one with the benthic species (Nitocra spinipes, Amphiascus tenuiremis and Tisbe 

battagliai) and one with the pelagic species Acartia tonsa. This was a consensus due to differences in 

biology leading to differences in methods, especially with regard to handling of animals, feeding and 

media, which made it difficult to include both harpacticoid and calanoid copepods in the same guideline. It 

was decided that the drafts should be ready by the end of 2003 and be commented within the first two 

months of 2004. A pre-validation test should start in spring 2004. OECD planned to finalise the prevalidation work by June 2004 followed by an inter-laboratory study on the two draft guidelines. The 

suggested time schedule could however not be met owing to a number of unforeseeable reasons. 

Revised drafts for the two test guidelines (with harpacticoid and calanoids) were prepared in 

December 2003 by Magnus Breitholtz, Stockholm University, and K. Ole Kusk and Leah Wollenberger, 

Technical University of Denmark, respectively, and sent out for commenting in January 2004 by OECD. 

The received comments on the method with harpacticoid were compiled and responded to in May 2004. 

Owing to a late response from one of the invertebrate experts, the draft was not fully responded to until 

December 2004. After the pre-validation small changes was made to the harpacticoid draft (assembled with 

some specific recommendations). The calanoid draft was also updated: some corrections were made in the 

validity criteria (e.g. mortality rate in controls was increased from 20% to 30%) and the egg production 

was observed by the end of the test (day 15-18 instead of day 7-12). 

1.4 Ring test 

OECD sent out an invitation letter (see Appendix C) to potential participants in the ring test. Labs 

from five countries (i.e. UK, USA, Denmark, Sweden and Germany; see below for details) have been 

involved in the ring test of the copepod Test Guideline. 

The actual ring-test started in the spring of 2006 and followed the schedule outlined below (see 

Table 1). 
 21

Table 1 

 2006  March April May June July August September Sending out 3,5-DCP x x x Nitocra Testing x x Sending raw data to ITM (deadline) x (May 31) Handling of raw data x x Sending test medium to ITM x x Analyses of test medium ITM Amphiascus Testing x x Sending raw data to ITM (deadline) x (June 10) Handling of raw data x x Sending test medium to ITM x Analyses of test medium ITM x x Acartia Testing x x Sending raw data to ITM (deadline) x (June 30) Handling of raw data x x Sending test medium to ITM x Analyses of test medium ITM x x Writing report: Harpacticoid part x x Writing report: Calanoid part x Writing report: Comparison x Sending report to Nord-Utte/OECD Potential revision Sending final report OECD 
ENV/JM/MONO(2007)26 

 22

1.4.1 Reference chemical 

At the expert meeting in OECD November 2003 (OECD 2003) it was decided to use two 

chemicals in the pre-validation work and perform the full test with both of them; but, according to the 

above-mentioned discussion (Section 1.2), only one reference substance was used in this ring-test activity: 

3,5-dichlorophenol (DCP) 

3,5-dichlorophenol is often used as a reference compound in various standardised toxicity test 

methods. It is water soluble, not ready biodegradable, non-volatile, and has a relatively low Kow. This 

means that the compound is easy to handle and is expected to stay in the water phase for the duration of the 

test period. 

Physical-chemical properties, toxicity and fate data of 3,5-DCP are presented below. 

Physical-chemical properties: 

CAS Number: 591-35-5 

Molecular weight: 163.0 

Water solubility: 5380 mg/L (25 °C) (Huyskens, P et al.1975) 

MP (deg C): 68 (Hansch, C et al. 1995) 

BP (deg C): 233 (Hansch, C et al.. 1995) 

Log Kow: 3.62 (Hansch, C et al. 1995) 

Vapor Pressure: 0.00842 mm Hg (extrapol.) (25 °C) (Shiu, WY et al.1994) 

pKa: 8.36 (SPARC on-line calculator) 

Toxicity data: (US EPA ECOTOX database except otherwise indicated) 

Acartia tonsa 48 h LC50: 0.51 mg/L 

Acartia tonsa 48 h LC50: 0.95 mg/L (ISO 14669) 

Acartia tonsa 48 h LC50: 1.00 mg/L (Bjørnestad et al. 1993) 

Crangon septemspinosa 96 h LC50: 1.50 mg/l 

Carassius auratus (Goldfish) 96 h LC50: 3-5 mg/L 

Zebra fish 24 h LC50: 2.9 mg/L (Zarorc-Konĉan et al 2002) 

Tisbe battagliai 24 h LC50: 10.74 μM (1.75 mg/L) 

Daphnia magna 48 h EC50: 2.48 mg/L (Zarorc-Konĉan et al 2002) 

Fate data: 

Test for ready biodegradability: 5% degradation in 28 days. (Zarorc-Konĉan et al 2002) 
ENV/JM/MONO(2007)26 

 23

1.4.2 Participants in the ring test 

The following laboratories participated in the ring test of the harpacticoid and calanoid draft TG: 

Harpacticoids 

Nitocra spinipes

• Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91 

Stockholm, Sweden. Contact: magnus.breitholtz@itm.su.se. 

• IVL Swedish Environmental Research Institute, Box 210 60, SE-100 31 Stockholm, Sweden. 

Contact: ann-sofie.allard@ivl.se. 

• Toxicon AB, SE-261 92 Landskrona, Sweden. Contact: anders.sjolin@toxicon.com. 

Amphiascus tenuiremis

• Department of Environmental Health Sciences, Arnold School of Public Health, University of 

South Carolina, SC-29208, USA. Contact: tchandler@sc.edu [The University of South Carolina 

participated as a lead laboratory for guidance because of their experience in developing the initial 

protocol for A. tenuiremis but did not perform an experiment in the Phase 1.] 

• Block Environmental Inc., Pleasant Hill, California, USA. Contact: dblock@blockenviron.com 

• The NOAA Center for Coastal Environmental Health and Biomolecular Research (CCEHBR), 

Charleston, South Carolina, USA. Contact: geoff.scott@noaa.gov

Calanoid 

Acartia tonsa

• Institute of Environment & Resources, Technical University of Denmark (E&R), Building 113, 

2800 Lyngby, Denmark. Contact: K. Ole Kusk; kok@er.dtu.dk

• DHI Water and Environment (DHI), Agern Allé 5, 2970 Hørsholm, Denmark. Contact Estelle 

Bjørnestad; esb@dhi.dk

• Opus Plus Ltd, (OPUS), Flotta, Stromness, Orkney, KW16 3NP, United Kingdom. Contact 

Brenda Hudson; brenda.hudson@opusplus.co.uk

• ECT Oekotoxikologie GmbH, Boettgerstr. 2-14 D-65439 Floersheim, Germany. Contact: 

Michael Meller; m-meller@ect.de
ENV/JM/MONO(2007)26 

 24

2. MATERIALS AND METHOD 

2.1 Full life cycle tests 

For the methods used we refer to the two draft harpacticoid test guidelines (OECD 2005a, b; See 

also the invitation letter in Appendix C). 

Before the ring test started the participating laboratories also received detailed ring test plans (see 

Appendix D and E). 

2.2 Chemical analysis 

Determination of chlorophenolics in water samples 

All test medium samples that were sent to ITM were shipped frozen. The samples were stored in 

freezer until analyses. 

For improved detection by GC-ECD the extracts were derivatised by extractive acylation with 

pentafluorobenzoyl chloride (PFBCl). The derivatisation method used is based on the methods reported by 

Renberg 1981; Kuch and K. Ballschmiter 2001. 

A surrogate standard (2,4-dichlorophenol) was added to the water sample (2mL). Then 50 μl of 

2M KOH and 10 μl of a 10 % (v/v) solution of pentafluorobenzoyl chloride (PFBCl) in toluene were 

added. The derivatives were generated and extracted by shaking for 2 min with 2 ml of n-hexane 

containing a volumetric standard (CB 189). The hexane phase was analysed by GC/ECD. 

Standard samples were prepared in the same way and standard solutions in five different 

concentrations (ranging from 5 to 40 pg/μl) were analysed in the beginning and at the end of each batch of 

samples. Concentrations were calculated using the surrogate standard. Calibration curves were linear over 

the concentration range studied with coefficients of correlation greater than 0.980. The regression plots 

appeared linear and the residual plots did not show any significant pattern. The residual sum squares were 

not greater than 0.178. 

The limit of quantification (LOQ), calculated as signal to noise ratio (S/N) of 10, was 3 pg/μL 

and the limit of detection (LOD), calculated as an S/N ratio of 3, was 1 pg/μL. 

The repeatability (inter-day) was calculated from a standard of 20 pg/μL 3,5-dichlorophenol that 

also was analysed as a part of the calibration curves analysed with every batch. The mean was 20 pg/μL, 

the standard deviation 3.2 and the relative standard deviation (RSD) 16%.
ENV/JM/MONO(2007)26 

 25

F

F

F

F

F

Cl

O

HO

Cl

Cl

F

F

F

F

F

O

O

Cl

Cl + <

pentafluorobenzoyl 

 chloride 3,5-dicholorophenol pentafluorobenzoyl ester 

2.3 Statistical analysis 

All data was delivered in Excel format from ITM and then transferred to, and analysed with the 

statistical software Stata version 8 (http://www.stata.com). Most of the statistical analyses chosen for each 

group of species and endpoint are discussed in the results and Discussion section. Still, specific 

explanations to some of the chosen statistical analyses are presented below. 

For the harpacticoid copepods, the development rates had to be estimated for the different 

development stages (nauplii to copepodite and copepodite to adult) separately. The developmental data 

should also be treated as survival data where the outcome variable is copepodite/adult (yes or no), and 

where censored observations are present since the time to copepodite/adult is not observed for every 

individual. A first approach to the analysis was to fit a parametric survival function to data (lognormal or 

gamma), but it did not fall out well. The reason for that could be that these particular data are not pure 

survival data; there may perhaps be individuals that never become copepods or adults even if we follow 

them to infinity. In order to take this into account, a more sophisticated model would have been needed. As 

a simpler alternative the analysis was based on the nonparametric Kaplan Meier survival estimate. 

In the analysis of development rate from nauplius to copepodite, an individual who turned out to 

be dead or is missing was handled as censored and a nauplius that did not develop to copepodite during the 

follow up time was also censored. The analysis of development rate from copepodite to adult was 

performed on individuals that had developed to copepodites during the follow up time. If an individual was 

dead, missing or did not become an adult within the follow up time, it was handled as censored at that 

specific time point. 

There are different measures of development rate which can be used, e.g. mean time, median 

time, proportion developed at a particular time point, and it is not straightforward how to choose the most 

suitable measure for this data. The first candidate is the median value, which is an ordinary measure for 

survival data. For these particular data, the median will be a quite rough measure. The reason for that is the 

low precision of the time measurements compared to the quite small exposure effect. The time 

measurements are rounded up to one whole day so consequently the median has a value in whole day 

increments. The second measure candidate is the mean value. The mean value is calculated as the area 

under the Kaplan Meier curve, and that measure should be less rough and more appropriate where we have 

pure survival data and the follow up time is longer. For data in this study, the mean value may be sensitive 

to the choice of endpoint time scales. For that reason the median (50th percentile) complemented with the 

25th percentile was chosen. In addition to that, a Log rank test was performed to test equality of the survival 

curves between an exposure and the control. 
ENV/JM/MONO(2007)26 

 26

3. RESULTS AND DISCUSSION 

Throughout the results and discussion section all the harpacticoid (i.e. both N. spinipes and A. 

tenuiremis) data have been treated in the same way as well as presented and discussed together, as if there 

were not two separate species that have been tested. The only calanoid species used in the ring test, A. 

tonsa was handled separately, both for statistics and presentation. 

For further information about within treatment variability raw data spreadsheets are also 

presented in Annex F for most of the endpoints. 

The following abbreviations were used to denote each laboratory in the figures and tables 

presented in the results and discussion section: 

Harpacticoids: 

Nitocra spinipes

• ITM (Department of Applied Environmental Science, Stockholm University, Sweden). 

• IVL (IVL Swedish Environmental Research Institute, Sweden). 

• TOXICON (Toxicon AB, Sweden). 

Amphiascus tenuiremis

• BEI (Block Environmental Inc., USA). 

• NOAA (The NOAA Center for Coastal Environ. Health and Biomolecular Research USA). 

Calanoid: 

Acartia tonsa

• DTU (Inst. of Environment & Resources, Technical University of Denmark, Denmark). 

• DHI (DHI Water and Environment, Denmark). 

• OPUS (Opus Plus Ltd, United Kingdom). 
ENV/JM/MONO(2007)26 

 27

3.1 Harpacticoids 

All five harpacticoid laboratories delivered developmental data, which have been analyzed and 

presented below. Adequate reproduction data were only available however for three of the laboratories, 

ITM, NOAA and Toxicon. 

3.1.1 Development 

In the developmental phase of the harpacticoid full life cycle test, the following endpoints were 

analysed: mortality, development rate and sex ratio. 

Mortality 

For a test to be valid, the mortality in the control must not exceed 20% at the end of the exposure 

period (OECD 2005b). Whether these requirements have been fulfilled was examined by the use of the 

non-parametric Kaplan Meier estimate of the survival function (1). In this analysis, an individual who 

turned out to be adult or missing was handled as censored at that specific time point. Also individuals still 

alive at the end of experiment were treated as censored. 

The length of the experiment period in the development phase differed between laboratories 

(NOAA 29 days, BEI 22 days, ITM 26 days, IVL 16 or 19 days1

, Toxicon 35 days) and the mortality was 

examined both at the last day (i.e., overall mortality) and at day 16. 

Table 2 shows the estimated survival proportions, over all, with 95% confidence intervals. Table 

3 shows the estimated survival proportions, at day 16, with 95% confidence intervals. The Kaplan Meier 

survival curves are shown in Figure 1 in appendix A. 

Table 2. Over all survival 

 Lab | Survival Lower_95 Upper_95 

---------+------------------------------ 

 NOAA | 0.861 0.757 0.923 A. tenuiremis

 BEI | 1.000 . . A. tenuiremis

 ITM | 0.724 0.154 0.946 N. spinipes

 IVL | 0.922 0.805 0.970 N. spinipes

 Toxicon | 0.965 0.865 0.991 N. spinipes

---------------------------------------- 

 

1

 19 days for concentration 180 and 540 
ENV/JM/MONO(2007)26 

 28

Table 3. Survival until day 16 

 Lab | Survival Lower_95 Upper_95 

---------+------------------------------ 

 NOAA | 0.861 0.757 0.923 A. tenuiremis

 BEI | 1.000 . . A. tenuiremis

 ITM | 0.965 0.867 0.991 N. spinipes

 IVL | 0.922 0.805 0.970 N. spinipes

 Toxicon | 0.965 0.865 0.991 N. spinipes

---------------------------------------- 

For ITM the overall mortality exceeds 20 % (27.6%) which exceeds the set validity criterion, but 

the mortality until day 16 is well within tolerance limits (i.e. 3.5 %). The other laboratories show a 

tolerable mortality for this particular experiment. An examination of the Kaplan Meier survival curve (see 

Figure 1 Appendix A) shows a massive mortality at day one (8 individuals out of 72) for the NOAA 

laboratory. It is difficult to ascertain whether this is an actual effect or if it is an artefact. Also, for NOAA 

we cannot be statistically certain that the “population mortality” is lower than 20 % (since the lower 95% 

confidence limit is below 80%). In this context it is important to notice that the protocols that were 

prepared for each laboratory to send in their detailed descriptions about the fate of each individual copepod 

were used differently between the laboratories. ITM apparently made much more detailed descriptions. For 

example, an animal that actually disappeared was classified as disappeared (“missing”) and not as dead. If 

a laboratory experienced problems finding an animal in a single well, it is not unlikely that this animal was 

denoted as “missing” instead of “dead”. For some laboratories this seems to have happened, which would 

of course have negatively skewed the mortality rates. 

The mortality among exposed was investigated in the same manner as the control (estimation of 

the Kaplan Meier survival function). Figure 2 -4 in Appendix A shows the survival curves for three of the 

concentrations; 6.6, 20 and 540 μg/L. For some of the laboratories there is a significantly higher mortality 

for exposed copepods compared to the controls. When observing Figure 2-4 it becomes relatively clear that 

some of the non dose-related trends depend on the problematic decline of 3,5-DCP concentrations. For 

some laboratories (e.g. ITM and Toxicon) the lowest concentration (6.6 μg/L) has higher mortality rates 

than for example the highest concentration (i.e. 540 μg/L). This could either depend on random factors or 

on the bioavailability of the test substance, which, according to the test medium analyses (see section 3.4 

below), for some reason was very low in the high concentration treatment(s). 

Mortality was also investigated separately for the different developmental stages (nauplius to 

copepodite and copepodite to adult). One peculiar example is shown in Figure 5; the survival in the 

copepodite to adult stage, for NOAA. Almost all deaths occur one day after reaching copepodite stage, 

which was surprising and difficult to explain. There are probably some explanations other than randomness 

for that, since the same trend was also seen in the control. The last nauplius stage to first copepodite stage 

is a major metamorphosis requiring more extensive genetic to physiological change. It is not uncommon to 

see this transition as the most sensitive developmental window for A. tenuiremis (Chandler published and 

unpublished observations). 
ENV/JM/MONO(2007)26 

 29

Development 

The development rates have to be estimated separately for the different developmental stages 

(nauplius to copepodite and copepodite to adult). The results for development are presented in Table 4 and 

Table 5, and the corresponding Kaplan Meier failure curves are shown in Figure 6 to 9 in Appendix A 

(only for the development from nauplii to copepodite). (For notification: Individuals that died on day 1 

from start were excluded from the study.) 

The lowest concentration giving significant differences from a control (LOEC) in mean time 

elapsed from the first nauplius stage to development of the first copepodite stage was 6.6 μg/L observed by 

the two Amphiascus laboratories NOAA and BEI. However, for NOAA there was a lack of increasing 

dose-response as only 6.6 μg/L and 540 μg/L resulted in significant effects. Also for BEI there was a lack 

of clear dose-response effects in the sense that in 20 μg/L no significant difference from the control was 

observed (but significant effects were seen in the others). For ITM and IVL there was a clear doseresponse but the LOECs were somewhat higher than for the two American Amphiascus laboratories; 60 

and 20 μg/L, respectively. For Toxicon, 20 and 60 μg/L gave significant effects from the control. Overall, 

relatively similar results were seen, but it is likely that the declining 3,5-DCP concentrations have 

introduced artefacts in the anticipated dose-dependent trends. 

The lowest concentration giving significant differences from a control (LOEC) in mean time 

elapsed from the first copepodite stage to development of the adult stage was 6.6 μg/L observed by ITM. 

There was however a lack of clear dose-response effects in the sense that in 60 μg/L no significant 

difference from the control was observed. (It should be noted, however, that at this concentration there was 

a significant effect for the earlier developmental stage (i.e. from nauplius to copepodite), which indicates 

that development overall was significantly affected for this laboratory). For NOAA and Toxicon, the 

LOEC was as high as 540 μg/L. It is noteworthy that for NOAA there was no significant difference from 

the control in any developmental phase in 20, 60 and 180 μg/L. For BEI there was a dose-related trend and 

the LOEC was 60 μg/L. For IVL there were significant differences from the control in 60 and 180 μg/L but 

not in 540 μg/L. 
ENV/JM/MONO(2007)26 

 30

Table 4. Time in days to copepodite, 25th and 50th percentile 

P-value of Log-rank test. Significant differences from the controls are marked by a light gray background. 

---------------------------------------------------- 

Lab and | 

conc. | p25 p50 p-value 

----------+----------------------------------------- 

NOAA | A. tenuiremis

 0 | 10 11 

 6.6 | 8 9 .0425 

 20 | 9 11 .6 

 60 | 9 10 .155 

 180 | 10 10 .258 

 540 | 11 12 .000137 

----------+----------------------------------------- 

BEI | A. tenuiremis

 0 | 8 10 

 6.6 | 9 10 .0265 

 20 | 9 11 .103 

 60 | 12 13 1.21e-08 

 180 | 11 15 4.30e-10 

 540 | 2.75e-08 

----------+----------------------------------------- 

ITM | N. spinipes

 0 | 9 9 

 6.6 | 9 10 .432 

 20 | 9 9 .967 

 60 | 9 11 .00437 

 180 | 9 10 .0146 

 540 | 10 12 .000208 

----------+----------------------------------------- 

IVL | N. spinipes

 0 | 8 9 

 6.6 | 8 9 .0834 

 20 | 8 9 .0469 

 60 | 8 9 .00503 

 180 | 12 12 .0272 

 540 | 12 14 2.08e-06 

----------+----------------------------------------- 

Toxicon | N. spinipes

 0 | 7 8 

 6.6 | 8 8 .601 

 20 | 6 7 .0000705 

 60 | 6 7 .000456 

 180 | 8 9 .539 

 540 | 7 8 .431 

---------------------------------------------------- 
ENV/JM/MONO(2007)26 

 31

Table 5. Time in days between copepodite and adult, 25th and 50th percentile. 

P-value of Log-rank test. Significant differences from the controls are marked by a light gray background. 

---------------------------------------------------- 

Lab and | 

conc. | p25 p50 p-value 

----------+----------------------------------------- 

NOAA | A. tenuiremis

 0 | 8 8 

 6.6 | 6 7 .136 

 20 | 6 7 .19 

 60 | 7 8 .825 

 180 | 7 8 .622 

 540 | 9 10 .000234 

----------+----------------------------------------- 

BEI | A. tenuiremis

 0 | 8 8 

 6.6 | 7 9 .155 

 20 | 6 8 .597 

 60 | 6 8 .0325 

 180 | 9 9 .0273 

 540 | - - - 

----------+----------------------------------------- 

ITM | N. spinipes

 0 | 6 7 

 6.6 | 7 8 .0121 

 20 | 7 8 .00129 

 60 | 6 8 .231 

 180 | 7 8 .0114 

 540 | 8 9 6.15e-06 

----------+----------------------------------------- 

IVL | N. spinipes

 0 | 4 5 

 6.6 | 4 7 .125 

 20 | 4 6 .145 

 60 | 4 7 .0353 

 180 | 4 7 .0265 

 540 | 4 5 .174 

----------+----------------------------------------- 

Toxicon | N. spinipes

 0 | 10 13 

 6.6 | 11 12 .58 

 20 | 11 13 .533 

 60 | 10 14 .272 

 180 | 10 11 .241 

 540 | 13 15 .0214 

---------------------------------------------------- 
ENV/JM/MONO(2007)26 

 32

Overall the LOECs are relatively similar among the five laboratories related to decrease in 

developmental rate. For NOAA, BEI and ITM the LOEC was μg/L and for IVL and Toxicon the LOEC 

was 20 μg/L. This indicates that Amphiascus may be somewhat more sensitive than Nitocra but too farreaching conclusions should not be made due to the problematic decline in DCP-levels (see section 3.4 

below), which was evident (but not necessarily identical) for all laboratories. 

It should be noted that the declining 3,5-DCP concentrations are likely to have introduced 

artifacts in the concentration-response relationship. Consequently, it is difficult to provide a valid 

interpretation of these data. Hence the derivation of LOEC and NOEC values and statements made about 

species sensitivity must be treated with caution. 

Sex ratio 

For a test to be valid, the proportion of females (or males) in the control should be within 40% to 

60% (OECD 2005b). 

Table 6 shows the female proportion for each laboratory and concentration, and for the four 

laboratories that have made sex determinations, the female proportion in the control is within the guideline 

limits. 

Table 6. Female proportion in adults 

------------------------------------------------------------ 

 | 

Laboratory 

 Conc. | NOAA BEI ITM IVL Toxicon 

----------+------------------------------------------------- 

 0 | 0.53 0.56 0.57 0.43 

 6.6 | 0.49 0.50 0.43 0.49 

 20 | 0.52 0.53 0.60 0.45 

 60 | 0.58 0.58 0.55 0.46 

 180 | 0.44 0.78 0.53 0.47 

 540 | 0.29 - 0.53 0.31 

------------------------------------------------------------ 

The following observations were made regarding development time ranges from copepodite to 

adult for the control, among individuals that became adults (this is not a survival analysis estimate): 

• For NOAA and BEI there were no significant differences between sexes. 

• For ITM there was a significant difference (mean time: female 7.9 days, male 6.4 days). For 

Toxicon there was a large significant difference (mean time: female 10.4 days, male 14.3 days). 

• For ITM this difference was due to difference from nauplii to copepodite but for BEI there was 

no difference from nauplii to copepodite. 
ENV/JM/MONO(2007)26 

 33

Sex ratios are often difficult to analyse in the sense that in these tests there is seldom information 

available about the sex of dead immature animals. Conclusions of sex-specific effects should as a 

consequence always be carefully made. In this context, one could regard the male:female ratio of 40:60 or 

60:40 as a prerequisite to be able to construct statistically reasonable n-size for male:female pairs for 

reproduction studies. This prerequisite has been met for all laboratories participating in the ring test. 

3.1.2 Reproduction 

Three laboratories performed the reproduction test (NOAA, ITM and Toxicon) and the outcomes 

analysed were fertilisation success, offspring production and time to first and second clutch. No survival 

analysis was made since information about the censored pairs was missing. Therefore we cannot exclude 

that an observed effect for an exposed group might be a consequence of differences in mortality for that 

group compared to the control. 

Fertilisation success 

Fertilization success was measured as the proportion of pairs able to produce two successive 

clutches within the follow up time, of all mating pairs at start2

. The follow up time differs between 

laboratories (NOAA 10 days, ITM 19 days and Toxicon 20 days), which possibly make the comparisons 

between species and laboratories more difficult. The results are shown in Tables 7 to 9; number of mating 

pairs at start, the proportion of pairs with two clutches together with exact 95 % confidence limits and a pvalue for testing the hypothesis of “similar proportions in the exposed and the control” (Fisher’s exact test). 

The lowest LOEC was observed in 6.6 μg/L for ITM but there was a lack of dose-response in the sense 

that there were no significant differences from the control at both 20 and 60 μg/L. It is obvious that for 

ITM the 6.6 μg/L treatment has been negative for most endpoints, which makes it doubtful that this LOEC 

is realistic. This also becomes evident when looking at the other two laboratories, having LOECs for this 

endpoint as high as 180 μg/L (Toxicon) and 540 μg/L (NOAA). So, if we consider the very low LOEC for 

ITM to be in error (i.e. an artefact), we would end up with an overall LOEC of 180 μg/L for this endpoint. 

Table 7. Proportion of pairs with two clutches, for NOAA (A. tenuiremis). 

Significant differences from the controls are marked by a light gray background. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 23 .74 .52 .9 . 

 6.6| 23 .65 .43 .84 .75 

 20 | 20 .50 .27 .73 .13 

 60 | 18 .50 .26 .74 .19 

 180 | 21 .52 .3 .74 .21 

 540 | 7 .14 .0036 .58 .0086 

------------------------------------------------------------ 

 

2

 ITM observed that several of their pairs consisted of two females. These pairs were excluded. 
ENV/JM/MONO(2007)26 

 34

Table 8. Proportion of pairs with two clutches, for ITM (N. spinipes). 

Significant differences from the controls are marked by a light gray background. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 18 .67 .41 .87 . 

 6.6| 20 .25 .087 .49 .021 

 20 | 18 .56 .31 .78 .73 

 60 | 10 .50 .19 .81 .44 

 180 | 15 .13 .017 .4 .004 

 540 | 11 .091 .0023 .41 .0057 

------------------------------------------------------------ 

Table 9. Proportion of pairs with two clutches, for Toxicon (N. spinipes). 

Significant differences from the controls are marked by a light gray background. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 17 .82 .57 .96 . 

 6.6| 21 .76 .53 .92 .71 

 20 | 22 .59 .36 .79 .17 

 60 | 22 .59 .36 .79 .17 

 180 | 18 .39 .17 .64 .015 

 540 | 16 .063 .0016 .3 .000013 

------------------------------------------------------------ 

The observed proportions of pairs able to produce two clutches are lower for the exposed 

compared to the control, for all concentrations in all three laboratories. If we use significance level3

 5%, 

every one of the laboratories shows a significant difference from the control for 540 μg/L, and ITM along 

with Toxicon also show significant differences from the control for 180 μg/L. In addition, ITM shows a 

significant difference from the control for 6.6 μg/L, which makes the data not following a dose related 

trend. This concentration did not respond as expected (i.e. being the lowest concentration it was expected 

to give little or no effects) for this laboratory. Care should thus be taken when setting a LOEC for this 

laboratory and endpoint. 

The probability that a statistical test of this kind will fall out as significant (the power of the test) 

depends on the size of the true effect and the sample size. Obviously, it was not possible to conclude, for a 

non-significant test, that there were no effects. The power might just have been too low to reveal it. For 

that reason, no attempt was made to estimate NOEC or LOEC with these data. 

An alternative approach is to assume a model for the dose response relationship. For these data, 

there seems to be a monotonic dose response trend (with an exception at 6.6 μg/L for ITM) so a logistic 

 

3

 not adjusted for multiple test 
ENV/JM/MONO(2007)26 

 35

regression model was fitted for each laboratory separately. The models are illustrated in Figure 10 in 

Appendix A, and except for the observation from ITM at 6.6 μg/L, the fit is quite good. 

In order to estimate an EC50 -value from these data, a non-linear model, like the logistic function, 

would have to be fitted. This is not a straightforward task (model choice, programming, model check etc.) 

and since there was limited time available for a major revision of the statistical analyses, no attempt was 

made to produce EC estimates for the ITM study. 

Offspring production 

For a test to be valid the average number of viable offspring per clutch in the controls should not 

be lower than 5 (OECD 2005b). This criterion was met for all three laboratories (average number was 

higher than 8 for all three, data not shown). For pairs that were able to produce two clutches, the total 

number of viable offspring was calculated. The results are displayed separately for each laboratory in 

Table 10 to 12. The tables show the frequency of mating pairs able to produce two clutches, and the mean 

value of total offspring production together with 95 % confidence limits (based on the t-distribution). A 

one-way ANOVA for each laboratory separately shows no significant differences (p-values4

; 0.10, 0.60 

and 0.74 for laboratory NOAA, ITM and Toxicon, respectively). The results are also illustrated with box 

plots in Figure 11 in Appendix A. It can be noted that in 540 μg/L (for all laboratories) almost no pairs 

were able to produce two clutches. 

Table 10. Total viable offspring production, for NOAA (A. tenuiremis). 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 17 17.4 15.3 19.4 

 6.6| 15 14.7 12.7 16.8 

 20 | 10 15.7 13.4 18 

 60 | 9 12.7 8.77 16.6 

 180 | 11 14.1 10.2 18 

 540 | 1 5 . . 

-------------------------------------------------- 

Table 11. Total viable offspring production, for ITM (N. spinipes). 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 12 22.3 16.3 28.2 

 6.6 | 5 18.6 11.5 25.7 

 20 | 10 23.7 18.2 29.2 

 60 | 5 20 9.32 30.7 

 180 | 2 28.5 9.44 47.6 

 540 | 1 29 . . 

-------------------------------------------------- 

 

4

 Concentration 540 with only one observation was excluded in this test. 
ENV/JM/MONO(2007)26 

 36

Table 12. Total viable offspring production, for Toxicon (N. spinipes). 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 14 16.9 13.3 20.4 

 6.6 | 16 19 15.7 22.3 

 20 | 13 19.8 16.1 23.4 

 60 | 13 17.6 13.3 21.9 

 180 | 7 20.3 10.6 30 

 540 | 1 25 . . 

-------------------------------------------------- 

Time to first and second clutch 

Tables 13 to 15 show the median time to the first clutch, estimated for the pairs that had produced 

a first clutch (not a survival analysis estimate). A Mann Whitney U-test was performed to test the 

hypothesis of equal time distributions for the exposed and the control test populations. P-values on these 

tests are shown in the table. Since we have ignored the censored observations, the interpretation of these 

results should be cautious. The results are also illustrated with box plots in Figure 12 in Appendix A. 

Observe that the lack of dose-response in Tables 13-15 (i.e., no significant difference from the control in 

the highest concentration) is likely due to the low number of observations at this concentration. The LOEC 

was 60 μg/L for all laboratories. 

Table 13. Time to production of first clutch, for NOAA (A. tenuiremis). 

Significant differences from the controls are marked by a light gray background. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 17 3 

 6.6 | 17 3 .79 

 20 | 11 3 .48 

 60 | 11 4 .029 

 180 | 13 4 .00027 

 540 | 1 7 + 

------------------------------------------------------- 
ENV/JM/MONO(2007)26 

 37

Table 14. Time to production of first clutch, for ITM (N. spinipes). 

Significant differences from the controls are marked by a light gray background. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 15 7 

 6.6 | 6 8.5 .5 

 20 | 13 10 .082 

 60 | 7 9 .016 

 180 | 4 10 .049 

 540 | 2 6.5 .55

Table 15. Time to production of first clutch, for Toxicon (N. spinipes). 

Significant differences from the controls are marked by a light gray background. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 15 4 

 6.6 | 18 6 .18 

 20 | 17 6 .21 

 60 | 18 7 .0041 

 180 | 12 9.5 .00056 

 540 | 4 5.5 .61 

------------------------------------------------------- 

Tables 16 to 18 show the median time between the first and second clutches, estimated for the 

pairs that had produced a second clutch (not a survival analysis estimate). A Mann-Whitney U-test was 

performed to test the hypothesis of equal time distributions for the exposed and control populations. Pvalues on these tests are shown in the table. Since we have ignored the censored observations, 

interpretation of the results should be cautious. 

Table 16. Time between first and second clutch, for NOAA (A. tenuiremis). 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 17 1 

 6.6 | 15 1 .65 

 20 | 10 1 .19 

 60 | 9 2 .59 

 180 | 11 2 .18 

 540 | 1 3 

------------------------------------------------------- 
ENV/JM/MONO(2007)26 

 38

Table 17. Time between first and second clutch, for ITM (N. spinipes). 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 12 3 

 6.6 | 5 5 .67 

 20 | 10 4 .17 

 60 | 5 3 .91 

 180 | 2 3 .84 

 540 | 1 3 

------------------------------------------------------- 

Table 18. Time between first and second clutch, for Toxicon (N. spinipes). 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 14 3.5 

 6.6 | 16 3 .35 

 20 | 13 3 .27 

 60 | 13 3 .42 

 180 | 7 4 .26 

 540 | 1 4 

------------------------------------------------------- 

Overall, the reproductive data are relatively consistent between the three harpacticoid 

laboratories, with lowest LOECs ranging from 20 to 60 μg/L. Also, the three endpoints related to 

reproduction follow the same trends for all three laboratories (with some small exceptions that may rather 

be credited to artefacts), with time to production of the first clutch being the most sensitive endpoint, and 

offspring production the least sensitive endpoint. In a population model, all of these three endpoints would 

lower e.g. population growth, which illustrates the importance to study other reproductive endpoints than 

solely production. Since the 3,5-DCP decline was probably most severely manifested in the end of the test 

and since the three laboratories’ test times were very different, the results still indicate a similar crossspecies sensitivity to the reference substance. The total offspring production data were calculated for pairs 

that were able to produce (a minimum of) two clutches. This approach ignores potential effects on 

reproduction whereby some pairs may only produce a single brood or egg sacs that fail to develop (female 

may be fertilized but eggs do not develop!). Consequently, it may be argued that the data analysis is 

‘biased’ towards the most fertile females in the ‘population’ of animals within each treatment. 
ENV/JM/MONO(2007)26 

 39

3.2 Calanoid 

3.2.1 Development 

Mortality of F0

For a test to be valid the average mortality5

 of animals in the controls shall not exceed 30% of the 

hatched animals (OECD 2005a). Table 18 presents the number of replicates used, and the mortality 

proportion, for each concentration and laboratory. The average mortality in the controls (Conc.=0) is lower 

than 30% for every one of the laboratories. 

Table 18. Number of replicates and mortality proportion. 

---------------------------------------------------- 

 | Conc. 

 Lab | 0 6.6 20 60 180 540 1620 

----------+----------------------------------------- 

 DHI | 9 4 4 4 4 

 | .21 .18 .17 .20 .40 

 | 

 DTU | 12 6 5 6 6 6 

 | .15 .21 .11 .15 .12 .17 

 | 

 OPUS | 12 5 5 5 5 5 

 | .071 .12 .17 .091 .11 .53 

----------------------------------------------------- 

LDR of F0 and F1 

LDR was measured as the proportionate copepodite stage of the total viable animals (number of 

copepodites/(number of copepodites + number of nauplii)) at the end of exposure. Because of overdispersion in the data (i.e., more variation between replicates within treatments than expected under the 

assumption of equal probability) the analysis of data was performed with a random effect logit model. It 

should be noted that the validity criterion (i.e. control LDR should be within 50 ± 20 %) for the LDR 

endpoint was not met for two of the laboratories (i.e. DHI and OPUS, in both generations), which weakens 

the lab-to-lab comparability of these results (Table 19). 

Tables 19 and 20 show the estimated LDR value from the logit model, together with a p-value for 

testing the hypothesis of equal LDR in the exposed and the control. No clear conclusion about the 

relationship between LDR and exposure can be made. For DHI an effect appears only for the highest 

concentration. For DHI an effect appears only for the highest concentration (the “extra” concentration 1620 

μg/L not included). For DTU the plot looks strange; LDR in the controls are surprisingly low, and the 

 

5

 Eggs at the end of exposure were excluded in the mortality calculation. 
ENV/JM/MONO(2007)26 

 40

observations for concentration 20 are clustered into two distinct groups. For OPUS there is a 

monotonically decreasing relationship with concentration (with some extreme outliers). 

Table 19. Estimated LDR of F0, p-value. 

Significant differences from the controls are marked by a light gray background. 

----------------------------------------------------------------------- 

 | conc 

 lab | 0 6.6 20 60 180 540 1620 

----------+------------------------------------------------------------ 

 DHI | .71 .61 .65 .33 .00 

 | - >.05 >.05 <.001 --- 

 | 

 DTU | .47 .58 .55 .45 .41 .04 

 | - .0186 .0674 .613 .15 <.0001 

 | 

 OPUS | .89 .65 .5 .22 .22 .03 

 | - .0001 <.0001 <.0001 <.0001 <.0001 

----------------------------------------------------------------------- 

The lowest LOEC for the F0-generation was observed at 6.6 μg/L for DTU and OPUS, but for 

DTU there was a lack of concentration-response in the sense that there were no significant differences 

from the control at 20, 60 and 180 μg/L. The LOEC for DHI was 540 μg/L for this endpoint. 

Table 20. Estimated LDR of F1, p-value. 

Significant differences from the controls are marked by a light gray background. 

---------------------------------------------------------------------- 

 | conc 

 lab | 0 6.6 20 60 180 540 

----------+----------------------------------------------------------- 

 DHI | .79 .75 .75 .74 .72 .31 

 | - .239 .101 .120 .0268 <.0001 

 | 

 DTU | .46 .51 .46 .5 .35 .21 

 | - .502 .99 .6 .135 .0003 

 | 

 OPUS | .89 .9 .76 .63 0 0 

 | - .921 .00208 <.0001 - - 

----------------------------------------------------------------------

The data show that for DHI, the LOEC for development in the F1-generation was 180 μg/L 

(Table 20). For OPUS, the F1-generation was less sensitive with a LOEC of 20 μg/L, as compared to 6.6 

μg/L for the F0-generation. However, the LDR validity criterion for OPUS was not met for either of the 

generations, so care should be taken when analysing these results.

Overall, the lowest “concentration-related” LOEC for development was 6.6 μg/L observed for 

OPUS in the F0-generation. The validity criterion that LDR should be within 50 ± 20 % was not met for 

two of the three laboratories (DHI & OPUS) for both generations (71 & 89% and 79 & 89% as shown in 

Table 19 and 20, respectively). Considering the limited number of data sets that met the validity criterion 

(i.e. non valid tests), it is difficult to provide a robust interpretation of the data. 
ENV/JM/MONO(2007)26 

 41

Length of male and females in F0

The mean length of males and females at the termination of the development of the F0-generation

in F0 is presented in Table 21. There were no obvious dose response trends in these data. To test for 

differences in mean length for each of the exposures compared to the control, t-tests with pooled variance 

estimates were performed. To control for compounded alpha error from multiple comparisons, Bonferroni 

corrections were made within each laboratory and sex dataset. With that correction a difference was 

deemed significant at the 0.05 level if the p-value was lower than 0.01. The p-values are shown in Table 

21. For DHI, the mean length at concentration 180 μg/L were significantly higher than the control, for both 

sexes. For DTU there were no significant differences in mean length between the exposed and the control. 

Overall, these data indicate that there are no obvious dose-related effects on length in Acartia 

tonsa exposed to 3,5-DCP up to 540 μg/L. Considering the time and effort required to produce the length 

data, and the accuracy of length measurements, it may be worth reviewing whether this endpoint should be 

included in the test method. 

Table 21. Number of observations, mean length (mm) of male and females in the development of the F0 

generation and p-value. 

-------------------------------------------- 

 | lab and sex 

 | ----- DHI ---- ----- DTU ---- 

 conc | Female Male Female Male 

----------+--------------------------------- 

 0 | 50 50 27 24 

 | .92 .75 .9 .76 

 | - - - - 

 | 

 6.6 | 25 25 38 25 

 | .91 .75 .88 .74 

 | .11 .13 .065 .11 

 | 

 20 | 25 25 25 37 

 | .92 .75 .88 .77 

 | .38 .27 .083 .15 

 | 

 60 | 25 25 25 40 

 | .93 .75 .88 .74 

 | .21 .27 .11 .06 

 | 

 180 | 25 25 25 51 

 | .95 .76 .9 .76 

 | .00071 .0065 .46 .46 

 | 

 540 | 25 25 31 25 

 | .93 .75 .89 .74 

 | .18 .33 .18 .045 

-------------------------------------------- 
ENV/JM/MONO(2007)26 

 42

Hatching success of F0 and F1 

For a test to be valid, the hatching success in the control must exceed 80% (OECD 2005a). The 

hatching success was measured as the proportion of hatched eggs at the end of each exposure and 

calculated as (1– number of unhatched eggs at end /number of eggs at start). The hatching success for DTU 

in the control was lower than 80% in 2 replicates out of 12 (the average was 86%). For DHI and OPUS the 

hatching success exceeded 80% in every one of the replicates. The hatching success in the F1-generation in 

the controls was within the limit for every one of the replicates. 

3.2.2 Reproduction 

Egg production 

For a test to be valid, the average control egg production should be higher than 30 

eggs/female/day at each of the three observation days from day 15 to day 18 (OECD 2005a). Table 22 

shows that this criterion was met for DHI and DTU, but not for OPUS (average 27, 28, and 29). There was 

no mortality in the controls (the mortality should be lower than 20%; OECD 2005a). In order to examine 

exposure effects on egg production the average egg production over the 3 days was calculated for each 

female. Figure 13 in Appendix A shows a scatter plot of average egg production against concentration. 

There are no obvious dose response trends in the data, except possibly for DTU. To test for differences in 

mean egg production for each of the exposures compared to the control, t-tests with pooled variance 

estimates were performed. To control for multiple tests, Bonferroni corrections were made within each 

laboratory. With that correction, a difference was significant at the 0.05 level if the p-value is lower than 

0.01. For DTU there is a significantly lower mean egg production for all exposures compared to the 

control. DHI shows a significantly higher mean egg production at 20 μg/L, and OPUS shows a 

significantly lower mean egg production at 540 μg/L. 

Table 22. Number of females, mean and standard deviation of egg production, in controls over 15, 16 or 17 

days. 

---------------------------- 

 | day 

 lab | 15 16 17 

----------+----------------- 

 DHI | 10 10 10 

 | 40 38 42 

 | 16 16 19 

 | 

 DTU | 12 12 12 

 | 45 40 39 

 | 7.6 8.6 14 

 | 

 OPUS | 7 7 7 

 | 27 28 29 

 | 6.7 9.5 6.4 

---------------------------- 
ENV/JM/MONO(2007)26 

 43

Table 23. Number of females, mean egg production and p-value. Significant differences from the controls are 

marked by a light gray background. 

---------------------------------------------- 

 | conc 

 lab | 0 6.6 20 60 180 540 

----------+----------------------------------- 

 DHI | 10 12 12 12 11 12 

 | 39.6 50.8 53.5 41.2 49 40.9 

 | - .019 .005 .381 .041 .405 

 | 

 DTU | 12 12 12 12 12 11 

 | 41.6 33.3 32.3 24.3 24 25.9 

 | - .004 .002 <.001 <.001 <.001 

 OPUS | 7 5 6 7 6 7 

 | 28.2 32.5 29.1 30.8 32.7 12 

 | - .241 .437 .321 .22 .003 

---------------------------------------------- 

3.3 Compilation of NOEC and LOEC for both groups of species 

Table 24 shows a compilation of all LOECs from the various tests conducted in the present ring 

test activity. For the three harpacticoid laboratories that completed full life cycle tests the lowest LOEC 

was observed for development (6.6 μg/L) whereas for the calanoids the picture was somewhat divided; for 

DTU reproduction was the most sensitive endpoint (6.6 μg/L), for DTU development and reproduction was 

equally sensitive (6.6 μg/L), and for OPUS development was far more sensitive than reproduction, 6.6 

μg/L compared to 540 μg/L. 

Table 24. Overview of LOEC-values (in μg/L) for all participating harpacticoid and calanoid laboratories. 

Laboratory Development Reproduction 

 Time to 1st 

clutch 

Proportion Production 

NOAA 6.6 60 540 a 

BEI 6.6 - - - 

ITM 6.6 60 180b a 

IVL 20 - - - 

Toxicon 20 60 180 a 

USCc

 18 d 200 d 

 Body length Production

DHI 180 180e 6.6f 

DTU 6.6g 540 6.6 

OPUS 6.6 - 540 

a No LOEC achieved owing to too few pairs in highest treatment. 

b Lowest observed LOEC was 6.6 μg/L but this is likely an artefact in the sense that all higher treatments resulted in no significant 

difference from the control. 

c Data from Pre-validation using Amphiascus tenuiremis (see OECD 2005c) 

d No significant difference from the control up to and including 400 μg/L 3,5-DCP. 

e Lack of concentration response since at 540 μg/L there were no response. 

f Lowest observed LOEC was 6.6 μg/L but there was a lack of concentration response. Also, this response was associated to an 

increased production of offspring. 

g Lowest observed LOEC was 6.6 μg/L but there was a lack of concentration response. 
ENV/JM/MONO(2007)26 

 44

3.4 Chemical analysis of 3,5-DCP – potential reasons for declining levels 

Measured 3,5-DCP concentrations follow a similar declining trend for all participating 

laboratories, but the trend is less steep for the Acartia labs (possibly a result of the larger test beakers). 

ITM has therefore conducted some follow-up tests where we have studied a) how fast the 3,5-DCP decline 

occurs and b) the fate of the parent compound. In brackish water at a pH of about 8-8.5 (which is the pH 

range most laboratories have reported) the disappearance from the water phase is significant within a 

couple of days (data not presented here). A first suggestion was that this problem was due to hydrolysis. 

After several experts’ comments on this, it seems likely that this is not the case. Since the pKa-value for 

3,5-DCP is about 8.2, it is obvious that the parent compound likely “oscillated” between a “protonated” 

and an ionized form during the test. It is common that pH decreases (may also increase) in the test medium 

as animals grow and/or debris accumulates in test beakers. A plausible hypothesis for the DCP-decline 

could be that pH-levels around the pKa-value may lead to ionization/protonation phenomena, which may 

explain the declining 3,5-DCP levels. Whether the ionized or the protonated form possibly “adheres” to 

test apparatus walls and becomes less bioavailable is currently not known. 

We have in two consequent experiments (with a relatively high pH = 8.5) shown that there is 

much more 3,5-DCP in the water than on the walls. In brief, the tests were made as follows: Brackish 

water was adjusted with 0.1 M NaOH to a pH of 8.5. A stock solution of 80 μg/L 3,5-DCP was prepared 

and added to 96-well glass coated microplates (300 μL total volume) in replicates of two for each sampling 

occasion. On days 3, 7, 10 and 14, water and acetone samples were collected (acetone was filled to the top 

of each well). The same method as described above was used for analyses. A calibration curve (5-40 μg/L) 

was used for all analyses. No animals and no algae were added in these experiments. The results from this 

analysis are presented in Table 25 below. Given some mild evaporation problems, these results give no 

indication that a 3,5-DCP decline due to chemical association with test vessel walls would have occurred 

and caused the “poor dose-response” problems observed in the ring tests. On the other hand, there were 

neither animals nor algae added in this analysis, and these “phases” may have affected 3,5 DCP fate and 

bioavailability. Further studies are therefore needed to investigate the cause(s) of declining DCP 

concentrations with time. 

Table 25. Two preliminary experiments showing the fate of 3,5-DCP in the test system used for the 

harpacticoid copepods. 

Food and algae were not added. “Acetone” denotes the fraction of the substances sorbed to the wall and “Water” the 

fraction that was freely dissolved in the water phase. 

 Test 12 Test 23

Day Water Acetone Total1 Water Acetone Total1

3 89 11 99 88 12 100.3 

 

7 79 1.3 80 74 1.4 75.5 

10 79 1.5 80 53 2.2 55.6 

14 72 2.4 74 87 4.7 91.6 1

 Should add up to 80 μg/L. Values higher than that likely owes to up-concentration due to evaporation 

2

 A stock solution was made in brackish water and was analysed day 3. Measured concentration was then 72 μg/L. 

3

 A stock solution was made in brackish and was analysed day 3. Measured concentration was then 66 μg/L. 
ENV/JM/MONO(2007)26 

 45

Figure 1 to 3 show that there was a steady decline of 3,5-DCP in the test medium in the 

harpacticoid tests. Interestingly, NOAA reported that they made new stock solutions from the crystalline 

powder each time the medium was renewed. As Figure 1a shows they still had declining levels, which may 

be explained by the fact that at most occasions the recovery of the test substance in the newly prepared 

medium was relatively low (on average 50%) (Figure 1b). This finding suggests that 3,5DCP is not stable 

in high ionic strength, basic pH seawater, and it should not be used as a reference compound in the future. 

For ITM, which had some difficulties dissolving the substance in distilled water, a distilled water 

stock solution was prepared in the beginning of the test and used to prepare new brackish water solutions at 

each test medium renewal until day 10, when a new batch was made. This 3,5-DCP volumetric cylinder 

was analysed (after it had been held cold, sealed and in darkness) after 21⁄2 months. The measured 

concentration was still approximately 50% of the nominal concentration, indicating that basic pH high 

ionic strength seawater drives 3,5DCP from its parent structure. 

Figure 1 a & b. Chemical analysis of NOAA’s test medium samples. 

Massive decline between day 0 and day 24. Observe that also the freshly made test medium have lowered 3,5-DCP 

levels, on average around 50%. 

b) NOAA (solution added)

0

25

50

75

100

125

150

175

day 0 day 6 day 12 day 18 day 24

Day of experiment

% nominal concentration

6.6μg/L

20μg/L

60μg/L

180μg/L

540μg/L

a) NOAA (solution removed)

0

10

20

30

40

50

60

70

80

90

100

day 0 day 6 day 12 day 18 day 24

Day of experiment

% nominal concentration

6.6μg/L

20μg/L

60μg/L

180μg/L

540μg/L
ENV/JM/MONO(2007)26 

 46

ITM 

-40

-20

0

20

40

60

80

100

120

140

dag 0 dag 2 dag 5 dag 7 dag 9 dag 12 dag 14 dag 16 dag 20

Day of experiment

% of nominal concentration

6.6

60

540

Figure 2. Chemical analysis of ITM’s test medium samples. 

Massive decline between day 0 and day 19.

Toxicon

0

10

20

30

40

50

60

70

80

90

100

0 2 5 9 14 16 19 23 30 35 40

Day of experiment

% nominal concentration

6.6μg/L

60μg/L

540μg/L

Figure 3. Chemical analysis of Toxicon’s test medium samples. 

Massive decline between day 0 and day 19. After that new and larger micro well plates were used. It should be noted 

that the same stock solution as was used during the first 19 days was used from day 10 and onwards. 
ENV/JM/MONO(2007)26 

 47

Figures 4 to 6 show that there was relatively consistent variability in 3,5-DCP concentrations in 

the test medium in the calanoid tests, but no clear trend in decline with time. In Figure 5 it is clear that the 

“disappearance” of 3,5-DCP is much more dramatic for the lower than for the higher concentrations. 

 OPUS

0

10

20

30

40

50

60

70

80

90

100

0 15 30 31 35

Day of experiment

% nominal concentration

6.6 ug/L

20 ug/L

60ug/L

540ug/L

750ug/l

Figure 4. Chemical analysis of OPUS test medium samples. 

DHI

0

10

20

30

40

50

60

70

80

90

100

0 14

Day of experiment

%nominal concentration

6.6μg/L

20μg/L

60μg/L

180μg/L

540μg/L

Figure 5. Chemical analysis of DHI’s test medium samples at start and at day 14. 
ENV/JM/MONO(2007)26 

 48

Figure 6. Chemical analysis of DTU’s test medium samples. 

For the lowest concentration there is likely an artefact day 0 since the measured levels were almost 3 times higher 

than the nominal. A plausible explanation could be that it is actually a 20 μg/L sample that has been denoted as a 6.6 

μg/L sample. 

Overall, the chemical analyses show that 3,5-DCP may not be the most suitable reference 

substance when conducting long-term experiments with marine species. Even if we today do not really 

know what caused the decline, it is likely that the problem is complex. Something happens both in the 

stock solution and in the test medium over time. Further, when animals and algae are not added, the 

problem is (as shown in Table 25 above) almost insignificant over a 14-day period. Whether this is an 

indication of metabolism in the animals, pH-changes and subsequent breakdown and/or accumulation in 

copepods and/or algae is not known. The importance to solve the 3,5-DCP problems as indicated in the 

present ring test should be of interest to many laboratories and organisations that use this substance as a 

reference molecule in standard ring tests. 

3.5 Water quality within test vessels 

Throughout the experiments the following water quality measurements were made in the control 

vessels: 

ITM 

• pH: 7.8 – 8.5 

• Salinity: 6.2 – 6.9 

• Dissolved O2: 91 – 107 % 

• Temperature: 21.1 – 23.1°C 

DTU

0

50

100

150

200

250

300

1 8 10 12 18 23

Day of experiment

% nominal concentration

6.6μg/L

60μg/L

540μg/L

DTU

0

50

100

150

200

250

300

1 8 10 12 18 23

Day of experiment

% nominal concentration

6.6μg/L

60μg/L

540μg/L
ENV/JM/MONO(2007)26 

 49

NOAA 

• pH: 8.0 – 8.3 

• Salinity: 30 – 31‰ 

• Dissolved O2: 88 -- 90 % 

• Temperature: 24.8 -- 25.3°C 

Toxicon 

• pH: 7.73 – 8.37 

• Salinity: 5.7 – 6.7 ‰ 

• Dissolved O2: 97 - 105% 

• Temperature: 19.4 – 24.3°C (extreme values, often around 21-22°C) 

OPUS 

• pH: 7.5 – 8.1 

• Salinity: 34 - 36 ‰ 

• Dissolved O2: 92 - 100% 

• Temperature: 19.1 – 21.9°C

DHI 

• pH: 7.8 - 8.4 

• Salinity: 20 ‰ 

• Dissolved O2: 97 - 99% 

• Temperature: 20°C 

DTU 

• pH: 8.0 - 8.3 

• Salinity: 20.1 – 20.4‰ 

• Dissolved O2: 8.4 – 8.7 mg/L 

• Temperature: 20 - 21°C 
ENV/JM/MONO(2007)26 

 50

4. SUMMARY AND CONCLUSIONS 

The present ring test activity shows that consultancy laboratories are able to run both the 

harpacticoid and calanoid full life cycle test, which both have relatively difficult and demanding 

experimental designs. However, the significant 3,5-DCP declines, as observed for all laboratories for 

which test medium was analyzed, obviously makes it difficult to draw thorough conclusions about exact 

effect levels. Nevertheless, similar declines were evident for all laboratories, which makes a comparison of 

LOECs possible on a nominal concentration basis. The developmental data were consistent among all the 

species used; normally the data also showed low variability. For the harpacticoids the LOEC range for 

“development” was between 6.6 and 20 μg/L whereas for the calanoids the range was somewhat larger and 

variable among the three participating laboratories (in the F0-generation); i.e. 6.6 to 180 (or 540 if only 

considering dose-related effects) μg/L. It should also be noted that for the calanoid development data (i.e. 

LDR), the validity criteria was not fulfilled for one or more laboratories (in both generations), which of 

course limits the comparability of these data. 

For the harpacticoids, the reproduction data was also consistent, at least for two out of three 

“reproductive” endpoints (i.e. “time to first clutch” and “offspring production”). The reproductive data for 

the calanoids varies more, with LOECs ranging from 6.6 to 540 μg/L. This might be due to a within test 

variability of biological tests which is well-known and normally corrected for via increased numbers of 

replicates. If we have a within test variability we consequently also have a between test variability. As 

complexity (and relevance) of the model increases, the reliability usually decreases since the large number 

of parameters interacting in the model will increase the rate of random errors and makes these tests less 

reproducible (Breitholtz et al. 2006). The judgement of the data from this ring test exercise should be made 

in light of the fact that there was no steady concentration level of the reference chemical for any treatment 

in any laboratory (which could be argued makes all tests invalid), and that there were perhaps too few 

participants per species to avoid possible negative impacts of random error and natural variation (on 

average three to four laboratories per species). 

Another finding from this ring test activity is that the LOECs for the developmental data are most 

often lower than the LOECs for reproduction. This is a finding that has been observed in several studies 

using e.g. harpacticoid copepods (e.g. Breitholtz and Wollenberger, 2003; Breitholtz et al. 2003; Bejarano 

et al. 2006). Whether this is actually reflecting the sensitivity of the species exposed to 3,5-DCP in the 

present ring test is difficult to say since the levels were not stable over time. On the other hand, the 

chemical analyses of the test medium samples for e.g. Toxicon shows that the measured concentrations of 

3,5-DCP seen on days 16-19 increased drastically as the reproductive phase was entered, but the LOEC for 

development was still lower for the developmental phase. Since especially for N. spinipes the total test 

time was over 40 days, these findings may call for application of partial instead of full life cycle tests. The 

only realistic full lifecycle alternative would otherwise be to continue with A. tenuiremis, for which the test 

time was more than 15 days shorter, as the only harpacticoid test species. Also, for this species there is 

already a standard test using the same experimental design available within ASTM (ASTM 2004) and 

numerous scientific articles has been published based on the current OECD draft TG experimental design 

(e.g. Chandler et al. 2004a, b). 
ENV/JM/MONO(2007)26 

 51

For future validation work of the two draft test guidelines, it is recommend that: 

• If all of the species used in the current ring test are to be included as a multi-species testing 

system in the future, then a partial life cycle test that focuses mainly on copepod development 

may be a better alternative if data comparability across all species is critical. 

• If the regulatory need for a full life cycle test is greater, then A. tenuiremis should be used as a 

single harpacticoid species, owing to its much shorter test time and better reproductive output and 

success, which drastically increases the statistical power of the test. 

• Other reference chemicals should be used until the reason(s) for the 3,5-DCP declines using the 

current brackish and marine species has been fully examined. 

• More laboratories should participate in the ring test activities to avoid random error and natural 

variability that can drastically influence the overall judgement on validity of ring test results and 

future recommendations. 

• In order to estimate EC50 values rather than traditional LOEC/NOEC’s (which are more stronglydependent on sample size and statistical power), non-linear regression-based models (e.g., Liber 

et al. 1992) should be fitted to the harpacticoid and calanoid development data because these 

models are more strongly dependent on numbers of test concentrations and organismal dose 

response along those test concentrations. 

• The Phase 2 validation work should consider the difficulties encountered in the Phase 1 studies in 

the planning of the test protocol and overall testing program. 
ENV/JM/MONO(2007)26 

 52

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

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

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

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steroids on the survival, development and reproduction of marine copepods (Tisbe battagliai). Sci. 

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Hutchinson, T.H., Pounds, N.A., Hampel, M., Williams, T.D. 1999b. Life-cycle studies with marine 

copepods (Tisbe battagliai) exposed to 20-hydroxyecdysone and diethylstilbestrol. Environ. Toxicol. 

Chem. 18(12), 2914-2920. 

Huys, R., Gee, J.M., Moore, C.G., Hamond, R. 1996. Marine and brackish water harpacticoid copepods 

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(New Series), The Dorset Press, Great Britain, ISBN: 1-85153-256-0. 

Huyskens, P., Mullens, J. and Gomez, A. 1975. Solubility of alcohols, phenols and anilines in water. Bull 

Soc Chimiq Belges 84 (3), 253-262 

Ingersoll, C.G., Hutchinson, T.H., Crane, M., Dodson, S., DeWitt, T., Geis, A., Huet, M.C., McKenney, 

C.L., Oberdörster, E., Pascoe, D., Versteeg, D.J., Warwick, O. 1999. Laboratory tests for evaluating 

potential effects of endocrine disrupting Componds. In: Endocrine disruption in invertebrates: 

SETAC Technical Publication (de Fur, P.J., Crane, M., Ingersoll, C.G., Tattersfield, L.J. Eds.), 

Brussels, Belgium, ISBN 1-880611-27-9, pp.107-109. 

ISO 1997. International Organization for Standardization. Water Quality - Determination of acute lethal 

toxicity to marine copepods (Copepoda, Crustacea). Draft International Standard ISO/DIS 14669. 

Geneve, Switzerland. 

Knuckey RM, Semmens GL, Mayer RJ, Rimmer MA. 2005. Development of an optimal microalgal diet 

for the culture of the calanoid copepod Acartia sinjiensis: Effect of algal species and feed 

concentration on copepod development. Aquaculture In press. 

Kuch H.M. and K. Ballschmiter 2001. Determination of endocrine-disrupting phenolic compounds and 

estrogens in surface and drinking water by HRGC-(NCI)-MS in the picogram per liter range Environ 

Sci Technol 35, 3201. 

Liber, K., N.K. Kaushik, K.R. Solomon and J.H. Carey. 1992. Experimental designs for aquatic mesocosm 

studies: A comparison of the "ANOVA" and "Regression" design for assessing the impact of 

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Mu X, LeBlanc GA. 2002. Environmental antiecdysteroids alter embryo development in Daphnia magna.

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Guidelines for Testing of Chemicals. 39 pp. 
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OECD (Organization for Economic Cooperation and Development). 2005b. Proposal for a New 

Guideline: Harpacticoid Copepod Development and Reproduction Test. OECD Draft Guidelines for 

Testing of Chemicals. 34 pp. 

OECD (Organization for Economic Cooperation and Development). 2005c. Pre-validation of Full LifeCycle test with the Harpacticoid Copepods Nitocra spinipes, Tisbe battagliai and Amphiascus 

tenuiremis (Authors: M. Breitholtz, T.D. Williams and G.

T. Chandler). September 2005. 

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pentafluorobenzoyl derivatives. Chemosphere 10(7), 767-73. 

Shiu, W.Y., Ma K.C., and Varhanickova, D. 1994. Chlorophenols and alkylphenols - a review and 

correlation of environmentally relevant properties and fate in an evaluative 

environment. Chemosphere 29 (6): 1155-1224. 

SIS 1991. Determination of acute lethal toxicity of chemical substances and effluents to Nitocra spinipes 

Boeck - Static procedure (in Swedish) Swedish Standard SS 02 81 06. SIS - 

standardiseringskommissionen i Sverige, Stockholm, Sweden. 17 pp. 

SPSF 2002. Standard Project Submission Form for a New Test Guideline on Copepod Development and 

Reproduction. Submitted by Sweden. 

Wesenberg-Lund, C. 1937. Ferskvandsfaunen, Biologiskt belyst, Invertebrata. Gyldendanske Boghandel - 

Nordiskt Forlag, Copenhagen, Denmark. 

Williams DR, Fisher MJ, and Rees HH. 2000. Characterization of ecdysteroid 26-hydroxylase: An enzyme 

involved in molting hormone inactivation. Arch. Biochem. Bipphys. 376, 389-398. 

Zarorc-Konĉan, J., Gotvajn, A Z and Tisler, T. 2002. Hazard idntification for 3,5-dichlorophenol in the 

aquatic environment. Cellular & Microbiol Lett 7 (2): 381-382. 
ENV/JM/MONO(2007)26 

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APPENDIX A 

Figure 1 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Control

Figure 2 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Conc 6.6
ENV/JM/MONO(2007)26 

 56

Figure 3 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Conc 20

Figure 4 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Conc 60
ENV/JM/MONO(2007)26 

 57

Figure 5 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Conc 180

Figure 6 

0.00 0.20 0.40 0.60 0.80 1.00

Survival Proportion

0 5 10 15 20 25 30 35

Days after start

BEI ITM

IVL NOAA

Toxicon

Kaplan-Meier Survival Estimates

Conc 540
ENV/JM/MONO(2007)26 

 58

Figure 7 

0.00 0.25 0.50 0.75 1.00

Survival Proportion

0 1 2 3 4 5 6 7 8 9 10 11 12

Days after C1

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier Survival Estimates

NOAA

Figure 8 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier Survival Estimates

NOAA
ENV/JM/MONO(2007)26 

 59

Figure 9 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier Survival Estimates

BEI

Figure 10 

0 .2 .4 .6 .8 0 .2 .4 .6 .8

0 10 20 30

0 10 20 30

ITM NOAA

Toxicon

pred obs

Proportion

konc^0.5

Graphs by lab
ENV/JM/MONO(2007)26 

 60

Figure 11 

0 10 20 30 40 0 10 20 30 40

0 6.6 20 60 180 540 0 6.6 20 60 180 540

0 6.6 20 60 180 540

ITM NOAA

Toxicon

Total offspring production

Graphs by lab

Figure 12 

0 5 10 15 20 0 5 10 15 20

0 6.6 20 60 180 540 0 6.6 20 60 180 540

0 6.6 20 60 180 540

ITM NOAA

Toxicon

Time to first clutch (days)

Graphs by lab
ENV/JM/MONO(2007)26 

 61

Figure 13. Average egg production (over 3 days) against concentration (Control=0). 

0 20 40 60 80 0 20 40 60 80

0 1 2 3 4 5

0 1 2 3 4 5

DHI DTU

OPUS

Egg production (average over 3 days)

concgr

Graphs by lab
ENV/JM/MONO(2007)26 

 62

APPENDIX B 

VALIDATION OF THE COPEPOD DEVELOPMENT AND REPRODUCTION TEST, PHASE 1 

DATA ANALYSIS 

Jan-Olov Persson 

Mathematical Statistics 

Stockholm University 
ENV/JM/MONO(2007)26 

 63

1) Harpacticoid Copepod Development and Reproduction Test 

DATA MANAGEMENT 

All data was delivered in Excel format from ITM and then transferred to, and analysed with the 

statistical software Stata version 8 (http://www.stata.com). 

There were development data from all five laboratories; Adriana6

, Chandler7, ITM, IVL and 

Toxicon, and reproduction data from Adriana, ITM, and Toxicon. 

Development test 

In the development test, the following outcome was analysed: mortality, development rate and 

sex ratio. 

Mortality 

a) Mortality in controls 

For a test to be valid, the mortality in the control must not exceed 20% at the end of the exposure 

period (Test protocol Appendix A). Whether these requirements have been fulfilled was examined by the 

use of the non-parametric Kaplan Meier estimate of the survival function (1). In this analysis, an individual 

who turned out to be adult or missing was handled as censored at that specific time point. Also individuals 

still alive at the end of experiment were treated as censored. 

The length of the experiment period differed between laboratories (Adriana 29 days, Chandler 22 

days, ITM 26 days, IVL 16 or 19 days8

, Toxicon 35 days) and the mortality was examined both the last day 

(over all mortality) and at day 16. 

Table 1 shows the estimated survival proportions, over all, with 95% confidence intervals. Table 

2 shows the estimated survival proportions, at day 16, with 95% confidence intervals. The Kaplan Meier 

survival curves are shown in Figure 1 in appendix. 

 

6

 In this report the name Adriana is used. The correct name is The NOAA Center for Coastal Environmental Health 

and Biomolecular Research 

7

 In this report the name Chandler is used. The correct name is Block Environmental Inc. 

8

 19 days for concentration 180 and 540 
ENV/JM/MONO(2007)26 

 64

Table 1. Over all survival 

 Lab | Survival Lower_95 Upper_95 

---------+------------------------------ 

 Adriana | 0.861 0.757 0.923 

Chandler | 1.000 . . 

 ITM | 0.724 0.154 0.946 

 IVL | 0.922 0.805 0.970 

 Toxicon | 0.965 0.865 0.991 

---------------------------------------- 

Table 2. Survival until day 16 

 Lab | Survival Lower_95 Upper_95 

---------+------------------------------ 

 Adriana | 0.861 0.757 0.923 

Chandler | 1.000 . . 

 ITM | 0.965 0.867 0.991 

 IVL | 0.922 0.805 0.970 

 Toxicon | 0.965 0.865 0.991 

---------------------------------------- 

For ITM the over all mortality exceeds 20 % (27.6%), but the mortality until day 16 is tolerable. 

The other laboratories show a tolerable mortality for this particular experiment. 

An examination of the Kaplan Meier survival curve (Figure 1) shows a massive mortality at day 

one (8 individuals out of 72). 

For Adriana we cannot be statistically certain that the “population mortality” is lower than 20 % 

(since the lower 95% confidence limit is below 80%). 

b) Mortality in exposed / comparison with control 

The mortality among exposed was investigated in the same manner as the control (estimation of 

the Kaplan Meier survival function). Figure 2 -4 shows the survival curves for three of the concentrations; 

6.6, 20 and 540. For some of the laboratories there is a significantly higher mortality for the exposed 

compared to the control (details to be reported by Magnus Breitholtz). 

The mortality was also investigated separately for the different development stages (nauplii to 

copepodite and copepodite to adult). One peculiar example is shown in Figure 5; the survival in the 

copepodite to adult stage, for Adriana. Almost all deaths occur one day after reaching copepodite stage, 

which was surprising and difficult to explain. There are probably some explanations other than randomness 

for that. 

Magnus Breitholtz will report more details and graphs on the stage specific mortality. 
ENV/JM/MONO(2007)26 

 65

Development 

The development rates have to be estimated for the different development stages (nauplii to 

copepodite and copepodite to adult) separately. The developments data should also be treated as survival 

data were the outcome variable is copepodite/adult (yes or no), and were censored observations are present 

since the time to copepodite/adult is not observed for every individual. A first approach to the analysis was 

to fit a parametric survival function to data (lognormal or gamma), but it did not fall out well. The reason 

for that could be that this particular data is not pure survival data; there may perhaps be individuals that 

never become copepods or adults even if we follow them to infinity. In order to take this in account a more 

sophisticated model would have been needed. As a simpler alternative the analysis was based on the 

nonparametric Kaplan Meier survival estimate. 

In the analysis of development rate from nauplii to copepodite, an individual who turn out to be 

dead or is missing, was handled as censored and a nauplii that did not develop to copepodite during the 

follow up time was also censored. 

The analysis of development rate from copepodite to adult was performed on individuals that had 

developed to copepodites during the follow up time. If an individual was dead, missing or did not become 

an adult within the follow up time, it was handled as censored at that specific time point. 

There are different measures of development rate which can be used, e.g. mean time, median 

time, proportion developed at a particular time point, and it is not straightforward how to choose the most 

suitable measure for this data. The first candidate is the median value, which is an ordinary measure for 

survival data. For this particular data, the median will be a quite rough measure. The reason for that is the 

low precision of the time measurements compared to the quite small exposure effect. The time 

measurements are rounded up to one whole day and consequently the median have a value in a whole day. 

The second measure candidate is the mean value. The mean value is calculated as the area under the 

Kaplan Meier curve, and that measure should be less rough and more appropriate, if we had pure survival 

data and if the follow up time was longer. For data in this study, the mean value may be sensitive to the 

choice of endpoint time. For that reason the median (50th percentile) complemented with the 25th 

percentile was chosen. In addition to that, a Log rank test was performed to test equality of the survival 

curve between an exposure and the control. The results are presented in Table 3 and Table 4 and the 

corresponding Kaplan Meier failure curves are shown in Figure 6 to 10 in appendix (only for the 

development from nauplii to copepodite). 

Note: Individuals that died day 1 from start were excluded from the study. 
ENV/JM/MONO(2007)26 

 66

Table 3. Time in days to copepodite, 25th and 50th percentile. 

P-value of Log-rank test 

---------------------------------------------------- 

Lab and | 

conc. | p25 p50 p-value 

----------+----------------------------------------- 

Adriana | 

 0 | 10 11 

 6.6 | 8 9 .0425 

 20 | 9 11 .6 

 60 | 9 10 .155 

 180 | 10 10 .258 

 540 | 11 12 .000137 

----------+----------------------------------------- 

Chandler | 

 0 | 8 10 

 6.6 | 9 10 .0265 

 20 | 9 11 .103 

 60 | 12 13 1.21e-08 

 180 | 11 15 4.30e-10 

 540 | 2.75e-08 

----------+----------------------------------------- 

ITM | 

 0 | 9 9 

 6.6 | 9 10 .432 

 20 | 9 9 .967 

 60 | 9 11 .00437 

 180 | 9 10 .0146 

 540 | 10 12 .000208 

----------+----------------------------------------- 

IVL | 

 0 | 8 9 

 6.6 | 8 9 .0834 

 20 | 8 9 .0469 

 60 | 8 9 .00503 

 180 | 12 12 .0272 

 540 | 12 14 2.08e-06 

----------+----------------------------------------- 

Toxicon | 

 0 | 7 8 

 6.6 | 8 8 .601 

 20 | 6 7 .0000705 

 60 | 6 7 .000456 

 180 | 8 9 .539 

 540 | 7 8 .431 

---------------------------------------------------- 
ENV/JM/MONO(2007)26 

 67

Table 4. Time in days between copepodite and adult, 25th and 50th percentile. P-value of Log-rank test 

---------------------------------------------------- 

Lab and | 

conc. | p25 p50 p-value 

----------+----------------------------------------- 

Adriana | 

 0 | 8 8 

 6.6 | 6 7 .136 

 20 | 6 7 .19 

 60 | 7 8 .825 

 180 | 7 8 .622 

 540 | 9 10 .000234 

----------+----------------------------------------- 

Chandler | 

 0 | 8 8 

 6.6 | 7 9 .155 

 20 | 6 8 .597 

 60 | 6 8 .0325 

 180 | 9 9 .0273 

 540 | 

----------+----------------------------------------- 

ITM | 

 0 | 6 7 

 6.6 | 7 8 .0121 

 20 | 7 8 .00129 

 60 | 6 8 .231 

 180 | 7 8 .0114 

 540 | 8 9 6.15e-06 

----------+----------------------------------------- 

IVL | 

 0 | 4 5 

 6.6 | 4 7 .125 

 20 | 4 6 .145 

 60 | 4 7 .0353 

 180 | 4 7 .0265 

 540 | 4 5 .174 

----------+----------------------------------------- 

Toxicon | 

 0 | 10 13 

 6.6 | 11 12 .58 

 20 | 11 13 .533 

 60 | 10 14 .272 

 180 | 10 11 .241 

 540 | 13 15 .0214 

---------------------------------------------------- 
ENV/JM/MONO(2007)26 

 68

Sex ratio 

For a test to be valid, the proportion of females (or males) in the control should be within 40% to 

60% (Test protocol Appendix A). 

Table 5 shows the female proportion for each laboratory and concentration, and for the four 

laboratories that have made sex determination, the female proportion in the control is within the limits. 

Table 5. Female proportion in adults 

------------------------------------------------------------ 

 | Lab. 

 conc. | Adriana Chandler ITM IVL Toxicon 

----------+------------------------------------------------- 

 0 | 0.53 0.56 0.57 0.43 

 6.6 | 0.49 0.50 0.43 0.49 

 20 | 0.52 0.53 0.60 0.45 

 60 | 0.58 0.58 0.55 0.46 

 180 | 0.44 0.78 0.53 0.47 

 540 | 0.29 0.53 0.31 

------------------------------------------------------------ 

A notification was made about the time range from copepodite to adult for the control, among 

individuals that had become adults (not a survival analysis estimate): 

For Adriana and Chandler there were no significant differences between sexes. For ITM there 

was a significant difference (mean time: female 7.9 days, male 6.4 days). For Toxicon there was a large 

significant difference (mean time: female 10.4 days, male 14.3 days). 

For ITM this difference was due to difference from nauplii to copepodite but for Chandler there 

was no difference from nauplii to copepodite. 

Reproduction test 

Three laboratories performed the reproduction test (Adriana, ITM and Toxicon) and the 

outcomes analysed was fertilisation success, offspring production and time to first and second clutch. No 

survival analysis was made since information about the censored pairs was missing. We cannot therefore 

exclude that an observed effect for an exposed group might be a consequence of a differences in mortality 

for that group compared to the control. 

Fertilisation success 

The fertilization success was measured as the proportion of pairs able to produce two successive 

clutches within the follow up time, of all mating pairs at start9

. The follow up time differs between 

 

9

 ITM observed that several of their pairs consisted of two females. These pairs were excluded. 
ENV/JM/MONO(2007)26 

 69

laboratories (Adriana 10 days, ITM 19 days and Toxicon 20 days), which possibly make the comparisons 

between laboratories invalid. The results are shown in Table 6 to 8; number of mating pairs at start, the 

proportion of pairs with two clutches together with exact 95 % confidence limits and a p-value of testing 

the hypothesis of similar proportions in the exposed and the control (Fisher’s exact test) . 

Table 6. Proportion of pairs with two clutches, for Adriana. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 23 .74 .52 .9 . 

 6.6| 23 .65 .43 .84 .75 

 20 | 20 .50 .27 .73 .13 

 60 | 18 .50 .26 .74 .19 

 180 | 21 .52 .3 .74 .21 

 540 | 7 .14 .0036 .58 .0086 

------------------------------------------------------------ 

Table 7. Proportion of pairs with two clutches, for ITM. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 18 .67 .41 .87 . 

 6.6| 20 .25 .087 .49 .021 

 20 | 18 .56 .31 .78 .73 

 60 | 10 .50 .19 .81 .44 

 180 | 15 .13 .017 .4 .004 

 540 | 11 .091 .0023 .41 .0057 

------------------------------------------------------------ 

Table 8. Proportion of pairs with two clutches, for Toxicon. 

 Conc | Freq Prop Lower_95 Upper_95 p-value 

---------+-------------------------------------------------- 

 0 | 17 .82 .57 .96 . 

 6.6| 21 .76 .53 .92 .71 

 20 | 22 .59 .36 .79 .17 

 60 | 22 .59 .36 .79 .17 

 180 | 18 .39 .17 .64 .015 

 540 | 16 .063 .0016 .3 .000013 

------------------------------------------------------------ 

The observed proportions of pairs able to produce two clutches are lower for the exposed 

compared to the control, for all concentrations in all three laboratories. If we use significance level10 5%, 

every one of the laboratories show significant effects of concentration 540, and ITM along with Toxicon 

 

10 not adjusted for multiple test 
ENV/JM/MONO(2007)26 

 70

also show significant effects of concentration 180. In addition, ITM show significant effect for 

concentration 6.6. 

The probability that a statistical test of this kind will fall out significant (the power of the test) 

depends on the size of the true effect and the sample size. Obviously, we cannot conclude, for a nonsignificant test, that there are no effects. The power might just have been to low to reveal it. Of that reason, 

no attempt was made to estimate NOEC or LOEC. 

An alternative approach is to assume a model for the dose response relationship. For this data, 

there seems to be a monotonic dose response trend (with exception on concentration 6.6 for ITM) and a 

logistic regression model was fitted for each laboratory separately. The models are illustrated in Figure 10 

in Appendix, and except for the observation from ITM on concentration 6.6, the fit is quite good. 

In order to estimate a EC50 -value a non-linear model, would have to be fitted. This is not a straightforward 

task (model choice, programming, model check etc.) and because of the rather low quality of data (we 

cannot exclude that the effects seen, depends on mortality) and shortage of time, no such attempt was 

made. 

Offspring production 

For a test to be valid the average number of viable offspring per clutch in the controls should not 

be lower than 5 (Appendix A). This criterion was met for all three laboratories (average number was higher 

than 8 for all three, data not shown). 

For pairs that were able to produce two clutches, the total number of viable offspring was 

calculated. The results are displayed separately for each laboratory in Table 9 to 11. The tables show the 

frequency of mating pairs able to produce two clutches, and the mean value of total offspring production 

together with 95 % confidence limits (based on the t-distribution). A one-way ANOVA for each laboratory 

separately shows no significant differences (p-values11; 0.10, 0.60 and 0.74 for laboratory Adriana, ITM 

and Toxicon respectively). 

The results are also illustrated with box plots in Figure 11 in Appendix. 

Table 9. Total viable offspring production, for Adriana. 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 17 17.4 15.3 19.4 

 6.6| 15 14.7 12.7 16.8 

 20 | 10 15.7 13.4 18 

 60 | 9 12.7 8.77 16.6 

 180 | 11 14.1 10.2 18 

 540 | 1 5 . . 

-------------------------------------------------- 

 

11 Concentration 540 with only one observation was excluded in this test. 
ENV/JM/MONO(2007)26 

 71

Table 10. Total viable offspring production, for ITM. 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 12 22.3 16.3 28.2 

 6.6 | 5 18.6 11.5 25.7 

 20 | 10 23.7 18.2 29.2 

 60 | 5 20 9.32 30.7 

 180 | 2 28.5 9.44 47.6 

 540 | 1 29 . . 

-------------------------------------------------- 

Table 11. Total viable offspring production, for Toxicon. 

 Conc | Freq Mean Lower_95 Upper_95 

---------+---------------------------------------- 

 0 | 14 16.9 13.3 20.4 

 6.6 | 16 19 15.7 22.3 

 20 | 13 19.8 16.1 23.4 

 60 | 13 17.6 13.3 21.9 

 180 | 7 20.3 10.6 30 

 540 | 1 25 . . 

-------------------------------------------------- 

Time to first and second clutch 

Table 12 to 14 shows the median time to the first clutch, estimated for the pairs that had produced 

a first clutch (not a survival analysis estimate). A Mann Whitney U-test was performed to test the 

hypothesis of equal time distribution in the exposed and the control. P-values on these tests are shown in 

the table. Since we have ignored the censored observations, the interpretation of the result should be 

careful. The results are also illustrated with box plots in Figure 12 in Appendix. 

Table 12. Time to production of first clutch, for Adriana. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 17 3 

 6.6 | 17 3 .79 

 20 | 11 3 .48 

 60 | 11 4 .029 

 180 | 13 4 .00027 

 540 | 1 7 

------------------------------------------------------- 
ENV/JM/MONO(2007)26 

 72

Table 13. Time to production of first clutch, for ITM. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 15 7 

 6.6 | 6 8.5 .5 

 20 | 13 10 .082 

 60 | 7 9 .016 

 180 | 4 10 .049 

 540 | 2 6.5 .55 

------------------------------------------------------- 

Table 14. Time to production of first clutch, for Toxicon. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 15 4 

 6.6 | 18 6 .18 

 20 | 17 6 .21 

 60 | 18 7 .0041 

 180 | 12 9.5 .00056 

 540 | 4 5.5 .61 

------------------------------------------------------- 

Table 15 to 17 shows the median time between the first and second clutch, estimated for the pairs 

that had produced a second clutch (not a survival analysis estimate). A Mann-Whitney U-test was 

performed to test the hypothesis of equal time distribution in the exposed and the control. P-values on these 

tests are shown in the table. Since we have ignored the censored observations, the interpretation of the 

result should be careful 

Table 15. Time between first and second clutch, for Adriana. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 17 1 

 6.6 | 15 1 .65 

 20 | 10 1 .19 

 60 | 9 2 .59 

 180 | 11 2 .18 

 540 | 1 3 

------------------------------------------------------- 
ENV/JM/MONO(2007)26 

 73

Table 16. Time between first and second clutch, for ITM. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 12 3 

 6.6 | 5 5 .67 

 20 | 10 4 .17 

 60 | 5 3 .91 

 180 | 2 3 .84 

 540 | 1 3 

------------------------------------------------------- 

Table 17. Time between first and second clutch, for Toxicon. 

------------------------------------------------------- 

 Conc | Freq Median p_value 

----------+-------------------------------------------- 

 0 | 14 3.5 

 6.6 | 16 3 .35 

 20 | 13 3 .27 

 60 | 13 3 .42 

 180 | 7 4 .26 

 540 | 1 4 

------------------------------------------------------- 
ENV/JM/MONO(2007)26 

 74

2) Calanoid Copepod Development and Reproduction Test12 

DATA MANAGEMENT 

All data was delivered in Excel and Word format from ITM and then transferred to, and analysed 

with the statistical software Stata. 

Data were from three laboratories: DHI, DTU and OPUS.

Development test 

In the development test mortality and Larval development ratio (LDR) of F0 was analysed. 

Analysis of LDR of F1 and Length of male and females will be reported by Magnus Breitholtz. 

Mortality of F0

For at test to be valid the average mortality of animals in the controls shall not exceed 30% of the 

hatched animals (Appendix B). Table 18 presents the number of replicates used, and the mortality 

proportion, for each concentration and laboratory. For the laboratories DTU and DHI, one replicate each 

was excluded due to disagreements in data (the number of units at the observation day was larger than at 

the start day). The average mortality in the controls (Conc.=0) is lower than 30% for every laboratory. 

Table 18. Number of replicates and mortality proportion. 

---------------------------------------------- 

 | Conc. 

 Lab | 0 6.6 20 60 180 540 

----------+----------------------------------- 

 DHI | 9 4 4 3 4 4 

 | .18 .25 .19 .11 .22 .39 

 | 

 DTU | 12 6 5 6 6 6 

 | .15 .21 .11 .15 .12 .17 

 | 

 OPUS | 12 5 5 5 5 5 

 | .071 .12 .17 .091 .11 .53 

---------------------------------------------- 

 

12 The analysis of the reproduction test will be reported by Magnus Breitholtz. 
ENV/JM/MONO(2007)26 

 75

LDR of F0 

LDR was measured as the proportion copepodite of the viable animals (number of 

copepodites/(number of copepodites + number of nauplii)). Table 19 shows the mean value and standard 

deviation of LDR for each concentration and laboratory. Figure 13 in Appendix shows a scatter plot of 

LDR against concentration. No clear conclusion about the relationship between LDR and exposition can be 

made. For DHI an effect appear only for the highest concentration. For DTU the plot looks strange; LDR 

in the controls are surprisingly low, and the observations for concentration 20 are clustered into two 

groups. For OPUS there is a monotonous decreasing relationship with some extreme outliers. 

Table 19. LDR of F0, mean value and standard deviation. 

---------------------------------------------------- 

 | konc 

 lab | 0 6.6 20 60 180 540 

----------+----------------------------------------- 

 DHI | .79 .74 .75 .76 .73 .29 

 | .037 .11 .057 .0049 .079 .11 

 | 

 DTU | .47 .58 .48 .45 .41 .043 

 | .049 .034 .18 .073 .037 .025 

 | 

 OPUS | .89 .65 .51 .24 .2 .033 

 | .071 .034 .21 .21 .14 .051 

---------------------------------------------------- 
ENV/JM/MONO(2007)26 

 76

APPENDIX 

Figure 1. Over all survival in the controls. 

0.00 0.20 0.40 0.60 0.80 1.00

Survival proportion

0 5 10 15 20 25 30 35

Days after start

Adriana Chandler

ITM IVL

Toxicon

Kaplan-Meier survival estimates

control
ENV/JM/MONO(2007)26 

 77

Figure 2. Over all survival in exposed, concentration 6.6. 

0.00 0.20 0.40 0.60 0.80 1.00

Survival proportion

0 5 10 15 20 25 30 35

Days after start

Adriana Chandler

ITM IVL

Toxicon

Kaplan-Meier survival estimates

conc 6.6

Figure 3. Overall survival in exposed, concentration 20. 

0.00 0.20 0.40 0.60 0.80 1.00

Survival proportion

0 5 10 15 20 25 30 35

Days after start

Adriana Chandler

ITM IVL

Toxicon

Kaplan-Meier survival estimates

conc 20
ENV/JM/MONO(2007)26 

 78

Figure 4. Over all survival in exposed, concentration 540. 

0.00 0.20 0.40 0.60 0.80 1.00

Survival proportion

0 5 10 15 20 25 30 35

Days after start

Adriana Chandler

ITM IVL

Toxicon

Kaplan-Meier survival estimates

conc 540

Figure 5. Survival in the copepodite to adult stage. Lab.: Adriana. 

0.00 0.25 0.50 0.75 1.00

0 1 2 3 4 5 6 7 8 9 10 11 12

Days after C1

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier survival estimates

Adriana
ENV/JM/MONO(2007)26 

 79

Figure 6. Development from nauplii to copepodite, Adriana. 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier survival estimates

Adriana

Figure 7. Development from nauplii to copepodite, Chandler. 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier survival estimates

Chandler
ENV/JM/MONO(2007)26 

 80

Figure 8. Development from nauplii to copepodite, ITM. 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier survival estimates

ITM

Figure 9. Development from nauplii to copepodite, IVL 

0.00 0.25 0.50 0.75 1.00

0 5 10 15 20

Days after start

konc = 0 konc = 6.6

konc = 20 konc = 60

konc = 180 konc = 540

Kaplan-Meier survival estimates

IVL

. 
ENV/JM/MONO(2007)26 

 81

Figure 10. Proportion of mating pairs able to produce two clutches; observed and predicted values. 

0 .2 .4 .6 .8 0 .2 .4 .6 .8

0 10 20 30

0 10 20 30

Adriana ITM

Toxicon

pred obs

prop

konc^0.5

Graphs by labnr

Figure 11.Total viable offspring production for pairs able to produce two clutches. 

0 10 20 30 40 0 10 20 30 40

0 6.6 20 60 180 540 0 6.6 20 60 180 540

0 6.6 20 60 180 540

Adriana ITM

Toxicon

Total offspring production

Graphs by lab
ENV/JM/MONO(2007)26 

 82

Figure 12. Time to production of first clutch. 

0 5 10 15 20 0 5 10 15 20

0 6.6 20 60 180 540 0 6.6 20 60 180 540

0 6.6 20 60 180 540

Adriana ITM

Toxicon

Time to first clutch (days)

Graphs by lab

Figure 13. LDR of F0 against increasing concentration (Control=0). 

0 .5 1 0 .5 1

0 1 2 3 4 5

0 1 2 3 4 5

DHI DTU

OPUS

LDR_F0

Conc_group

Graphs by lab
ENV/JM/MONO(2007)26 

 83

APPENDIX C (THIS VERSION OF THE INVITATION LETTER DOES NOT INCLUDE 

ANNEXES) 

Invitation for participation in

Ring test of the draft OECD13 Test Guideline 

“Harpacticoid and Calanoid Copepod 

Development and Reproduction Test” 

ABOUT THE OECD TEST GUIDELINES PROGRAMME (TGP) 

Test guidelines, including those produced by the OECD, are used by governments, industry and 

independent laboratories to determine the hazard or safety of chemicals. The use of test guidelines that are 

based on validated test methods promotes the generation of dependable data for human and animal health 

and environmental hazard assessment (OECD, 2005)14. 

In recognition of the advantages of internationally agreed test methods, OECD Member countries 

have developed the OECD Guidelines for the Testing of Chemicals in order to: 

• enhance the validity and international acceptability of test data; 

• make the best use of available resources in both governments and industry; 

• avoid the unnecessary use of laboratory animals; 

• minimise non-tariff trade barriers. 

 

13 OECD – Organisation for Economic Co-operation and Development 

14 OECD Series on Testing and Assessment No 34: Guidance Document on the Validation and 

International Acceptance of New and Updated Test Methods for Hazard Classification, 2005 
ENV/JM/MONO(2007)26 

 84

In addition, OECD countries have adopted the Decision of the Council concerning the Mutual 

Acceptance of Data in the assessment of chemicals. This Decision, which is binding on Member countries, 

requires that data generated in an OECD Member country in accordance with OECD Test Guidelines and 

Principles of Good Laboratory Practice (GLP) will be accepted in other OECD countries for purposes of 

health, safety and environmental assessment (OECD web site). 

Test method validation is a process based on scientifically sound principles by which the 

reliability and relevance of a particular test, approach, method, or process are established for a specific 

purpose. Reliability is defined as the extent of reproducibility of results from a test within and among 

laboratories over time, when performed using the same standardized protocol. The relevance of a test 

method describes the relationship between the test and the effect in the target species and whether the test 

method is meaningful and useful for a defined purpose, with the limitations identified. In brief, it is the 

extent to which the test method correctly measures or predicts the (biological) effect of interest, as 

appropriate. Regulatory need, usefulness and limitations of the test method are aspects of its relevance. 

New and updated test methods need to be both reliable and relevant, i.e., validated (OECD, 2005)2

.

RING TEST OF THE DRAFT OECD TEST GUIDELINE “HARPACTICOID AND CALANOID 

COPEPOD DEVELOPMENT AND REPRODUCTION TEST” 

Background 

At the moment there are no internationally harmonised (i.e. OECD) chronic test methods for 

marine invertebrates, although they represent an ecologically important and large group of organisms that 

need to be protected to secure transfer of energy from the primary producers to higher levels in the 

ecosystems, because this group of animals serves as food for fish and marine mammals. Copepods, 

subdivided into seven groups, of which the calanoids and harpacticoids are two, has the largest number of 

species of any of the lower crustacean groups. They are enormously abundant as individuals and constitute 

perhaps the most important group in the plankton of the world’s oceans. They are also perhaps the 

ecologically most important of the crustaceans in that they are herbivores, grazing on the phytoplankton 

and thus forming the basis of most food chains in the sea. Therefore, the development of a test guideline on 

development and reproduction of marine copepods has been conducted under the work plan of the Error! 

Hyperlink reference not valid.. The copepod reproduction test is needed in several regulatory systems 

and will be of wide applicability and as such will facilitate international harmonisation of risk and hazard 

assessments. It addresses important endpoints and environmental compartments 

(marine/estuarine/brackish) that are poorly covered at present. Four common and regularly used species in 

toxicity tests are presented as model test organisms, namely the harpacticoid copepods Nitocra spinipes, 

Amphiascus tenuiremis and Tisbe battagliai as well as the calanoid copepod Acartia tonsa. 

The work was initially started by Swedish members at the Department of Applied Environmental 

Science Error! Hyperlink reference not valid., Stockholm University. In 2002 a first unofficial draft 

including both harpacticoid and calanoid copepods (which were written by the Swedish members together 

with colleagues from England and Denmark) were sent to the members of the OECD expert group on 

invertebrates. Since then, the draft guideline has been divided into two separate documents, which have 

been circulated to the OECD expert group on invertebrates two times. In September 2005 the two drafts 

have been revised according to the results and experiences of the pre-validation round. These revised drafts 

are found in Annex A (harpacticoid draft) and Annex B (calanoid draft), respectively. 
ENV/JM/MONO(2007)26 

 85

Sweden (i.e. ITM together with the Swedish Chemicals Inspectorate) will lead the validation of 

the copepod tests, which includes sending out reference chemicals, preparation of the protocols to be used 

in the ring tests, data treatment and writing of a final validation report (see Table 1 below). The two test 

protocols are currently separate to reflect the differences in biology and thus in the testing conditions 

between calanoid species and harpacticoid species. However, protocols are intended to be merged into one 

OECD Test Guideline in the future, when the validation has demonstrated the reliability of the protocols, 

including common parts valid for both groups and two separate parts reflecting the difference in 

experimental set up between the calanoid and harpacticoid species (see Test design).

Test design 

The proposed harpacticoid and calanoid tests focus on identifying effects on development and 

reproduction, with emphasis on potential endocrine disruptive effects. A brief overview of the respective 

test is given below. Harpacticoid and calanoid copepods are closely related phylogenetically, but they 

differ ecologically. Harpacticoids are almost always benthic or epibenthic and have strong thigmotaxes 

that make them want to be in contact with structure (e.g., sediments or the walls of microwell test 

chambers). Calanoids on the other hand are much stronger swimmers, usually negatively thigmotactic and 

seek suspension in larger volumes of test media. Owing to these differences the experimental setup of the 

two tests also differs. 

Harpacticoida 

The test is a full life cycle test, where newly hatched larvae (termed nauplia or metanauplia), 

aged less than 24 hours at the start of the test, are exposed individually in microwell (300-5000 μL total 

volume) test chambers to the test substance added to seawater at a range of concentrations. Mandatory test 

variables to investigate in the F0 generation are mean development rates to the copepodite and adult stages, 

respectively, fertilization success (i.e. number of mating pairs able to produce two successive clutches), 

total viable offspring production per mating pair (i.e. through two clutches averaged over the number of 

mating pairs created and tested), time to production of first clutch, time interval between successive 

clutches, sex ratio and survival of the parent animals. The test duration of the full life cycle test will vary 

according to life cycle rate of the test species, but it should extend to hatching of > 80% of the second 

clutches in control pairs at 20oC (termed the F1 generation). When the copepods reach adulthood, which 

normally takes 10-20 days for the present species, the sex of each copepod is determined by light 

microscopy. Individual male:female mating pairs are constructed and allowed to mate for 7-14 days in new 

isolated micro well test chambers containing seawater and the test substance added to seawater at the same 

range of concentrations as during maturation/development. The mating pairs are followed until the control 

females have been fertilized and have released at least two clutches of offspring. The reproductive output 

of the animals exposed to the test substance is compared to that of the control(s) over a fixed time period in 

order to determine the lowest observed effect concentration (LOEC) and hence the no observed effect 

concentration (NOEC). In addition, and as far as possible, the data are analyzed using a regression model 

in order to estimate the concentration that would cause an x% reduction in reproductive output, i.e. ECx

(e.g. EC10). 

Calanoida 

The test is a full life-cycle test, where the organisms are exposed to various concentrations of the 

test substance from the egg stage to the juvenile stages of the next generation (F1 generation). Mandatory 

test variables to investigate in the F0 generation are mean development of early life stages (i.e. larval 

development ratio also called LDR), survival of early stages and egg producing females, egg production

and length of animals. The next generation (F1) is exposed during its early life stages and mean larval 

development ratio and survival are investigated. The naupliar (larval) and copepodite (juvenile) stages are 
ENV/JM/MONO(2007)26 

 86

morphologically distinct, therefore, the transition from the last naupliar to the first copepodite stage is 

easily observed. Larval development ratio is normally recorded after 5-7 days, when about 50% of the 

control animals have reached a copepodite stage, and is expressed as the ratio of copepodites to the total 

number of surviving early life stages (nauplii + copepodites) at the end of the LDR test. Hatching success 

of eggs and mortality of the early stages are presented along with the LDR. Egg production of females, 

which have been exposed through their whole life from egg to adult, is also studied. At the proposed test 

condition this takes about 12-14 days. Such eggs may bear a chemical burden transferred from the exposed 

females to the eggs and this can in some cases result in a more sensitive response of the next (F1) 

generation LDR test (relative to the F0 test), because the animals are also exposed in their embryonic stage 

before hatching. The observed variable of the animals exposed to the test substance is compared to that of 

the control(s). As far as possible, the data are analyzed using a regression model in order to estimate the 

concentration that would cause an x% reduction in reproductive output, i.e. ECx (e.g. EC10). Also the 

lowest observed effect concentration (LOEC) and hence the no observed effect concentration (NOEC) may 

be found using ANOVA statistics. 

A short compilation of the harpacticoid and calanoid full life cycle test design (test duration, test 

solution renewal, feeding, etc.) is presented in Annex C. 

Reference chemicals 

At the OECD invertebrate expert meeting in Paris 2003 it was decided that two reference 

chemicals should be used in order to cover both general toxicity and specific endocrine activities. 3,5-

dichlorophenol (DCP) was chosen as a reference substance because of its non-specific toxic mode of 

action, since it is a regularly used reference substance in biological tests and is relatively easy to handle 

and analyze chemically. This substance has also been used in a pre-validation round in 2004-2005. As a 

reference substance with a specific mode of action we will use bisphenol-A, which has been shown to 

delay development of young stages in calanoids, harpacticoids and cladocerans. A disrupting mode of 

action has been proposed for all three groups of organisms; e.g. in cladocerans bisphenol-A was supposed 

to exert its activity via an enhancement of the activity of endogenous juvenile hormone analogs that 

decrease larval and juvenile development. 

Both chemicals are relatively stable in water over short time periods. DCP will partly dissociate 

at the test pH, because of its pKa of 8.2. Bisphenol A has a pKa of 10.8 and is not supposed to dissociate at 

the test pH. Further information about the reference substances is presented in Annex D. 

Both reference substances will be available free of charge from ITM to ensure that all 

participants are using the same batch of test chemicals. 

Chemical analysis 

The project has received about 10.000 euro to fund parts of the chemical analysis. Efforts will be 

made to analyse the respective test substances from all participating laboratories at the same laboratory in 

order to avoid what may become a confounding factor of unreasonable lab-to-lab analytical variability, and 

to minimize the costs for test analyses. 

Laboratories involved in the development of the copepod guideline 

The laboratories that have been participating in the development and pre-validation of the 

copepod guideline are listed below (main species in brackets). Cultures of both copepods and microalge 

will be available from these laboratories on request. It is desirable that the participating laboratories have 

former experience from working with copepods in controlled laboratory experiments. Information on how 

to get into contact with the responsible scientists at each lab is presented at the end of this invitation. 
ENV/JM/MONO(2007)26 

 87

Harpacticoid copepods:

• Department of Applied Environmental Science (ITM), Sweden (Nitocra spinipes) 

• Department of Environmental Health Sciences, University of South Carolina, USA (Amphiascus 

tenuiremis) 

• AstraZeneca, Brixham, UK (Tisbe battagliai) 

Calanoid copepod

• Environment & Resources DTU, Technical University of Denmark (Acartia tonsa) 

• DHI Water & Environment, Hørsholm, Denmark (Acartia tonsa) 

Planned exercise for 2005-2006 

Each participating laboratory should use the existing drafts (annex A and B, respectively) to run 

full life-cycle experiments on both 3,5-DCP and bisphenol-A. However, a more thorough experimental 

validation plan will be sent out as soon as a laboratory has shown interest in participating in the validation. 

The experiments should at the most take 2 months each, including pre-treatment, range finding 

and after-treatment. This means that each laboratory should be able to finalise tests on both reference 

chemicals and report effect values and other findings to ITM in April 2006. This allows ITM sufficient 

time to compile the results and write a validation report, which should be finished and sent to OECD no 

later than 31st of June 2006. 

Table 1. Planned validation activities for 2005 and 2006. 

 2005 2006 

Laboratories interested in participating in the validation should contact Dr Magnus Breitholtz at 

ITM no later than 15th of October 2005, preferably via e-mail. Please, indicate whether the laboratory has 

earlier experience working with copepods life cycle tests. Also, to be able to direct further inquiries about 

cultures of the different species, the following information should be included in the letter of interest: 

Activity 

Sept Oct Nov Dec Jan Febr March April May June 

Send out invitation x 

Answer on invitation x 

Delivery of cultures x x 

Order/deliver ref 

subst. 

 x 

Optimize cultures x x 

3,5-DCP test x x 

Bisphenol-A test x x 

Send data to ITM x 

Data treatment ITM x x 

Write report x x 

Report to OECD x 
ENV/JM/MONO(2007)26 

 88

• Which species the laboratory is interested in working with; 

• Need for cultures of both copepods as well as micro algae for feed. 

Yours sincerely, 

Dr. Magnus Breitholtz (Nitocra spinipes) 

Stockholm University

Department of Applied Environmental Science (ITM) 

SE-106 91 Stockholm

Sweden

Tel. +46-8-674 7241 

Fax +46-8-674 7636

e-mail: magnus.breitholtz@itm.su.se

Professor G. Thomas Chandler (Amphiascus tenuiremis) 

Dept. of Environmental Health Sciences 

Arnold School of Public Health 

University of South Carolina 

Columbia, SC 29208 

e-mail: tchandler@sc.edu 

Dr Tim D. Williams (Tisbe battagliai) 

AstraZeneca 

Brixham Environmental Laboratory 

Freshwater Quarry 

Brixham 

Devon 

TQ5 8BA 

Tel : +44 (0) 1803 882882 

Fax : +44 (0) 1803 882974 

Professor Kresten Ole Kusk (Acartia tonsa) 

Technical University of Denmark 

Institute of Environment & Resources DTU 

Building 115, DK-2800 Lyngby 

Denmark 

Tel +45-4525 1569 

Fax +45-4593 2850 

e-mail: kok@er.dtu.dk

Estelle Bjørnestad (Acartia tonsa)

DHI Water & Environment 

Hørsholm 

Denmark 

esb@dhi.dk 
ENV/JM/MONO(2007)26 

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APPENDIX D 

Harpacticoid Copepod Life-cycle

Ring-test Protocol

1. Background 

A draft test guideline for a harpacticoid (three species) life cycle protocol has been developed. 

Pre-validation has been conducted and reported and the draft has been revised accordingly. 

OECD has sent out an invitation letter to potential participants in the coming ring test. So far, 

labs from three countries are involved in the ring test of the harpacticoid copepod Test Guideline. 

However, owing to a low number of participating laboratories for Tisbe battagliai, the ring test will only be 

conducted with Nitocra spinipes and Amphiascus tenuiremis. 

The actual ring-test will start in early 2006. However, it was decided at 2nd OECD Invertebrate 

Expert Group meeting in Paris to only use 3,5-DCP as a reference chemical, since it was concluded that the 

aim of the copepod test is not to detect a specific mode of action, but to evaluate test procedures and 

adverse consequences of chemicals on development and reproduction. This means that the ring-test follows 

the scheduled outlined in the invitation letter (see Table 1). 

For more thorough background information, we refer to the ‘Invitation Letter’ that was 

distributed by OECD in September 2005 to national coordinators and interested laboratories. 

2. Ring test activities 

2.1. Laboratories participating in the ring test 

The laboratories that are presently involved in the ring test are presented below. 

Nitocra spinipes 

• Department of Applied Environmental Science (ITM), Stockholm University, SE-106 91 

Stockholm, Sweden. Contact: magnus.breitholtz@itm.su.se. 

• IVL Swedish Environmental Research Institute, Box 210 60, SE-100 31 Stockholm, Sweden. 

Contact: ann-sofie.allard@ivl.se. 

• Toxicon AB, SE-261 92 Landskrona, Sweden. Contact: anders.sjolin@toxicon.com. 
ENV/JM/MONO(2007)26 

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Amphiascus tenuiremis 

• Department of Environmental Health Sciences, Arnold School of public Health, University of 

South Carolina, SC-29208, USA. Contact: Chandlgt@gwm.sc.edu

• Block Environmental Inc., Pleasant Hill, California, USA. Contact: dblock@blockenviron.com 

• The NOAA Center for Coastal Environmental Health and Biomolecular Research (CCEHBR), 

Charleston, South Carolina, USA. Contact: geoff.scott@noaa.gov 

2.2. Schedule 

The general schedule for the ring test of the harpacticoid copepod guideline is outlined in Table 1 

below. 

Each participating laboratory should use the existing draft guideline to run full life-cycle 

experiments on the reference chemical 3,5-DCP. 

The experiments should at the most take 2 months, including pre-treatment, range finding and 

after-treatment. This means that each laboratory should be able to finalize the test and report raw data and 

other findings to ITM in late April 2006. This allows ITM sufficient time to compile the results and write a 

validation report, which should be finished and sent to OECD no later than 31st of June 2006.

2.3. 

Principle of the test 

The test is a full life cycle test, where newly hatched larvae (termed nauplia or metanauplia), 

aged less than 24 hours at the start of the test, are exposed individually in microwell (300 μL total volume) 

test chambers to the test substance added to seawater at a range of concentrations. Mandatory test variables 

to investigate in the F0 generation are mean development rates to the copepodite and adult stages, 

respectively, fertilization success (i.e. number of mating pairs able to produce two successive clutches), 

total viable offspring production per mating pair (i.e. through two clutches averaged over the number of 

mating pairs created and tested), time to production of first clutch, time interval between successive 

clutches, sex ratio and survival of the parent animals. The test duration of the full life cycle test will vary 

Table 1. Ring test activities for 2005 and 2006.

 2005 2006 

Activity 

Nov Dec Jan Febr March April May June 

-Delivery of cultures x x 

Order/deliver ref subst. x 

Optimize cultures x x x 

3,5-DCP test x x x 

Send data to ITM x x 

Send test medium to 

ITM 

 x x 

Chemical analysis ITM x x 

Data treatment ITM x x 

Write report x 

Report to OECD x 
ENV/JM/MONO(2007)26 

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according to life cycle rate of the test species, but it should extend to hatching of > 80% of the second 

clutches in control pairs (termed the F1 generation). When the copepods reach adulthood, which normally 

takes 10-20 days for the present species, the sex of each copepod is determined by light microscopy. 

Individual male:female mating pairs are constructed and allowed to mate for 7-14 days in new isolated 

micro well test chambers containing seawater and the test substance added to seawater at the same range of 

concentrations as during maturation/development. The mating pairs are followed until the control females 

have been fertilized and have released at least two clutches of offspring. The reproductive output of the 

animals exposed to the test substance is compared to that of the control(s) over a fixed time period in order 

to determine the lowest observed effect concentration (LOEC) and hence the no observed effect 

concentration (NOEC). In addition, and as far as possible, the data are analyzed using a regression model 

in order to estimate the concentration that would cause an x% reduction in reproductive output, i.e. ECx

(e.g. EC10).

2.4. Reference chemical 

3,5-dichlorophenol (3,5-DCP) has been chosen as a reference chemical because of its nonspecific toxic mode of action, since it is a regularly used reference substance in biological tests and is 

relatively easy to handle and analyze chemically. This substance has also been used in a pre-validation 

round in 2004-2005. 3,5-DCP is relatively stable in water over short time periods but may partly dissociate 

at the test pH, because of its pKa of 8.2. 

The reference substance will be available free of charge from ITM to ensure that all participants 

are using the same batch of test chemical. 

2.5. Chemical analysis 

ITM has received about 10.000 euro to fund parts of the chemical analysis, which will be made 

by ITM on all participating laboratories test medium samples in order to avoid what may become a 

confounding factor of unreasonable lab-to-lab analytical variability, and to minimize the costs for test 

analyses. 

Test medium should be sampled and pooled across microplates within treatments from each well 

on each test medium renewal occasion (< 15 ml). Fresh test solution from the full concentration range 

should also be sampled each time new stock solution are prepared including start of the test (if a stock 

solution of 200 mL is prepared this volume should be sufficient throughout the test). All test medium 

samples should be stored at -20°C until chemical analyses. 

The general procedure for the chemical analyses is based on a dervatization method for phenolic 

substances. In brief, after addition of NaOH and fluorine-reagents directly to the test medium water, it is 

possible to extract the fluorine esters that are formed to hexane and then use GC/ECD and MS/NICI for 

quantification. 

Owing to a high cost (both analyses and shipping) as well as difficult handling procedures, the 

chemical analyses will only be made on specific treatments, i.e. in the lower end of the dose-range (e.g. 

without an effect) and in the high end (e.g. were significant mortality has occurred).Test medium samples 

will at least be analyzed at the beginning, in the middle and in the end of the exposure. This means that 

participating laboratories should not send all test medium samples to ITM but instead those that will be 

identified after all laboratories have reported their raw data. 

Old test medium should therefore be sampled and pooled (replicates within each treatment) for 

the following concentrations: 6.6, 60 and 540 μg/L. 
ENV/JM/MONO(2007)26 

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2.6. Procedure 

General 

2.6.1 Cultures 

The culture procedures for A. tenuiremis and N. spinipes are described in Annex 2 and 3 of the 

harpacticoid draft guideline. 

2.6.2 Conditions of exposure 

Test vessels – microplates 

Microwell test chambers and other test vessels and apparatus, which will come into contact with 

the test solutions, should, if possible, be made entirely of glass or other materials chemically inert to the 

test chemical. Microwell plates are typically made of polystyrene plastic. Polystyrene is not well suited to 

testing with hydrophobic organic chemicals because of non-specific plastic:organic binding, which can 

reduce chemical bioavailability to copepods. Therefore, microplates with microwells pre-coated with glass 

or polyacrylamide gel are better suited for copepod testing of hydrophobic chemicals than microplates with 

naked polystyrene wells. For N. spinipes it is recommended to use glass coated microwell chambers or to 

silanize the walls (during exposure of individuals) because of problematic surface tension. 

• For A. tenuiremis, 96 well microplates (each well containing 250 μL seawater test solution) are 

used during the development phase. After sexing and creation of sexed pairs, adult pairs are 

exposed in new, unused microwells of each 96-well microplate. 

• For N. spinipes, 96 well microplates (each containing 270 μL) are used during the development 

phase. After sexing and creation of sexed pairs, adults are exposed in 24 well microplates (each 

containing 2 mL). 

Duration 

The test duration of the full life cycle test will vary according to life cycle rate of the test species, 

but it should extend to hatching of > 80% of the second clutches in control pairs (termed the F1

generation). 

Animal loading 

Newly hatched larvae (termed nauplia or metanauplia), aged less than 24 hours at the start of the 

test, are exposed individually (> 60 replicate nauplii per treatment) in microwell (300 μL total volume; see 

above for details) test chambers to seawater (i.e. control) and to the test substance added to seawater at a 

range of concentrations. Treatments should be dispersed randomly among separate replicate (n=3) 

microplates, as should all subsequent handling, loading, observation and feeding. Failure to do this may 

result in bias that could be construed as being a chemical-dependent effect. In particular, if experimental 

units are handled in treatment or concentration order, then some time-related effect, such as operator 

fatigue or other error, could lead to greater or lesser effects at the higher or lower concentrations. Only one 

test chemical concentration should be tested per microplate to reduce chances for microwell cross 

contamination. Twenty or more nauplii should be tested per microplate. Where possible, nauplii should be 

placed within the microwell array away from any end or edge microwells. Microwells located on ends and 

edges of the microplate are most susceptible to temperature and evaporation. Furthermore, if the test 

results are likely to be affected by an initial or environmental condition of the test, such as position in the 
ENV/JM/MONO(2007)26 

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laboratory or environmental chamber, then consideration should be given to blocking the test, or using a 

Latin-square experimental design. 

Feeding 

Species-specific information on feeding of individual copepods in test chambers during 

maturation/development and reproduction is presented below: 

• Amphiascus tenuiremis

Each copepod is fed 2μl of a 1:1:1 volume mixed-algal cell suspension (107 cells/mL) of a 

chlorophyte (e.g., Dunaliella tertiolecta), a chrysophyte (e.g., Isochrysis galbana) and a cryptophyte 

(e.g., Rhodomonas salina) every six days during both maturation/development and reproduction. The 

concentration added to each test chamber at each feeding is ~ 2 x 104 cells/microwell; i.e., ~2 x 105

cells/mL test solution in a 250 uL microwell. 

• Nitocra spinipes 

Each copepod is fed a cell suspension (stock suspension 5 x 107 cells/mL) of a cryptophyte, preferably 

Rhodomonas salina or Rhodomonas baltica, every third day during both maturation/development and 

reproduction. Copepods are fed on test solution renewal occasions hence algae is added to the bulk 

volume of prepared test solution prior to dispensing into test chambers. The concentration added to 

each test chamber at each feeding is ~ 5 x 105 cells/microwell; e.g., ~ 2.5 x 105

 cells/mL test solution 

in a 2 mL microwell 

Light and temperature 

During testing, microplates are covered and placed in a clear/opaque humidity chamber (a 

covered plastic tub holding several sponges soaked in distilled water works well for this) in a temperatureregulated incubator, or a room set at 22°C. Amphiascus requires a 12-h light and 12-h dark test 

photoperiod. N. spinipes should be reared/tested in darkness throughout the test duration. 

Aeration 

The test vessels must not be aerated during the test, but dilution seawater should be aerated for 24 

h prior to filtration at 0.45 μm and subsequent mixing with test chemical stocks. 

Test concentrations and test medium renewal 

The following concentration range should be used for the testing of 3,5-DCP: 

6.6, 20, 60, 180 and 540 μg l-1 3,5-DCP 

Since 3,5-DCP is soluble in water at the given range, a carrier/solvent is not needed to prepare 

the stock solution(s) in water. 

Natural or synthetic seawater may be used as the test medium. If natural seawater is used, it 

should be collected from a location distant from any known sources of pollution and filtered to remove 

indigenous organisms. If synthetic seawater is used, it should be prepared by dissolving reagents of 

recognised analytical grade, or a commercially available formulation, in distilled or deionised water. 

However, for these copepod species, there is insufficient information on the use of synthetic seawater to 
ENV/JM/MONO(2007)26 

 94

allow a particular brand or type to be recommended. The suitability of synthetic seawater for culture and 

testing should, therefore, be evaluated in trials. Any water in which the copepods show suitable long-term 

survival with normal development and fecundity may be used as the test medium. 

The salinity of the test medium should be the same as the culture medium for the respective 

species. Each species may be used at a range of salinities but in this ring test the following salinities should 

be used: 

• ~6.5‰ for N. spinipes. 

• 30 ‰ for A. tenuiremis. 

Whichever salinity is employed, the test organisms must be cultured or maintained at the same 

salinity (±3‰) for at least 7 days before the start of the test. The dilution water must have a dissolved 

oxygen concentration above 90% of the air saturation value, and a pH of 8.0 ± 0.3 before being used to 

prepare the test solutions. It is recommended that all dilution water be filtered at 0.45 μm prior to 3,5 DCP 

test solution preparation to minimize bacterial contamination. 

The dilution water used for testing should be from the same source as water that has been found 

to support culture of the organisms for at least two generations. If, however, the medium to be used in the 

test is different from that used for routine culture, it is a good practice to include an adequate pre-test 

acclimation period (i.e. sufficient time to culture the copepods through at least two generations) to avoid 

stressing the animals. 

Potential evaporation losses of media from the test chambers should be observed daily and, if 

needed, be corrected by the addition of deionised or distilled water. When using microwell test chambers 

(300-2000 μL total volume), which are normally held in temperature-regulated incubators or rooms, care 

should be taken so that the evaporation losses do not exceed 7% - on a daily basis. To prevent such a 

scenario, microplates may be placed over a water bath in a humidity chamber in the incubator or room. 

When the medium is renewed, a second series of microwell test chambers or other appropriate 

vessels are prepared and the animals transferred to them by, for example, a glass Pasteur pipette of suitable 

diameter. The volume of medium transferred with the copepods should be minimised. Alternatively, a 

fixed volume (preferable 70-90% of the total test medium volume) of the medium may be removed from 

each chamber using either automatic pipette or a syringe of suitable diameter. Both procedures should be 

performed under dissection stereomicroscopic observation, and care should be taken to use a pipette or a 

syringe of suitable diameter so as not to damage the copepods when they are aspirated into the pipette or 

syringe. If, after medium renewal, any copepods are observed to have been damaged during this procedure, 

the appropriate test chamber should be disqualified from the test. 

Observations 

As summarized in Table 2 below, observations include sex ratio, larval development ratio (LDR)

of nauplius versus first copepodite stage on day 5-7 of the bioassay, mean development rates of nauplius to 

copepodite, copepodite to adult, and nauplius to adult stages, respectively, fertilization success (i.e. 

number of mating pairs able to produce two successive clutches), total viable offspring production per 

mating pair (i.e. through two clutches averaged over the number of mating pairs created and tested), time 

to production of first and second clutches, and survival of the FO parent animals. Observations should also 

be made for any abnormal behaviour (relative to the controls), including uncoordinated swimming, loss of 

equilibrium, change in pigmentation and atypical quiescence. Sex ratio is determined by observations of 
ENV/JM/MONO(2007)26 

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male and female secondary sexual characteristics (see draft guideline annexes), which in copepods 

normally can be distinguished in the 5th copepodite stage at the earliest. 

• Development: The number of days it takes for development of each individually isolated 

copepod, from a first stage nauplius to a juvenile copepodite to a reproductively mature adult, is 

checked daily. For some harpacticoid species, morphological identification of the different stage 

durations is extremely difficult. In this case, precise determination of time to reach individual 

stage durations is best attempted by recording individual moults to adult (e.g. there are 5 

copepodite stages [moults] to the adult). For all species within a given treatment or control 

microplate, once adulthood is achieved, all males should be pooled briefly in a 60-mm Petri dish, 

and all females within a separate 60-mm Petri dish. Random within-treatment individual pairs 

are then made by selecting one male and one female adult from each pooled collection of same 

treatment individuals. As many pairs as can be made per treatment (< 30 maximum possible 

pairs) are placed one each into new unused microwells loaded with the appropriate 3,5 DCP test 

concentration. 

• Offspring production: After pairing of male:female mating pairs, observations are made daily for 

the presence of females carrying their first and second egg sacs, and offspring are counted as they 

hatch. Live offspring (nauplii) will usually be released from the egg sac after an incubation 

period of two to several days. If necessary, in order to count the number of offspring released 

from second broods, pairs may be placed in new unused microwells until hatch of the second 

brood. The development of egg sacs, the presence of abnormal eggs or aborted unhatching egg 

sacs, the release of offspring and the number of live and dead offspring are recorded at each 

observation point during the approximately 25 day test. Renewal of test medium should be 

continued as described above through hatching of brood two. 

At the end of the exposure period, all remaining copepods (i.e. including unfertilised females and 

males) are either discarded or fixed in formaldehyde (4%) for determination of sex. 

• Mortality: Survival of larvae (nauplii), juveniles (copepodites) and adults is recorded daily during 

maturation/development/mating up to and including the last day of the test. Mortality of mating 

pairs also should be checked and recorded daily. 
ENV/JM/MONO(2007)26 

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Table 2. Summary of endpoints to be reported from the life cycle test.

Detail Endpoints 

Test variables to be reported 

Development 

Reproduction 

Other 

- Mean development rates for full lifecycle and for each life-stage window 

- Fertilization success 

- Total viable offspring production 

- Time to production of first clutch 

- Time interval between successive 

clutches. 

- Sex ratio of F0

- Survival rate of F0 

Regular observations of F0-generation nauplii and copepodites as well as counting of F1-

generation nauplii must be facilitated by the use of light microscopy. Upon termination of the test, staining 

in Lugol’s solution may facilitate counting of released nauplii and aborted egg sacs before using light 

microscopy. 

The results of the observations made during the test should be recorded on the data sheets 

provided below. 

Physical-chemical parameters 

During the test, dissolved oxygen, pH and temperature should be measured in the controls and all test 

concentrations each time test medium is renewed. As a minimum, these measurements shall be made in the 

control(s) and in the highest test concentration. Temperature should preferably be monitored continuously 

in at least one test vessel.

2.6.3. Reporting 

Raw data should be compiled in the attached raw data sheets and sent to ITM as soon as the 

testing has been finalized for each participating laboratory. 
ENV/JM/MONO(2007)26 

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APPENDIX E 

Detailed Calanoid Copepod Life-cycle 

Ring-test Protocol 

1. Background 

A draft test guideline for a calanoid life cycle protocol has been developed. Pre-validation has 

been conducted and reported and the draft guideline has been revised accordingly. 

OECD has sent out an invitation letter to potential participants in the coming ring test. So far, 

four labs from three countries are involved in the ring test of the Calanoid Copepod Test Guideline. At the 

2nd OECD Invertebrate Expert Group meeting in Paris, 3rd to 4th of November 2005, it was decided within 

the coming weeks, once again to take a direct contact with potential participants. 

The actual ring-test will start in spring 2006. However, it was decided at the meeting in Paris to 

use 3,5-DCP as the only reference chemical, since it was concluded that the aim of the copepod test is not 

to detect a specific mode of action, but to evaluate test procedures and adverse consequences of chemicals 

on development and reproduction. This means that the ring-test follows the scheduled outlined in the 

invitation letter (see Table 1). 

For more thorough background information, we refer to the ‘Invitation Letter’ that was 

distributed by OECD in September 2005 to national coordinators and interested laboratories. 

2. Ring test activities 

2.1. Laboratories participating in the ring test with Acartia tonsa 

The laboratories that are presently involved in the ring test are presented below (main species in 

brackets). 

• Institute of Environment & Resources, Technical University of Denmark (E&R), Building 113, 

2800 Lyngby, Denmark (Contact: K. Ole Kusk; kok@er.dtu.dk) 

• DHI Water and Environment (DHI), Agern Allé 5, 2970 Hørsholm, Denmark (Contact Estelle 

Bjørnestad; esb@dhi.dk) 

• Opus Plus Ltd, (OPUS), Flotta, Stromness, Orkney, KW16 3NP, United Kingdom (Contact 

Brenda Hudson; brenda.hudson@opusplus.co.uk) 

• ECT Oekotoxikologie GmbH, Boettgerstr. 2-14 D-65439 Floersheim, Germany (Contact: 

Michael Meller; m-meller@ect.de) 
ENV/JM/MONO(2007)26 

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2.2. Schedule 

The general schedule for the ring test of the copepod guideline is outlined in Table 1 below. 

Each participating laboratory should use the existing draft guideline to perform a full life-cycle 

experiment with the reference chemical 3,5-DCP. 

The experiment should at the most take 2 months, but can be performed in about three-four 

weeks. This means that each laboratory should be able to finalise the test and report raw data, effect values 

and other findings to Department of Applied Environmental Science (ITM), Stockholm University, SE-106 

91 Stockholm, Sweden (Contact: magnus.breitholtz@itm.su.se) in August 2006 at latest and preferably 

earlier. 

2.3. 

Principle of the test 

The test is a full life cycle test, where the organisms are exposed to various concentrations of the 

test substance from the egg stage (F0 generation) to the developing stages of the next generation (F1) 

including adult egg production (F0). Compulsory test variables to investigate in the F0 generation are 

hatching success, development of early life stages (mean larval development ratio also called LDR), 

survival of early stages and egg producing females, egg production and length of animals. The next 

generation (F1) is exposed during its early life stages and hatching success, mean larval development ratio 

and survival are investigated. Optional/Recommended test variables to investigate are acute toxicity for 

range finding purposes, sex ratio in the F0 generation and egg diameter. The total test duration at 20 °C and 

20 ‰ salinity is about three to four weeks (5-7 days for F0 generation LDR test + 17-18 days for F0

generation life cycle exposure + 5-7 days F1 generation LDR test), which is sufficient time to investigate 

the development of the first generation (F0) - and in the life-cycle test for the control animals to reach 

adulthood and produce eggs, as well as for the second generation (F1) to partly develop into copepodites.

The nauplii (larval) and copepodite (juvenile) stages are morphologically distinct, therefore, the transition 

from the last naupliar to the first copepodite stage is easily observed. Larval development ratio is normally 

recorded after 5-7 days, when about 50% of the control animals have reached a copepodite stage, and is 

expressed as the ratio of copepodites to the total number of surviving early life stages (nauplii + 

copepodites) at the end of the LDR test. Hatching success of eggs and mortality of the early stages are 

presented along with the LDR. Egg production of females, which have been exposed through their whole 

Table 1. Ring test activities for 2006.

 2006 

Activity 

Jan Feb Mar Apr May Jun Jul Aug 

-Delivery of cultures x x x 

Order/delivery of 

reference substance 

 x 

Optimize cultures x 

3,5-DCP test x x x x 

Send data to ITM x x x 

Send water samples to 

ITM 

 x x 

Chemical analysis ITM x x 

Data treatment ITM x x 

Write report x 

Report to OECD x 
ENV/JM/MONO(2007)26 

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life from egg to adult, is also studied. At the proposed test condition it takes about 12-14 days for the 

animals to reach adulthood. Eggs produced by exposed females may bear a chemical burden, which might 

result in a more sensitive response of the next (F1) generation in the LDR test (relative to the F0

generation), because the animals are also exposed during their embryonic stage.. Observed variables of 

exposed groups are compared to that of the control(s) in order to determine the lowest observed effect 

concentration (LOEC) and hence the no observed effect concentration (NOEC) by using ANOVA 

statistics. As far as possible, the data are also analyzed using a regression model in order to estimate the 

concentration that would cause an x% reduction in observed variable, i.e. ECx (e.g. EC10). 

2.4. Reference chemical 

3,5-dichlorophenol (3,5-DCP) has been chosen as a reference chemical because of its nonspecific toxic mode of action, its regularly use as reference substance in biological tests and because it is 

relatively easy to handle and analyze chemically. This substance has also been used in a pre-validation 

round in 2004-2005. 3,5-DCP is relatively stable in water over short time periods but may partly dissociate 

at the test pH because of its pKa of 8.2. 

The reference substance will be available free of charge from ITM to ensure that all participants 

are using the same batch. 

2.5. Chemical analysis 

ITM has received about 10.000 euro to fund parts of the chemical analysis, which will be made 

by ITM on all participating laboratories test medium samples in order to avoid what may become a 

confounding factor of unreasonable lab-to-lab analytical variability, and to minimize the costs for test 

analyses. 

Test medium should be sampled and pooled across the replicates of three exposure 

concentrations on each test medium renewal occasion (10-15 ml in total of the composite sample is enough 

for analysis). Fresh test solution from the same three concentrations should also be sampled each time new 

stock solutions are prepared including start of the test. All test medium samples should be stored at -20°C 

until chemical analyses. Concentrations sampled should be 6.6, 60 and 540 μg/L. Samples from more 

concentrations may be collected, but may not necessary be analysed at the end (see below). 

The general procedure for the chemical analyses is based on a derivatization method for phenolic 

substances. In brief, after addition of NaOH and fluorine-reagents directly to the test medium water, it is 

possible to extract the fluorine esters that are formed to hexane and then use GC/ECD and MS/NICI for 

quantification. 

Owing to a high cost (both analyses and shipping) as well as difficult handling procedures, the 

chemical analyses will only be made on specific treatments, i.e. in the lower end of the dose-range (e.g. 

without an effect) and in the high end (e.g. were significant mortality has occurred). Test medium samples 

will at least be analyzed at the beginning, in the middle and in the end of the exposure. This means that 

participating laboratories should not send all test medium samples to ITM but instead those that will be 

identified after all laboratories have reported their raw data. 
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2.6. Procedure 

2.6.1 Culture 

The culture procedures for Acartia tonsa are described in Annex 7 of the calanoid draft guideline. 

2.6.2. Conditions of exposure 

Test vessels and other equipment 

Test vessels, other equipment and apparatus which will come into contact with the test solutions 

should, if possible, be made entirely of glass or other materials chemically inert to the test chemical. 

Duration 

The duration of the full life cycle test with Acartia tonsa at the specified conditions will be about 

4 weeks including LDR tests on F0 and F1. 

Animal loading 

Larval development ratio (LDR) tests (F0 and F1) are started with 50-80 eggs per test vessel. The 

life cycle test (basic or mass exposure) is started with 500-600 eggs per test vessel. Eggs are counted on a 

filter before addition. In Annex 3 of the Calanoid guideline is given an exemplary flow diagram of the test 

procedure. 

Feeding 

Rhodomonas salina (Chryptophyceae) is used as feed. Feeding should be done daily or just after 

media changes. In the flow diagram (Annex 3 of the Calanoid guideline) are given the recommended algal 

densities. 

Light and temperature 

Test vessels shall be covered and placed in a temperature-regulated incubator or room set at 20 

oC with a 16-h light and 8-h dark photoperiod at low light intensity. 

Aeration 

In the life cycle test, vessels are aerated from day 7. In the LDR test vessels are not aerated. 

Test media and media renewal 

Synthetic seawater with a salinity of 20 ‰ is used as medium. Synthetic seawater should be 

prepared by dissolving reagents of recognised analytical grade or a commercially available formulation in 

distilled or deionised water. Any synthetic seawater of known composition in which the copepods show 

suitable long-term survival with normal development and fecundity may be used as the test medium. 

The same type of media should be use as culture media, test media and dilution water. The 

media must have a dissolved oxygen concentration above 70% of the air saturation value, and a pH 

variation of maximally ± 0.3 from the initial pH. The initial pH is typically 8.1-8.3 in the synthetic media 

used at DTU (see annex 7 in the Calanoid guide). 
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Potential evaporation losses of media from the test chambers should be observed regularly and, if 

needed, be compensated by the addition of deionised or distilled water. 

Media is renewed three times a week. - In the calanoid guideline (paragraph 32 and footnote 2 

and 3 and in the flow diagram of Annex 3) are given details on media renewal for the different parts of the 

test. 

Test concentrations and test medium renewal 

The following concentration range should be used for the testing of 3,5-DCP with Acartia tonsa: 

6.6, 20, 60, 180 and 540 μg l-1 

Since 3,5-DCP is soluble in water at the given range, no carrier/solvent is needed to prepare the 

stock solution(s). 

Media should be renewed as described in the draft guideline. An example of how to perform the 

test in practice is given in Annex 3 of the draft guideline for calanoid copepods. 

Observations 

As summarized in Table 2 below, observations include hatching success, larval survival and 

larval development ratio in F0 and F1 generation, fecundity (i.e. number of eggs per female per day), and 

length of males and females. Observations should also be made for any abnormal behaviour (relative to the 

controls), including uncoordinated swimming, loss of equilibrium, change in pigmentation and atypical 

quiescence. Sex ratio may be determined by observations of male and female secondary sexual 

characteristics (see draft guideline annexes), which in copepods normally can be distinguished in the 5th

copepodite stage at the earliest. 

• Hatching, survival and development of early stages: The exact number of eggs transferred to each 

vessel is counted. At the end of the exposure number of unhatched eggs, number of nauplii and 

number of copepodites are counted. These numbers are used for calculation of hatching success, 

survival and LDR. 

The LDR tests are stopped when the fraction of copepodites in the controls is as close to 50 % as 

possible. To determine this point of time several additional control vessels shall be set up. On day 5 control 

vessels are sacrificed with time intervals by adding lugols solution and numbers of nauplii and copepodites 

are counted. If the copepodite fraction is close to 50 % the test is stopped. If the test is started with a time 

interval between the separate vessels, the same time interval should be observed when stopping the test. 

One validity criteria for the LDR test is a copepodite fraction of 50 ± 20 %. If a 30 % copepodite 

fraction is not reached by the end of day 5 the test may be continued at a dark place at 15 °C and the 

examinations may then be resumed the following morning. 

Fecundity: At the end of the basic exposure 12 females from each exposure group are placed 

individually in vessels with an inner chamber and a bottom of stainless steel net (180-200 μm mesh size). 

Eggs will pass through this net but not the female. The females are transferred to new test vessels every 24 

hours. Eggs are not counted after the first 24 hours but the production over the successive three days is 

counted. (See Annex 3 in the calanoid guide for more details). 

• Length of animals: The body length of 25 males and 25 females is measured under a microscope. 

The median axis from the front of the head to the end of the prosome is measured. The urosome 
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(or tail) is NOT considered. The urosome is often bended upwards and it is thus difficult to 

measure the total length. (See Annex 2 of the calanoid guide for more details). 

Survival of 50 males and 50 females in the control of the basic exposure: To secure a sufficient 

number of animals for the egg production test and for measuring lengths this validity criterion has been 

defined. 

Physical-chemical parameters 

During the test, dissolved oxygen, pH and temperature should be measured in the controls and all 

test concentrations each time medium is renewed. As a minimum, these measurements shall be made in the 

control(s) and in the highest test concentration. Temperature should preferably be monitored continuously 

in at least one test vessel.

Table 2. Summary of endpoints to be reported from the life cycle test.

Detail Endpoints 

Test variables to be reported 

Development F0

Reproduction 

Development F1

Other 

- Hatching success 

- Larval survival 

- Larval development ratio (LDR) (count number of eggs at 

start, number of unhatched, number of nauplii and number 

of copepodites at end) 

Length of 25 males and 25 females 

Total number of eggs per female per day (count number of 

eggs over three separate days and number of females) 

- Hatching success 

- Larval survival 

- Larval development ratio (LDR) (count number of eggs at 

start, number of unhatched, number of nauplii and number 

of copepodites at end) 

- Sex ratio of F0

- Survival rate of F0 

(count all egg at start, count and determine sex on all 

surviving animals at end) 

- Egg diameter 

2.6.3 Reporting 

Raw data should be compiled in the attached raw data sheets and sent to ITM as soon as the testing has 

been finalized for each participating laboratory preferably as electronic files or by using the data sheets in 

Annex 5 of the draft guideline.