DRAFT

Invertebrate Two-Generation ISR May 28, 2013

Validation of the Mysid Two-generation Toxicity Test and Harpacticoid 

Copepod Development and Reproduction Test:

Integrated Summary Report

U.S. Environmental Protection Agency

Endocrine Disruptor Screening Program

Washington, D.C.

Prepared by

Battelle

Columbus, Ohio

Gradient

Cambridge, Massachusetts

Contract No. EP-W-11-063

Task Order 4

28 May 2013
DRAFT

Invertebrate Two-Generation ISR ii May 28, 2013

1 Contents

2 Page

3 EXECUTIVE SUMMARY ........................................................................................................VI

4 Currently, the MTTT provides a number of clear advantages over the HCDRT as 

5 an aquatic arthropod test to evaluate chemical effects on invertebrate 

6 growth, reproduction, and survival. Therefore, the MTTT is proposed as a 

7 Tier 2 test to be used in EPA’s EDSP.................................................................. viii

8 OBJECTIVE ................................................................................................................................. 1

9 1 INTRODUCTION............................................................................................................. 1

10 1.1 Endocrine Disruptor Screening Program................................................................ 2

11 1.2 Validation................................................................................................................ 3

12 2 HISTORICAL OVERVIEW OF THE INVERTEBRATE TESTS............................. 6

13 2.1 Purpose of Invertebrate Testing in EDSP ............................................................... 6

14 2.2 Invertebrate Life-cycle and Multi-generational Toxicity Tests for the 

15 Detection of Possible Endocrine Disruption........................................................... 8

16 2.2.1 Relevance of the Mysid Test Model ........................................................... 8

17 2.2.2 Relevance of the Copepod Test Model..................................................... 10

18 3 MYSID AND COPEPOD TEST PROTOCOL DEVELOPMENT............................ 12

19 3.1 Summary of the Mysid Two-Generation Toxicity Test (MTTT) Protocol.......... 12

20 3.1.1 Principle of the Test.................................................................................. 12

21 3.1.2 Endpoints .................................................................................................. 14

22 3.1.3 Statistical Analyses................................................................................... 17

23 3.2 Summary of the Harpacticoid Copepod Development and Reproduction 

24 Test (HCDRT) Protocol........................................................................................ 18

25 3.2.1 Principle of the Test.................................................................................. 18

26 3.2.2 Endpoints .................................................................................................. 20

27 3.2.3 Statistical Analyses................................................................................... 21

28 4 DEMONSTRATION AND OPTIMIZATION OF THE MYSID AND 

29 COPEPOD TEST METHODS ...................................................................................... 23

30 4.1 Mysid Two-Generation Toxicity Test (MTTT) Guideline Performance.............. 23

31 4.1.1 Demonstration Studies Using the 2004 Draft MTTT Guideline .............. 23

32 4.1.2 Optimization Studies Using the 2004 Draft Guideline ............................. 35

33 4.1.3 Lessons Learned from Demonstration and Optimization Studies 

34 Using the 2004 Draft MTTT Guideline .................................................... 38

35 4.2 Proposed Harpacticoid Copepod Development and Reproduction Test 

36 Protocol Performance............................................................................................ 44

37 4.2.1 Demonstration and Optimization Studies Using Proposed HCDRT 

38 Protocol..................................................................................................... 44

39 4.2.2 Lessons Learned from Demonstration and Optimization Studies 

40 Using Proposed HCDRT Protocol............................................................ 47

41 5 INTERLABORATORY EVALUATION OF MYSID AND COPEPOD 

42 TEST METHODS........................................................................................................... 48

43 5.1 Lindane ................................................................................................................. 49
DRAFT

Invertebrate Two-Generation ISR iii May 28, 2013

1 5.1.1 Exposure Levels........................................................................................ 49

2 5.1.2 Sensitive Endpoints................................................................................... 53

3 5.1.3 Conclusions............................................................................................... 55

4 5.2 Atrazine................................................................................................................. 55

5 5.2.1 Sensitive Endpoints................................................................................... 56

6 5.2.2 Conclusions............................................................................................... 56

7 5.3 Vinclozolin............................................................................................................ 57

8 5.3.1 Exposure Levels........................................................................................ 57

9 5.3.2 Sensitive Endpoints................................................................................... 58

10 5.3.3 Conclusions............................................................................................... 60

11 5.4 4-tert-octylphenol.................................................................................................. 60

12 5.4.1 Exposure Levels........................................................................................ 60

13 5.4.2 Sensitive Endpoints................................................................................... 61

14 5.4.3 Conclusions............................................................................................... 62

15 6 DISCUSSION AND SUMMARY OF THE MTTT AND HCDRT METHODS....... 64

16 6.1 Reproducibility of the Test Methods .................................................................... 64

17 6.2 Data Interpretation ................................................................................................ 66

18 6.3 Strengths and Limitations of Each Test................................................................ 67

19 6.3.1 Strengths and Limitations of the MTTT ................................................... 67

20 6.3.2 Strengths and Limitations of HCDRT ...................................................... 69

21 6.4 Rationale for the Use of Mysid Versus Copepod Test ......................................... 70

22 7 REFERENCES................................................................................................................ 72

23 8 LIST OF APPENDICES ................................................................................................ 88

24 APPENDIX A. VALIDATION OF SCREENING AND TESTING ASSAYS 

25 PROPOSED FOR THE EDSP (DRAFT JULY 2007)................................................. 88

26 APPENDIX B. DETAILED REVIEW PAPER ON AQUATIC ARTHROPODS IN 

27 LIFE CYCLE TOXICITY TESTS (OECD NO. 55, JULY 2006).............................. 88

28 APPENDIX C. REPORTS ON THE INTER-LABORATORY VALIDATION 

29 TRIALS FOR THE MITT............................................................................................. 88

30 APPENDIX D. REPORTS ON THE INTER-LABORATORY VALIDATION 

31 TRIALS FOR THE HCDRT ......................................................................................... 88

32 D.6 COPEPOD STUDY WITH LINDANE (C5069), CEFAS, 2011................................. 89

33 APPENDIX E. MYSID TWO-GENERATION TEST GUIDELINE (DRAFT 

34 PROPOSAL FOR A STANDARDIZED PROTOCOL AS AN IN VIVO 

35 TIER 2 TEST), APRIL 2013.......................................................................................... 89

36 APPENDIX F. HARPACTICOID COPEPOD DEVELOPMENT AND 

37 REPRODUCTION TEST WITH AMPHIASCUS TENUIREMIS

38 (STANDARDIZED OECD DRAFT PROTOCOL FOR THE HCDRT AS A 

39 POTENTIAL ALTERNATIVE TO THE MTTT), MAY 2009.................................. 89

40 APPENDIX G. INTRA-LABORATORY STATISTICAL ANALYSIS REPORTS 

41 FOR TWO-GENERATION INVERTEBRATE STUDIES........................................ 89

42
DRAFT

Invertebrate Two-Generation ISR iv May 28, 2013

1 Tables

2

3 Table 1-1. Assays recommended for consideration for the Tier 1 screening battery............... 3

4 Table 3-1. Test Conditions for the Mysid Two-Generation Test (MTTT) Guideline............ 14

5 Table 3-2. Test Conditions for the Harpacticoid Copepod Development and 

6 Reproduction Test (HCDRT) guideline................................................................ 19

7 Table 4-1. Results of the EPA demonstration study of the MTTT with fenoxycarb. ............ 25

8 Table 4-2. Results of the EPA demonstration study of the MTTT with 3,5-DCP. ................ 27

9 Table 4-3. Results of the EPA demonstration study of the MTTT with fipronil. .................. 28

10 Table 4-4. Results of the EPA demonstration study of the MTTT with prochloraz. ............. 30

11 Table 4-5. Results of the EPA demonstration study of the MTTT with flutamide................ 32

12 Table 4-6. Results of the EPA demonstration study of the MTTT with ketoconazole. ......... 33

13 Table 4-7. Results of the EPA demonstration study of the MTTT with 4-tert-Octylphenol. 34

14 Table 4-8. Results of the EPA optimization study of the MTTT with lindane. ..................... 36

15 Table 4-9. Results of the EPA optimization study of the MTTT with prochloraz................. 37

16 Table 4-10. Results of the EPA optimization study of the MTTT with atrazine. .................... 38

17 Table 4-11. Results of the EPA optimization study of the MTTT with 

18 perfluorodecanoic acid.......................................................................................... 39

19 Table 4-12. Results of the Chandler et al. demonstration study of the HCDRT

20 with fipronil. ......................................................................................................... 44

21 Table 4-13. Results of the Bejarano and Chandler demonstration study of the HCDRT 

22 with atrazine.......................................................................................................... 45

23 Table 4-14. Results of the OECD demonstration study of the HCDRT with 3,5-DCP. .......... 46

24 Table 5-1. Survival and growth results of the EPA interlaboratory validation study 

25 of the MTTT with lindane..................................................................................... 50

26 Table 5-2. Reproduction results of the EPA interlaboratory validation study of the 

27 MTTT with lindane............................................................................................... 51

28 Table 5-3. Results of the EPA interlaboratory validation study of the HCDRT 

29 with lindane........................................................................................................... 52

30 Table 5-4. Results of the OECD interlaboratory validation study of the HCDRT 

31 test with lindane. ................................................................................................... 53

32 Table 5-5. Results of the OECD interlaboratory validation study of the HCDRT 

33 with atrazine.......................................................................................................... 56

34 Table 5-6. Survival and growth results of the EPA validation study of the MTTT 

35 with vinclozolin. ................................................................................................... 57

36 Table 5-7. Reproduction results of the EPA validation study of the MTTT with 

37 vinclozolin............................................................................................................. 58
DRAFT

Invertebrate Two-Generation ISR v May 28, 2013

1 Table 5-8. Results of the EPA validation study of the HCDRT with vinclozin..................... 59

2 Table 5-9. Reproduction results from the EPA validation study of the MTTT 

3 using 4-tert-Octylphenol. ...................................................................................... 61

4 Table 5-10. Survival and growth results from the EPA validation study of the MTTT 

5 using 4 tert-Octylphenol. ...................................................................................... 62

6 Table 6-1. Ranges of coefficient of variation (CV) in the F0 and F1 generations 

7 based on six studies carried out at four laboratories with three 

8 chemicals (see Section 5)...................................................................................... 65

9

10
Invertebrate Two-Generation ISR vi May 28, 2013

1 EXECUTIVE SUMMARY

2 This Integrated Summary Report (ISR) provides a validation record of the Endocrine 

3 Disruptor Screening Program (EDSP) Tier 2 Mysid Two-generation Toxicity Test (MTTT) and 

4 the Harpacticoid Copepod Development and Reproduction Test (HCDRT), both of which are 

5 aimed at characterizing potential disruption of the endocrine system by putative Endocrine 

6 Disrupting Chemicals (EDCs). Pertinent literature was previously summarized in an in-depth 

7 review completed by the Organisation for Economic Co-Operation and Development (OECD) in 

8 2006. Relevant additional literature has been reviewed, as described the ISR. 

9 MTTT. Results from an inter-laboratory test provide insights into sources of variability 

10 likely to arise in the conduct of a multi-generation test at different testing laboratories. The 

11 MTTT was used in an inter-laboratory validation study consisting of six studies carried out at 

12 four laboratories with three representative endocrine-active chemicals (i.e., lindane, vinclozolin, 

13 and 4-tert-octylphenol). Results of the MTTT validation studies can be summarized as follows:

14  Except for minor deviations from the guidelines test conditions, the different 

15 laboratories were able to successfully execute the proposed MTTT

16  Laboratory-to-laboratory variability was generally observed to be less than 30 percent 

17 in the reproduction and growth endpoint data collected for the control groups

18  Significant differences were observed in the technical proficiency (as estimated by 

19 the variability of the endpoint measurements in the control groups) among labs that 

20 participated in the inter-laboratory validation study, indicating difficulties in 

21 transferability of the MTTT between these labs

22  Considerable variability was observed within laboratories between studies for the 

23 reproductive and survival endpoints in the control groups, whereas that variability 

24 was smaller for the growth endpoints (length and weight)

25  Where the same chemical was tested in two or more laboratories (i.e., lindane and 

26 4-tert-octylphenol), dose-response relationships were not generally consistent (based 

27 on comparisons of significantly affected endpoints at each treatment level) across all 

28 affected endpoints among the laboratories.

29  Endpoint responses between the F0 and F1 generations were not consistent and were 

30 significantly different in several instances.

31 Advantages of the MTTT

32  Many environmental testing laboratories have established experience with A. bahia

33 due to its long-standing use as a standard test model for regulatory toxicity testing

34  Mysids have been used as a test model for evaluating reproductive toxicity for several 

35 decades and an increasing body of literature is available regarding their use for 

36 evaluating endocrine toxicity

37  The test can be conducted in continuous or intermittent flow

38  The control variability of the survival and several of the reproduction and growth 

39 endpoints indicate that these should be able to detect significant adverse effects with 

40 adequate statistical power

41  Common population modeling approaches can be employed. 
Invertebrate Two-Generation ISR vii May 28, 2013

1 Limitations of the MTTT

2  A lot of endpoints are included, and there is some redundancy between endpoints

3  Several endpoints require animals to be sacrificed, resulting in reduced replication for 

4 other endpoints

5  Several aspects of the MTTT, such as the collection of newly hatched offspring, 

6 pairing of mature males/females, etc. are time-consuming and resource-intensive 

7  Significant variability was observed in endpoint responses between different 

8 laboratories, resulting in reduced power to detect significant differences 

9  Some of the endpoints were consistently non-responsive

10  The addition of a second generation significantly adds to the time and resources 

11 required to perform the MTTT. The value of the additional information obtained from 

12 the second generation was not obvious based on the results of the inter-laboratory 

13 validation studies. 

14 HCDRT. Chandler and colleagues conducted a range of studies with the harpacticoid 

15 copepod Amphiascus tenuiremis, which served as initial development studies for the HCDRT. A 

16 draft OECD guideline was used in two rounds of OECD validation studies, and an EPA-led 

17 inter-laboratory validation study and results can be summarized as follows:

18  Based on the pre-validation study (OECD 2007), in which two harpacticoid (N. 

19 spinipes and A. tenuiremis) and one calanoid copepod (A. tonsa) were tested, the A. 

20 tenuiremis assay was recommended for further validation due to its shorter test time, 

21 better reproductive output and success, its focus on reproductive and developmental 

22 endpoints, its greater statistical power due to high replication, and its availability as 

23 an existing ASTM method

24  Lindane was tested at the same concentrations in three different laboratories as part of 

25 the EPA-led interlaboratory validation study and in four laboratories as part of the 

26 OECD validation study. Laboratories in the OECD validation study had difficulties 

27 obtaining the desired chemical concentrations. Lindane generally caused reduced 

28 survival, developmental delay, and reduced fecundity, although differences were 

29 observed in the concentrations at which these effects were reported by different labs. 

30 In addition, some labs did not find the same endpoints to be significantly affected. 

31  Atrazine was tested in three different laboratories as part of the OECD interlaboratory 

32 validation study. One of the laboratories was not able to achieve the required mating 

33 success in the controls; however the other two labs were able to perform the test 

34 within acceptable guidelines and reported consistent biological responses 

35 (i.e., reduced number of nauplii hatched over two clutches in two highest test 

36 concentrations with none of the other endpoints significantly affected).

37  Only one laboratory tested vinclozolin as part of the EPA validation study. While the 

38 test was conducted within test method guidelines, all measured vinclozolin 

39 concentrations were undetected.

40 Advantages of HCDRT

41  Relative to other harpacticoid copepod studies in the literature, A. tenuiremis is more 

42 chronically sensitive than all other species published to date where there is 

43 comparative data
Invertebrate Two-Generation ISR viii May 28, 2013

1  Laboratories were generally able to successfully culture A. tenuiremis, harvest gravid 

2 copepods, and hatch healthy nauplii needed to conduct the HCDRT 

3  Microplate test allows for easy (individual-based) tracking of endpoints and high 

4 replication resulting in strong statistical power

5  Relatively consistent concentration-dependent delays in development rates, increased 

6 naupliar-specific mortality, mating-success depressions, and reduction of fecundity 

7 were observed during the lindane interlaboratory validation study

8  A. tenuiremis has a rapid life cycle and can be easily cultured in water-only and 

9 sediment:water matrices

10 Limitations of the HCRDT

11  Commercial testing laboratories generally have no prior experience with this species

12  Issues of proficiency in the successful application of the test method were identified 

13 as an issue with the HCDRT

14  Microwell plate design results in difficulties for continuous monitoring of water 

15 quality and difficulties producing consistently accurate and precise measured 

16 concentrations

17  Test results need to be reported in nominal concentrations rather than measured 

18 concentrations 

19  Absence of effects on most of the endpoints reported in the atrazine study

20  Possible lack of sensitivity of the population of A. tenuiremis used in the study

21  Influence of mortality (males more sensitive than females) on the sex ratio 

22  Manpower required in the study

23  Limited knowledge of the copepod endocrine system.

24

25 Currently, the MTTT provides a number of clear advantages over the HCDRT as an 

26 aquatic arthropod test to evaluate chemical effects on invertebrate growth, reproduction, and 

27 survival. Therefore, the MTTT is proposed as a Tier 2 test to be used in EPA’s EDSP.
Invertebrate Two-Generation ISR 1 May 28, 2013

1 OBJECTIVE

2 The objective of this integrated summary report is to provide a detailed account of the 

3 validation process for the Mysid Two-generation Toxicity Test (MTTT) and the Harpacticoid 

4 Copepod Development and Reproduction Test (HCDRT) so that Federal Insecticide, Fungicide 

5 and Rodenticide Act (FIFRA) Scientific Advisory Panel members may address the 

6 accompanying charge questions.

7 1 INTRODUCTION

8 Section 408(p) of the Federal Food Drug and Cosmetic Act (FFDCA) requires the

9 USEPA to:

10

11 develop a screening program, using appropriate validated test systems and other

12 scientifically relevant information, to determine whether certain substances may 

13 have an effect in humans that is similar to an effect produced by a naturally 

14 occurring estrogen, or other such endocrine effect as the Administrator may 

15 designate [21 U.S.C. 346a(p)].

16

17 Subsequent to passage of the Food Quality Protection Act in 1996, which amended 

18 FFDCA and FIFRA, the EPA formed the Endocrine Disruptor Screening and Testing Advisory 

19 Committee (EDSTAC), a committee of scientists and stakeholders that was charged by the EPA 

20 to provide recommendations on how to implement its Endocrine Disruptor Screening Program 

21 (EDSP). The EDSP is described in detail at the following website: 

22 http://www.epa.gov/scipoly/oscpendo/. Upon recommendations from the EDSTAC (EDSTAC 

23 1998), the EPA expanded the EDSP using the Administrator’s discretionary authority to include 

24 the androgen and thyroid hormonal systems as well as the endocrine systems of wildlife. 

25 Following broader international concerns and the creation of similar programs in other countries, 

26 the Organisation for Economic Co-operation and Development (OECD) established the 

27 Endocrine Disrupters Testing and Assessment (EDTA) Task Force in 1998 within its Test 

28 Guidelines Programme. EDTA is charged with developing an internationally harmonized testing 

29 strategy for the screening and testing of endocrine disrupting chemicals, taking into account the 

30 consequences of such a testing strategy on the development and validation of Test Guidelines, 

31 and on existing regulatory systems for new and existing substances.

32 The MTTT is one of the assays proposed as part of the EDSP Tier 2 and the HCDRT is 

33 considered as a potential alternative or option. These tests are the subject of this report. This 

34 report complements the attached supporting materials and is meant to provide the FIFRA 

35 Scientific Advisory Panel reviewers with the necessary information, and/or direction to 

36 necessary information, to address the peer review charge questions. It introduces the purpose of 

37 the MTTT and HCDRT and how this fits into the EDSP, the scientific rationale for the assays, 

38 and an historical account of the development and optimization of the assay protocols. It also 

39 synthesizes the information gained during the validation process and addresses the advantages 

40 and limitations of the MTTT and HCDRT based on their strengths and weaknesses, practicality, 

41 reproducibility, reliability, and protocol transferability.
Invertebrate Two-Generation ISR 2 May 28, 2013

1 1.1 Endocrine Disruptor Screening Program 

2 To comply with its mandate, the EPA chartered a Federal advisory committee (EDSTAC) 

3 to provide advice and guidance on the development of a screening and testing program. In 1998, 

4 it recommended to the EPA a conceptual two-tiered approach that involved screening and testing 

5 chemical compounds for effects on the estrogen, androgen, and thyroid (EAT) hormone axes. 

6 The ultimate goal of these assays is to provide input into hazard identification to assess risk of 

7 adverse consequences to humans and wildlife (EDSTAC 1998).

8 The EPA submitted a proposal of the EDSP for public review and comment (FRN 1998) 

9 as well as peer review by a joint subcommittee of the EPA Science Advisory Board and FIFRA 

10 Scientific Advisory Panel (SAB, SAP 1999). A complete description of the program proposal 

11 can be found in the Federal Register Notice (FRN 1998). Briefly, the EDSP proposed by 

12 EDSTAC allows for: 1) initial sorting and prioritization of chemical compounds, 

13 2) identification of chemicals for further testing using a Tier 1 screening battery that includes in 

14 vitro and in vivo mammalian, amphibian and fish assays, and 3) characterization of adverse 

15 consequences resulting from possible endocrine disruption and establishment of dose-response 

16 relationships for hazard identification using Tier 2 testing. 

17 In comparison to the more refined, detailed, and definitive tests in Tier 2, the EDSTAC 

18 indicated that the in vitro and in vivo screening assays in the Tier 1 battery should:

19  be relatively fast and efficient

20  be standardized and validated

21  be more sensitive than specific to minimize false negatives without an unreasonable 

22 rate of false positives

23  be comprised of multiple endpoints that reflect as many modes of endocrine action as 

24 possible

25  have a sufficient range of taxonomic groups among test organisms represented 

26  yield data that can be interpreted as either negative or positive for determining the 

27 necessity and manner in which to conduct Tier 2 tests.

28 Together, the suite of Tier 1 assays form a battery in which some endocrine axis 

29 redundancy is incorporated (e.g. two different assays may cover some similar aspects of the 

30 estrogenic response). This redundancy allows for a weight-of-evidence approach, as 

31 recommended by EDSTAC, to determine whether a chemical shall undergo further, more 

32 definitive, testing.

33 The assays listed in Table 1-1, recommended by EDSTAC, have undergone validation 

34 and have been adopted as the EDSP Tier 1 screening battery. These assays are meant to detect 

35 chemicals that may affect the estrogen, androgen and thyroid hormone axes through any known 

36 modes of action.

37
Invertebrate Two-Generation ISR 3 May 28, 2013

1 Table 1-1. Assays recommended for consideration for the Tier 1 screening battery.

Assay Reason for Inclusion

Estrogen receptor binding or transcriptional 

activation assay

An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the estrogen receptor.

Estrogen receptor transcriptional activation 

assay

An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the estrogen receptor.

Androgen receptor binding assay An in vitro test to detect chemicals that may affect the endocrine 

system by binding to the androgen receptor.

In vitro steroidogenesis assay An in vitro test to detect chemicals that interfere with the 

synthesis of the sex steroid hormones

Placental Aromatase Assay An assay to detect interference with aromatase.

Uterotrophic Assay An in vivo assay to detect estrogenic chemicals. 

Hershberger Assay An in vivo assay to detect androgenic and antiandrogenic 

chemicals. 

Pubertal Male An in vivo assay to detect chemicals that act on androgen or 

through the HPG axis that controls the estrogen and androgen 

hormone systems. It is also enhanced to detect chemicals that 

interfere with the thyroid system. This assay could in part 

substitute for the female pubertal assay.

Pubertal female assay An in vivo assay to detect chemicals that act on estrogen or 

through the HPG axis that controls the estrogen and androgen 

hormone systems. It is also enhanced to detect chemicals that 

interfere with the thyroid system.

Amphibian metamorphosis assay An in vivo assay for detection of chemicals that interfere with the 

thyroid hormone system. 

Fish screening assay An in vivo assay for detection chemicals that interfere with the 

HPG axes.

2

3 A weight-of-evidence (WOE) review of the Tier 1 screening battery and other 

4 scientifically relevant information (OSRI), will be made to ascertain if a chemical is deemed 

5 sufficiently endocrine active to prompt requirements for Tier 2 testing (US EPA 2011). The 

6 following tests, as generally recommended by EDSTAC, are being considered as part of the 

7 EDSP Tier 2:

8  Two-generation Rat Reproduction Test

9  Medaka Multi-generation Test

10  Japanese Quail Two-generation Test

11  Mysid Two-generation Toxicity Test

12  Larval Amphibian Growth and Development Test.

13 The Two-generation Rat Reproduction Test (OCSPP 870.3800) has already been adopted. 

14 The remaining four Tier 2 tests are the subject of the FIFRA Scientific Review Panel for which 

15 the MTTT and the HCDRT, as a potential alternative or option, are reviewed here.

16 1.2 Validation

17 Validation has been defined as “the process by which the reliability and relevance of a 

18 test method are evaluated for a particular use” (NIEHS 1997; OECD 1996).

19 Reliability is defined as the reproducibility of results from an assay within and between 

20 laboratories. 
Invertebrate Two-Generation ISR 4 May 28, 2013

1 Relevance describes whether a test is meaningful and useful for a particular purpose 

2 (OECD 1996). For Tier 1 EDSP assays, relevance can be defined as the ability of an assay to 

3 detect the effects of chemicals with the potential to interact with the endocrine system.

4 Validation is generally recognized as necessary for the regulatory acceptance of new and 

5 revised test methods, and is now an integral component of the international development and 

6 acceptance of these methods (OECD 2005a). The criteria used to guide the validation process for 

7 the MTTT were based on the principles of validation developed by the U.S. Interagency 

8 Coordinating Committee for the Validation of Alternative Methods (ICCVAM) (NIEHS 1997)

9 and the OECD (OECD 2005a). These criteria as stated by ICCVAM (NIEHS 1997) are as 

10 follows:

11 1. The scientific and regulatory rationale for the test method, including a clear 

12 statement of its proposed use, should be available.

13 2. The relationship of the endpoints determined by the test method to the in vivo 

14 biologic effect and toxicity of interest must be addressed.

15 3. A formal detailed protocol must be provided and must be available in the public 

16 domain. It should be sufficiently detailed to enable the user to adhere to it and should 

17 include data analysis and decision criteria.

18 4. Within-test, intra-laboratory and inter-laboratory variability and how these 

19 parameters vary with time should have been evaluated.

20 5. The test method’s performance must have been demonstrated using a series of 

21 reference chemicals preferably coded to exclude bias.

22 6. Sufficient data should be provided to permit a comparison of the performance of a 

23 proposed substitute test to that of the test it is designed to replace.

24 7. The limitations of the test method must be described (e.g., metabolic capability).

25 8. The data should be obtained in accordance with Good Laboratory Practices (GLPs).

26 9. All data supporting the assessment of the validity of the test methods including the 

27 full data set collected during the validation studies must be publicly available and, 

28 preferably, published in an independent, peer-reviewed publication. 

29 The EPA has adopted these various validation criteria for the EDSP as described in 

30 Appendix A (EDSP 2007) which were considered to have been met in the review of the Tier 1 

31 assays. Validation of these assays according to the criteria proved a challenge because these are 

32 not replacement assays (Validation Criterion No. 6). Many of them are novel assays; 

33 consequently, large data bases do not exist as a reference to establish their predictive capacity 

34 (e.g., determination of false positive and false negative rates).

35 In general, the EPA uses a five-part or stage validation process outlined by ICCVAM 

36 (NIEHS 1997). The EPA also believes it is important to recognize that this process was 

37 originally developed for alternative assays intended to replace or refine existing assays. A 

38 rudimentary problem confronting the EPA is how to adapt and work with this process for the 

39 rodent and ecological in vivo assays in Tiers 1 and 2 that are considered the standards and not as 

40 alternatives to other assays. Nonetheless, the stages of the process outlined by the ICCVAM are 

41 as follows:

42 The first stage of the process was test development, an applied research function which 

43 culminated in an initial protocol. As part of this phase, EPA drafted a Detailed Review Paper 

44 (DRP) to explain the purpose of the test method, the context in which it will be used, and the 
Invertebrate Two-Generation ISR 5 May 28, 2013

1 scientific basis upon which the assay’s protocol, endpoints, and relevance rest (Appendix B). The 

2 DRP reviews the scientific literature for candidate protocols and evaluates them with respect to a 

3 number of considerations, such as whether candidate protocols meet the assay’s intended 

4 purpose, costs, and other practical considerations. The DRP also identified the developmental 

5 status and questions related to each protocol; the information needed to answer the questions; 

6 and, when possible, recommends an initial protocol for the initiation of the second stage of 

7 validation, standardization and optimization. During standardization and optimization, studies 

8 were performed geared toward refining, optimizing, and standardizing the protocol, and initially 

9 assessing protocol transferability and performance. In inter-laboratory validation, studies were 

10 conducted in several independent laboratories with the refined protocol. The results of these 

11 studies were used to determine inter-laboratory variability and to set or cross-check performance 

12 criteria. The reports on the inter-laboratory trials for the MTTT are provided in Appendix C. The 

13 reports on the inter-laboratory trials for the HCDRT are provided in Appendix D. Inter14 laboratory validation is followed by peer review, an independent scientific review by qualified 

15 experts, and by regulatory acceptance, adoption for regulatory use by an agency. EPA has 

16 developed extensive guidance on the conduct of peer reviews because the Agency believes that 

17 peer review is an important step in ensuring the quality of science that underlies its regulatory 

18 decisions (US EPA 2006).

19 The purpose of this ISR is to provide an historical summary of the development and 

20 validation of a standardized protocol for the MTTT (Appendix E) proposed as an in vivo Tier 2 

21 test and the HCDRT as a potential alternative or option (Appendix F). The reasoning and 

22 judgments leading to the various studies, and conclusions concerning the strengths and 

23 weaknesses of these assays in their current form, are presented.

24
Invertebrate Two-Generation ISR 6 May 28, 2013

1 2 HISTORICAL OVERVIEW OF THE INVERTEBRATE TESTS

2 Invertebrates (especially arthropods such as insects and crustaceans) constitute the vast 

3 majority of animal species on earth. Many invertebrate toxicity test protocols are routinely used 

4 in regulatory testing; however, few have been designed with endocrine-specific endpoints in 

5 mind. Although growth, reproduction, development, and other aspects of invertebrate physiology 

6 and life cycle are known to be under endocrine control, the endocrine systems and the hormones 

7 produced and used by invertebrates are not directly analogous to those of vertebrates. For 

8 example, ecdysozoans account for more than 75% of all known animal species, and they rely 

9 largely on ecdysteroid and juvenile hormones to regulate their physiology (Chang 1993; 

10 deFur et al. 1999; Subramoniam 2000). Ecdysteroids are steroid hormones that regulate growth 

11 and molting in arthropods, and they exhibit some functional and structural similarities to 

12 estrogen. 

13 It has been reported that vertebrate steroids, such as the androgen testosterone, can

14 interfere with ecdysteroid regulation in a crustacean (Mu and LeBlanc 2002a). Also, the 

15 aromatase inhibitor fenarimol, which prevents the conversion of testosterone to estrogen, has 

16 been demonstrated to inhibit ecdysteroid synthesis and interfere with normal molting processes 

17 in a crustacean (Mu and LeBlanc 2002b). Still, reported effects of vertebrate-active hormones on 

18 crustaceans are limited, and several studies have found little or no effects of vertebrate steroids in 

19 crustaceans related to endocrine disruption (Caspers 1998; Bechmann 1999; Hutchinson et al. 

20 1999a, 1999b; Breitholtz and Bengtsson 2001 in OECD 2006). Many crustacean species hold 

21 key ecological functions and several crustacean species are of economic importance. Further, 

22 crustaceans (and other arthropods) are the only invertebrates that have true endocrine glands 

23 derived from epithelial tissue and functioning similar to vertebrate glands (deFur 2004) and their

24 endocrine systems are relatively well understood compared to those of other invertebrates

25 (Oehlmann and Schulte-Oehlmann 2003; LeBlanc 2007). Given that endocrine disruption has 

26 been reported in crustaceans (OECD 2006), an invertebrate test method that uses crustaceans for 

27 evaluating potential effects of EDCs is relevant.

28 2.1 Purpose of Invertebrate Testing in EDSP

29 Invertebrates comprise 95% of the world’s animal species (Wilson 1988), and certainly a 

30 larger percentage of the Earth’s total animal abundance. Hormones are important in controlling 

31 physiological processes in invertebrates (Lafont 2000; Oehlmann & Schulte-Oehlmann 2003). 

32 However, until recently, invertebrates received little attention regarding potential endocrine 

33 disruption, the main exception being studies of imposex in gastropods. Invertebrate endocrine 

34 systems, except perhaps those of arthropods, are generally poorly studied compared to those of 

35 vertebrates, and this limits ability to evaluate potential EDCs (Lafont 2000; Oehlmann and 

36 Schulte-Oehlmann 2003). Because many invertebrate endocrine systems are not very well 

37 understood, some changes to endocrine systems after exposure to a chemical may not be detected 

38 or may not be measurable (Oehlmann and Schulte-Oehlmann 2003). However, the situation is 

39 improving, with many studies being done within the last few years on such groups as sponges, 

40 corals, polychaetes, and echinoderms, adding to what has been learned about arthropod 

41 endocrinology (Environment Agency 2007). Nonetheless, to gain a full understanding of the 

42 complex endocrine disruption issue, invertebrates must be included in a tiered testing approach 

43 (Vandenbergh et al. 2003).
Invertebrate Two-Generation ISR 7 May 28, 2013

1 Many anthropogenic pollutants eventually end up in the world’s oceans, carried there 

2 through riverine and estuarine pipelines (Nimmo and Hamaker 1982). Since the mid-1990s, there 

3 has been an increased awareness that many sewage constituents or chemicals associated with 

4 industrial production that enter the environment can disrupt endocrine systems, and that these 

5 compounds will likely affect marine organisms (Depledge and Billinghurst 1999; Oberdörster 

6 and Cheek 2001). Although early concern over EDCs focused on vertebrates, attention has 

7 broadened to include invertebrates, because they are ecologically important and have distinctive 

8 endocrine systems that differ from those of vertebrates. Estuaries, which are intrinsically and 

9 commercially important ecosystems, are among the earliest recipients of EDCs. Among the 

10 many estuarine organisms that could be adversely affected by these compounds, crustaceans are 

11 good candidates for study of potential impacts. Attention here is focused on aquatic arthropods 

12 (insects and crustaceans), because they are often among the most abundant organisms in 

13 freshwater and estuarine systems, they form vital links in estuarine food webs, and they are 

14 known to be susceptible to the effects of EDCs.

15 Hutchinson et al. (2000) offered a counter argument to the inclusion of an arthropod, 

16 specifically a daphnid or mysid, in the endocrine screening program. The principal objection 

17 applicable to both taxa was the general data gap in the basic endocrinology of mysids and 

18 daphnids. They argued that the assertion by EDSTAC that vertebrate estrogen or androgen 

19 disruptors could interfere with ecdysteroid activity was only speculation and that any effects on 

20 reproduction or development by known vertebrate EDCs have not been directly linked to 

21 ecdysteroid or juvenile hormone activity. Hutchinson et al. (2000) also expressed concern that 

22 the gender identification of mysids in the laboratory was technically difficult and that the test 

23 results might be confounded by cannibalism of offspring by parents. However, these three 

24 concerns can be addressed. 

25 First, the understanding of invertebrate endocrine systems is improving rapidly and the 

26 links between hormones and physiological processes in crustaceans are increasingly established

27 (Verslycke et al. 2007). For example, in companion studies, Mu and LeBlanc (2002a,b) showed 

28 that ecdysteroids regulate the embryonic development of Daphnia and that this development 

29 could be interrupted by testosterone and fenarimol, a fungicide used in agriculture. Testosterone 

30 did not display ecdysteroidal activity, but interfered with ecdysteroids by outcompeting them to 

31 occupy the ecdysteroid receptor (Mu and LeBlanc 2002a). Fenarimol acted by reducing body 

32 levels of ecdysone in individual daphnids (Mu and LeBlanc 2002b). Vandenbergh et al. (2003) 

33 found that the synthetic estrogen 17α-ethinylestradiol altered sexual development in individuals 

34 of the amphipod Hyalella azteca. Males did not develop secondary sex features, reproductive 

35 system morphology was altered, and sex ratios in exposed groups favored females. Further, 

36 recent studies of crustaceans have identified that methyl farnesoate has a role in regulating larval 

37 development in lobsters (summarized by Chang et al. 2001) and stimulates gonad development 

38 in a freshwater prawn (Nagaraju et al. 2003). Also, driven by increased availability of crustacean 

39 genomic sequences, our understanding of crustacean hormone receptors (including the 

40 ecdysteroid receptor), hormone receptor signaling, and its chemical disruption is rapidly 

41 increasing (Dhadialla et al. 1998; Tarrant et al. 2011). 

42 Second, female and male mysids have anatomical differences that can be used to indicate 

43 maturity and gender. In females, the appearance of the oostegites that form the marsupium are 

44 one indication of gender and maturity. While maturity is more difficult to recognize in males, 

45 mature males have one pair of elongated abdominal pleopods that is modified to assist in 
Invertebrate Two-Generation ISR 8 May 28, 2013

1 copulation and that can be used to identify gender. Males of the most commonly used mysids test 

2 species, Americamysis bahia, are also significantly smaller than females at maturity. Molt 

3 staging could also be used to alert the laboratory staff of the potential appearance of mature 

4 individuals because A. bahia reaches maturity at the fourth molt (Touart 1982). Ultimately, 

5 trained staff using a dissecting microscope should be able to discern sex reliably and quickly.

6 Third, the susceptibility of young to being eaten by adult mysids is also well-known 

7 (e.g., Johnston and Ritz 2001; Quirt and Lasenby 2002). Premolt newborns are particularly 

8 susceptible. Testing protocols call for newly released young to be removed from the tanks as 

9 soon as possible and placed into separate rearing chambers (or new test chambers if they are to 

10 be exposed to a potential toxicant. Mysids are also typically fed liberal quantities of food 

11 (Artemia nauplii) to lessen the likelihood that adults will eat the young. Taken together, 

12 considerable evidence exists to counter the primary concerns raised by Hutchinson et al. (2000)

13 regarding the use of mysids for testing of potential EDCs.

14 Below are some of the general research findings to date that are important to consider: 

15  Chronic assays often reveal impacts by a chemical at doses that are much lower than 

16 those eliciting effects during acute exposures. This emphasizes the importance of 

17 chronic assays in an EDC evaluation program (e.g., Watts et al. 2001, 2002)

18  Potential EDCs may have effects in invertebrates other than those anticipated by 

19 knowledge of their actions in vertebrates (e.g., Hahn et al. 2002, Watts et al. 2001) 

20  Pesticides that target certain aspects of insect physiological process may affect 

21 nontarget organisms in ways not predicted by the pesticide’s desired action 

22 (e.g., Hahn et al. 2001, 2002) 

23  Males and females may react differently to chemicals that are not thought to cause 

24 gender-specific responses (e.g., Hahn and Schulz 2002) 

25  Chemicals that have very similar effects on toxicity, and have similar octanol/water 

26 partition coefficients (Kow), could have very different effects on endocrine-controlled 

27 processes (e.g., Andersen et al. 2001)

28  Some of the endpoint responses measured in a study of a potential EDC may also be 

29 caused by non-EDC stressors

30  Finally, chemical mixtures may show synergistic impacts that are not evident in the 

31 actions of the individual components (e.g., Mu and LeBlanc 2004). In nature, most 

32 animals will most certainly be exposed to chemical mixtures rather than to single 

33 compounds; thus considering the potential effects of mixtures is important to an EDC 

34 evaluation program. However, to be able to predict the possible outcomes of mixtures, 

35 at a minimum the individual impacts and mechanisms of action of the component 

36 chemicals need to be understood.

37 2.2 Invertebrate Life-cycle and Multi-generational Toxicity Tests for the 

38 Detection of Possible Endocrine Disruption

39 2.2.1 Relevance of the Mysid Test Model

40 Mysid crustaceans have been used in regulatory (and other) toxicity testing for more than 

41 30 years. Standard testing protocols have been developed for some species. Despite that little is 

42 known about general endocrine functions in mysids (Ingersoll et al. 1999) and there have been 

43 few direct links between potential EDCs, beyond certain insect growth regulators (IGRs), and 
Invertebrate Two-Generation ISR 9 May 28, 2013

1 endocrine disruption in mysids, they have been suggested as providing a useful model of the 

2 hormonal control of crustacean molting (Cuzin-Roudy and Saleuddin 1989). 

3 Advances in biological control agents to control insect pests have inspired the synthesis 

4 of IGRs, which find their way into the estuarine environment by either direct or indirect 

5 application. Crustaceans, which along with insects are in the phylum Arthropoda, could also be 

6 sensitive to these compounds. The mysid group has been shown to be among the most sensitive 

7 members of the estuarine community (McKenney 1982, 1985, 1986, 1996; Nimmo and Hamaker 

8 1982; Nimmo et al. 1981). One study focused on exposure of Americamysis bahia to methoprene, 

9 a juvenile hormone (JH) analog used to control mosquitoes (McKenney and Celestial 1996). The 

10 goal was to determine whether typical application rates shown to control mosquito larvae also 

11 cause problems for nontarget organisms. The results showed a significant effect during the mysid 

12 life cycle test. Total lethality occurred at 125 μg/L in a 4-day test. Similar concentrations caused 

13 significant mortality in the larvae of an estuarine crab and shrimp. Mysids grown at sublethal 

14 concentrations were smaller (lower weight), had a longer time to the production of the first brood, 

15 and produced fewer young per female than control animals. McKenney and Celestial (1996)

16 suggested that the effects shown were likely from the interruption of endocrine function by 

17 methoprene.

18 These results suggest that methoprene could interfere with the mysid endocrine system, 

19 which uses hormones that act like JH. Retarded growth rates were also accompanied by 

20 bioenergetic disruption, resulting in lower net growth efficiency values. This suggested that 

21 increased metabolic demands reduced the amount of assimilated energy available for new tissue 

22 production (McKenney 1982, 1985). The delays in mysid first brood production could be the 

23 result of slowing sexual maturity and/or embryogenesis. Diminished reproductive success could 

24 be the result of inhibited vitellogenesis, modifications in ovarian development, or disruption of 

25 successful embryogenesis. More recently, methoprene was also shown to disrupt molting, 

26 vitellogenesis, and marsupial development in the mysid Neomysis integer (Ghekiere et al. 

27 2006a, b; Ghekiere et al. 2007). Verslycke et al. (2002, 2003a, 2003b, 2004b) studied 

28 testosterone metabolism in Neomysis integer and examined the changes in energy and 

29 testosterone metabolism after exposure to potential EDCs. Further work is required with the 

30 mysid to determine a more conclusive cause-and-effect relationship between potential EDCs and 

31 their effects, as observed by test measurement endpoints.

32 Nimmo and Hamaker (1982) advocated the use of mysids in toxicity testing, including 

33 acute and chronic life-cycle testing. Nimmo and Hamaker compared acute and chronic toxicity 

34 data for a variety of taxa and concluded that mysids were more sensitive than most of the taxa, 

35 including grass shrimp, an estuarine fish, and 17 freshwater species. Nimmo and Hamaker 

36 foresaw a role for mysids in the evaluation of the effects of pesticides and metals on successive 

37 generations, in part because the life cycle of mysids is short. This role is being fulfilled by the 

38 inclusion of mysids in an EDC evaluation program. Verslycke et al. (2004a, 2007) evaluated the 

39 potential for using mysids in an endocrine disruption evaluation program. They reviewed aspects 

40 of the biology and ecology of mysids in the context of this potential use and summarized the 

41 literature on toxicity studies done using mysids. Their review also included a discussion of many 

42 endpoints that may be useful when using mysids to test for endocrine disruption. Verslycke et al. 

43 (2004a, 2007) concluded that mysids have the ecological relevance and sensitivity to stressors 

44 required of a taxon that would be suitable for evaluation of endocrine disruption in marine and 

45 estuarine invertebrates and could serve as a surrogate for other crustacean species. 
Invertebrate Two-Generation ISR 10 May 28, 2013

1 As a candidate test species, A. bahia has many strengths and few weaknesses. Its primary 

2 advantages include its widespread availability and ease of culture. Because animals can be 

3 obtained from commercial cultures, the likelihood of misidentifying the species is very low. Its 

4 relatively short generation time makes it desirable in life cycle testing (i.e., life cycle duration is 

5 17 to 20 days). The species is widely used in toxicological testing, and appropriate test 

6 conditions are well known. Standardized life cycle test protocols have been developed (OCSPP 

7 850.1350; ASTM 1997) and applied (e.g., McKenney 1982, 1985, 1986, 1994; McKenney et al. 

8 1991; McKenney and Celestial 1996) or evaluated (Lussier et al. 1999). These standardized test 

9 protocols contain several EDC-related endpoints; others appear in peer-reviewed publications 

10 (Ingersoll et al. 1999). McKenney (1998) synthesized the results of several of his earlier studies 

11 on the effects of chronic exposures to various pesticides on A. bahia and concluded that 

12 life-cycle endpoints (slowed juvenile growth and reduced production of young) were more 

13 sensitive than survival. More recently, studies showed that the results from toxicity tests, which 

14 included standard and multi-generational tests using A. bahia, could be used to extrapolate from 

15 laboratory to population effects (Kuhn et al. 2000; 2001). McKenney (2005) also demonstrated 

16 that A. bahia can be used to evaluate multi-generational toxicity in a two-generational exposure 

17 protocol. Verslycke et al. (2004a, 2007), in their reviews of the use of mysids for endocrine 

18 disruptor evaluations, favored A. bahia as a reasonable test species. One criticism of the 

19 widespread use of A. bahia in toxicological testing is that because it is a warm-temperate or 

20 subtropical species, it may not be ecologically relevant to colder-water materials testing. A 

21 proposed Mysid Two-Generation Toxicity Test (MTTT) using A. bahia is discussed further in 

22 Section 3.1.

23 2.2.2 Relevance of the Copepod Test Model

24 Copepods occur widely in almost every marine, brackish, and freshwater ecosystem. 

25 They represent important prey items for the larvae and juveniles of many fish and larger 

26 invertebrates, and are increasingly used as a live food source in aquaculture. A number of 

27 copepod species (predominantly marine and brackish water species) have been used for many 

28 years to evaluate the acute and chronic toxicity of single chemicals and complex mixtures in 

29 water and sediments (Chandler and Green 2001). Copepod development is characterized by the 

30 accomplishment of several molt cycles. In the estuarine copepod Amphiascus tenuiremis, a 

31 species that has been put forward for life cycle toxicity testing of potential EDCs, juveniles pass 

32 through six naupliar and five copepodite stages before turning into the adult (12th) stage (Hicks 

33 and Coull 1983). The time to develop for copepods from hatch to adult as well as the generation 

34 time (hatch to hatch) varies with temperature, food availability, population density, etc. In 

35 general, however, time from hatch to adulthood takes 10 to 20 days and the generation time is 15

36 to 40 days at 15 to 25oC. A. tenuiremis possesses a generation time of 21 days at 20oC (Chandler 

37 and Green 1996). The shape of the egg, presence and number of egg sacs, number of eggs within 

38 each egg sac, as well as the number of broods (from a single fertilization) differ widely between 

39 species (Hicks and Coull 1983).

40 Harpacticoid copepods comprise more than 3,000 species, are mostly free-living benthic

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

42 marine benthic communities (Huys et al. 1996). They almost always dominate the gut contents of 

43 bottom or phytal feeding larval and juvenile fish (Hicks and Coull 1983). Several authors have 

44 described toxic effects of single substances, oils, complex effluents, etc. on harpacticoid 

45 copepods, such as A. tenuiremis (Chandler et al. 2004a,b; Bejarano and Chandler 2003), Tisbe 
Invertebrate Two-Generation ISR 11 May 28, 2013

1 battagliai (Hutchinson et al. 1999a, b), Nitocra spinipes (Bengtsson 1978; Renberg et al. 1980; 

2 Tarkpea et al. 1999; Breitholtz and Bengtsson 2001; Breitholtz and Wollenberger 2003), Nitocra 

3 affinis (Ustach 1979) and Tigriopus japonicus (D’Agostino and Finney 1974).

4 Zooplankton communities in the Baltic Sea and other waters are often dominated by 

5 calanoid copepods, such as Eurytemora spp. and Acartia spp. (Voipio 1981), which provide an 

6 important link between phytoplankton and higher communities (e.g., fish) in the pelagic food 

7 chain (e.g., Størrup et al. 1986). For instance, Acartia tonsa has been widely used for 

8 ecotoxicological testing owing to its ecological importance and since it is easy to cultivate in the 

9 laboratory (Bushong et al. 1990; Kusk and Petersen 1997). A sensitive larval inhibition test has 

10 been developed and used to study effects of environmental chemicals, such as tributyltin (TBT) 

11 (Kusk and Petersen 1997) and estrogens (Andersen et al. 2001). In short, this test covers the 

12 period of development from egg until approximately 50% of the control organisms reach a 

13 copepodite stage (after 5 to 8 days depending on temperature). 

14 Two common and regularly used copepod species were originally proposed for an OECD

15 copepod development and reproduction test, namely the harpacticoid copepods Nitocra spinipes

16 and Tisbe battagliai. Later, one further harpacticoid species, A. tenuiremis, and the calanoid 

17 copepod Acartia tonsa were included. The proposed guidelines were for evaluating long-term 

18 effects (including endocrine-disruptive effects) of various types of chemicals to aquatic 

19 invertebrates (OECD 2007). In 2007, based on results of a first intercalibration study, it was 

20 decided to proceed with the harpacticoid A. tenuiremis for a second intercalibration study 

21 (OECD 2011). The draft Harpacticoid Copepod Development and Reproduction Test (HCDRT) 

22 is discussed further in Section 3.2.

23
Invertebrate Two-Generation ISR 12 May 28, 2013

1 3 MYSID AND COPEPOD TEST PROTOCOL DEVELOPMENT

2 As discussed in Section 2, the focus of the ISR is on life-cycle and multi-generational 

3 studies conducted using the mysid and copepod test model. This section describes the 

4 recommended Mysid Two-Generation Toxicity Test (MTTT) guideline and the recommended

5 Harpacticoid Copepod Development and Reproduction Test (HCDRT). Full versions of the 

6 recommended protocols are included as appendices (Appendices E and F), and summarized 

7 below.

8 3.1 Summary of the Mysid Two-Generation Toxicity Test (MTTT) Protocol

9 3.1.1 Principle of the Test

10 The MTTT protocol describes a two-generation toxicity test with an estuarine mysid that 

11 considers reproductive fitness in parents and offspring as an integrated measure of toxicant 

12 exposure. It also enables measurement of a suite of other endpoints that allow for diagnostic and 

13 definitive evaluation of endocrine disrupting chemicals (EDCs) or other types of reproductive 

14 toxicants (Verslycke et al. 2004a). This guideline is intended to be applicable to the mysid, A. 

15 bahia, but could be adapted to other mysid species. The MTTT is a relatively long-term assay 

16 (normally 60 days or longer) that assesses early development, growth, and reproduction in two 

17 generations (F0 and F1). The MTTT is an extension of existing standard practice for conducting a 

18 mysid life-cycle test (ASTM 2004; McKenney 1986, 1998; and Nimmo et al. 1977, 1978). 

19 The MTTT is intended to serve as a higher tier test with an aquatic arthropod for 

20 collecting definitive concentration-response information on adverse effects suitable for use in 

21 ecological risk assessment. Specifically, the design enables the collection of invertebrate 

22 hormone-regulated endpoint data (e.g., molt-related endpoints) and information concerning 

23 various aspects of the reproductive biology and life-stage viability. The former is useful as a 

24 basis for better understanding comparative toxicological relationships across biological groups 

25 and/or extrapolation across species (McKenney 1986, 1996), while the latter are critical to 

26 application of population models to risk assessment scenarios (Raimondo and 

27 McKenney 2005a,b).

28 The MTTT is conducted with a minimum of five chemical concentrations, as well as 

29 appropriate controls and with a minimum of three experimental units (replicates) per treatment. 

30 The MTTT is initiated with newly released <24 hours old mysids (F0 generation) that are grown 

31 to maturity (~13 to 16 days) and then their offspring are collected (F1 generation). The F1 

32 generation is also grown to maturity and their offspring enumerated (F2 generation). In general, 

33 each replicate tank contains two retention baskets, each holding 20 mysids per basket (one basket 

34 housing animals used for reproductive observations and a second basket for growth observations 

35 and optional biochemical measurements). With the appearance of a marsupial pouch on F0 

36 females (sexual maturity), mature females are isolated as pairs with F0 males (smaller individuals 

37 without a visual marsupial pouch) in separate brood cups. Optimally, seven breeding pairs are 

38 selected in each replicate reproduction basket. The exposure of these F0 mysids is terminated 

39 following release of the second brood or 7 days past the median second brood release in the 

40 controls. Toxicant and control treatments are delivered to the mysids in water via a continuous

41 flow apparatus (e.g., infusion pumps) (McKenney 1982) or an exposure apparatus using 

42 intermittent flow (e.g., proportional diluter) (McKenney 1998). Offspring (F1) from the parental 
Invertebrate Two-Generation ISR 13 May 28, 2013

1 generation (F0) are collected each morning from the brood cups and transferred immediately to 

2 baskets, which are housed within separate tanks for each of the F0 exposure conditions. 

3 The F1 generation is begun with newly hatched offspring. Offspring are collected each 

4 morning from the brood cups and transferred immediately to baskets (labeled with the date), 

5 which are housed within separate tanks for each of the F0 exposure conditions. Optimally, two 

6 groups of 15 F1 juvenile mysids are selected and maintained within two separate baskets for each 

7 of the F0 exposure conditions (one basket housing animals for reproductive observations and a 

8 second basket for growth or optional biochemical measurements). With the appearance of a 

9 marsupial pouch on F1 females (sexual maturity), mature females are isolated as pairs with F1 

10 males in separate brood cups. Optimally, seven breeding pairs are selected in each replicate 

11 reproduction basket. The exposure of these F1 mysids is terminated after the second brood release 

12 is complete (or 7 days past the median second brood release in the control group). F1 adults may 

13 be discarded or saved for subsequent measurements (e.g., if stage-specific survival is measured 

14 for population modeling) after release of their second brood. Offspring (F2) from the parental 

15 generation (F1) are collected each morning from the brood cups and enumerated. 

16 Measurements are made of a number of endpoints in both F0 and F1 generations reflective 

17 of functional endocrine regulation of development, growth, reproduction, and survival. They 

18 include sex ratio, time to maturity, time to brood release for the first two broods, and interbrood 

19 duration between the first two broods. Observations are recorded daily for each individual on 

20 mortality, sex determination, reproductive condition of female, (marsupial with or without 

21 developing embryos), and number of young released in each of the first two broods. A total of 45

22 Day 0 mysids are collected from the stock culture and their length determined. These mysids are 

23 separated into nine groups of five mysids for weight determination. At two time intervals, Day 7 

24 and Day 14, animals from the growth baskets are subsampled to determine length and weight. 

25 An overview of the recommended MTTT protocol is provided in Table 3-1. The full 

26 recommended MTTT protocol (Appendix E), includes details regarding test animals (i.e., care, 

27 handling, feeding, acclimation, culture water) and assay system, test substance concentration 

28 selection (i.e., range-finding and definitive testing), test substance administration and analytical 

29 determination, performance of the test and performance criteria, data reporting, and exposure. 

30 The MTTT includes study parameters (i.e., study day, exposure duration, and pairing day) and 

31 three categories of biological endpoints (i.e., reproduction, survival, growth), which are

32 described further below. Changes incorporated into the recommended MTTT protocol since the 

33 proposed 2004 draft guideline are discussed further in the demonstration and optimization 

34 section (Section 4). 

35
Invertebrate Two-Generation ISR 14 May 28, 2013

1 Table 3-1. Test Conditions for the Mysid Two-Generation Test (MTTT) Guideline.

Recommended species Mysid (Americamysis bahia)

Test type Continuous or intermittent flow-through

Water temperature 26 ± 1oC 

Illumination quality Fluorescent bulbs (wide spectrum)

Salinity 20 ± 2 o/oo 

Photoperiod 14 h light, 10 h dark, with 15-30 minute transition period

Loading rate 40 (min. 30) animals per replicate tank (each replicate tank contains two baskets 

with 20 [min. 15] animals, one for reproductive and one for growth measurements) 

Test replicate chamber size Minimum of 98x15x18 cm 

Test replicate solution volume 21 liters (when water depth is at its greatest)

Volume exchanges of test 

solutions

Provide no less than 90% water replacement in 8-12 hours (Sprague 1969) for 

continuous-flow systems or a minimum of 70% water displacement every 5 hours 

using an intermittent-flow system with self-starting excurrent siphons (McKenney 

1986)

Age of test organisms at 

initiation

< 24 hours

No. of mysids per test vessel F0: 40 (min. 30)/replicate; 20 (min. 15)/retention chamber; 2 (1 male & 1 female)/ 

brood chamber

F1: 30/replicate; 15/retention chamber; 2 (1 male & 1 female)/ brood chamber

No. of treatments  5 (plus appropriate controls)

No. replicates per treatment  3 

No. of mysids per test 

concentration (start)

40 (min. 30)/replicate; 3 replicates/treatment; 120 (min. 90) mysids per test 

concentration

Feeding regime 24-hour old brine shrimp nauplii at recommended densities (refer to section 10 of 

MTTT for details)

Aeration None unless DO reaches <60% saturation

Dilution water Natural or artificial seawater acceptable to saltwater mysids; uniform quality during 

test; should not affect test outcome; same source as culture water.

If natural, must use 0.25 micron-filtered

Must allow satisfactory survival, growth, and reproduction in controls

If solvent used, < 0.05 ml/L concentration; use solvent controls

Termination Date Trans-generational test: after release of second brood or 7 days after median day 

of release of second brood in controls (F0 only)

Two-generation test: 7 days after median day of release of second brood in F1 

controls

Primary endpoints (Annex 4 of 

MTTT)

Survival (F0, F1); 

Growth - length & dry weight, (Days 0, 7 and 14 for F0 and F1);

Time to maturation (F0, F1)

Time to first brood release (F0, F1)

Time to second brood release (F0, F1)

Interbrood duration (F0, F1)

Number of young per female in each of the first two broods (F0, F1)

Number of total young in the first two broods in each replicate population (F0, F1)

Sex ratio (F0, F1)

Percentage of females that are reproductively active (F0, F1)

Optional endpoints Optional biochemical measurements

Test acceptability Dissolved oxygen >60% of saturation; mean temperature of 26 ± 1oC; mean 

salinity of 20 o/oo S ± 2 o/oo S; 70% survival of each generation of mysids in 

control treatments; successful reproduction in at least 75% of control females with 

an average total of at least 8 young in the first two broods

2 3.1.2 Endpoints

3 A detailed description of assay endpoints reflective of reproductive and developmental 

4 impairment in aquatic arthropods, including mysids, is provided in a Detailed Review Paper on 

5 Aquatic Arthropods with an Emphasis on Developmental, Reproductive and Endocrine 

6 Disruptive Effects (OECD 2006). The MTTT has more than 20 endpoints, as shown in Annex 4 
Invertebrate Two-Generation ISR 15 May 28, 2013

1 of the MTTT protocol – Appendix E). Some of the endpoints are recorded per mysid or 

2 composite of mysids, some are recorded per breeding pair, and some are recorded per replicate 

3 tank. Endpoints measured in the MTTT are presented in more detail in the following subsection.

4 Survival (F0, F1)

5 Survival is assessed daily for F0 and F1 individuals throughout the MTTT to provide a 

6 basis for expression and interpretation of reproductive output and to quantify chemically induced 

7 mortality. Survival rate for reproduction animals is measured at days 7, 14, and termination 

8 (reproduction survival). Survival rate for all animals including both growth and reproduction 

9 animals is measured at days 7, 14, and termination (total survival). Survival rates should be 

10 adjusted for non-treatment related deaths (e.g., handling accidents, impingement) and animals 

11 removed from growth baskets for growth measurements. Unless acceptable water quality 

12 excursions and/or disease occur, observed mortality in untreated control animals during the 

13 chemical exposure phase of the MTTT is generally <25%. There should be at least 70% survival 

14 of control animals in all test phases over the duration of the chemical exposure in the MTTT.

15 Growth (F0, F1)

16 Crustacean growth is characterized by the periodic shedding (molting) and replacement 

17 of the exoskeleton. Molting is either directly or indirectly involved in the expression of various 

18 other endpoints that are examined in the MTTT. Because growth can only occur as a result of 

19 molting, any disruption of molting could result in alterations in growth (OECD 2006). 

20 Crustaceans and other molting animals utilize ecdysteroids (molting hormones) as major 

21 signaling molecules to regulate molting, reproduction, and development (Huberman 2000). The 

22 actions of ecdysteroids are mediated by a ligand-dependent transcription factor, the ecdysteroid 

23 receptor, which is present only in invertebrates. The insect ecdysteroid receptor is known to play 

24 a central role in regulating expression of a vast array of genes during insect development, and 

25 this regulatory function is intentionally targeted by many pesticides. A similar central role for the 

26 ecdysteroid receptor is expected, but presently unknown, in crustaceans, making ecdysteroid 

27 signaling a potential effective and unintended target of environmental chemicals. While the 

28 ecdysteroid receptor of the mysid Americamysis bahia has been sequenced (Yokota et al. 2005; 

29 Hirano 2010), its functional role needs to be further elucidated (Verslycke et al. 2007). 

30 Two direct measures of growth may be used in the assessment of sublethal effects on 

31 crustaceans, dry weight and body length. The most common response to exposure to toxicants is 

32 reduced growth, although it may not be a particularly sensitive endpoint (Lussier et al. 1999). 

33 Reduced growth has strong implications for reproductive success in mysids, as several studies 

34 (e.g., Mees et al. 1994; Turpen et al. 1994; Winkler and Greve, 2002) have shown that fecundity 

35 is directly related to female body size. 

36 In the MTTT, growth is evaluated by measuring weight (to the nearest 0.1 μg) and length 

37 (to the nearest 0.1 mm) in the F0 and F1 generation. Growth observations should be recorded 

38 separately for males, females, and immature individuals. Data analyses should also be conducted 

39 relative to each sex. 

40 Reproduction (F0, F1)

41 There are several measures of reproductive performance that can be used to assess 

42 sublethal responses in mysids. Despite the well-established use of mysid reproductive endpoints 

43 such as fecundity, egg development time, and time to first brood release in standard toxicity 
Invertebrate Two-Generation ISR 16 May 28, 2013

1 testing, information on the hormonal regulation and basic biology of these processes is still 

2 limited (Verslycke et al. 2004a). For example, while much attention has been given to 

3 vitellogenin (the precursor to the yolk protein vitellin in egg-laying vertebrates and invertebrates)

4 as an indicator of exposure to xeno-estrogens in egg-laying vertebrates, such studies were 

5 lacking in mysids until recently (Ghekiere et al. 2005). In general, studies on the effects of 

6 vertebrate-type endocrine disruptors on crustacean vitellogenesis and reproduction remain 

7 fragmented and are often contradictory (Ghekiere et al. 2005). There is, however, a growing 

8 weight-of-evidence that (xeno)estrogens (and potentially other vertebrate-type endocrine 

9 disruptors) are less effective in causing disruption of normal vitellogenesis in crustaceans than 

10 they are in oviparous vertebrates. Most likely, this is a result of the different hormonal control 

11 strategies for vitellogenesis and reproduction in crustaceans compared with oviparous vertebrates 

12 (Ghekiere et al. 2005).

13 Time to maturation (F0, F1)

14 While vitellogenesis and gonad maturation are known to be under the control of molting 

15 hormones (e.g., ecdysone) and a number of peptide hormones in crustaceans (Ghekiere et al. 

16 2005 and references therein), our mechanistic understanding of its disruption by environmental 

17 chemicals remains poorly understood. Khan et al. (1992) asserted that sexual maturity, which 

18 they described as the presence of gonads or a brood pouch, is a feasible endpoint for 

19 reproductive tests, because gonad maturation is essentially the first step toward reproductive 

20 output. 

21 In the MTTT, time to maturation is determined for the F0 and F1 generation and typically 

22 occurs at approximately 13 to 16 days for the recommended species, Americamysis bahia

23 (McKenney 2005). 

24 Time to first brood release, Time to second brood release, and Interbrood duration (F0, F1)

25 In uncontaminated systems, the time to the release of the first brood is primarily related 

26 to environmental temperature, with some influence of salinity and an interaction between the two 

27 factors (McKenney 1996). At the MTTT recommended temperature (26 ± 2 ̊C) and salinity 

28 (20 ± 2‰), the release of the first brood in the controls during the six interlaboratory validation 

29 studies occurred at about 19 days (weighted average across studies; full range 16 to 26 days;

30 Appendix G).

31 In the MTTT, time to first and second brood release (i.e., number of days after exposure 

32 initiation to release of young in the first and second brood), as well as interbrood duration 

33 (i.e., time between release of first and second brood) is determined for the F0 and F1 generation. 

34 Note that median day of brood release should be based on values from reproductive pairs that 

35 survived to produce a brood and produced some offspring. 

36 Sex ratio in broods 1 and 2 (F0, F1)

37 Exposure to endocrine disruptors has been associated with changes in sex ratio in aquatic 

38 arthropods (e.g., Olmstead and LeBlanc 2002; Tatarazako et al. 2003). Intersexuality has also 

39 been observed in natural populations of mysids (e.g., Mees et al. 1995) and other aquatic 

40 arthropods (e.g., Moore and Stevenson 1991; 1994; Ford et al. 2004a,b). The origin of 

41 intersexuality in natural populations of crustaceans is therefore not necessarily known or 

42 attributable to EDCs

43 In the MTTT, sex ratio is determined for the F0 and F1 generation.
Invertebrate Two-Generation ISR 17 May 28, 2013

1 Fecundity (brood size; F0, F1)

2 Because it is an important measure of reproductive success, any reduction in brood size 

3 can be interpreted as an indication of reproductive toxicity (Khan et al. 1992). However, brood 

4 size is also related to female size in mysids (e.g., Mees et al. 1994; Turpen et al. 1994; Winkler 

5 and Greve, 2002). Reduced mysid fecundity following contaminant exposure is commonly 

6 reported as a sensitive endpoint (e.g., Hollister et al. 1980; Lussier et al. 1985).

7 In the MTTT, fecundity is evaluated through measurement of number of young per 

8 female in each of the first two broods for the F0 and F1 generation, and total number of young in 

9 the first two broods in each replicate population for the F0 and F1 generation. In addition, the 

10 number of reproductively active females is determined for the F0 and F1 generation, total 

11 reproduction days in the first two broods, and number of young per female per reproductive day 

12 in the first two broods. 

13 In the MTTT, mortality among mating pairs must be distinguished from mating pairs that 

14 produced no offspring. If a male member of a reproductive pair dies before production of the 

15 second brood, he is replaced by a male from the growth retention basket if there are sufficient 

16 animals. If a female member of a pair dies before release of her second brood, she is not replaced 

17 and the data record for the second brood should be identified as “NA” (not applicable) and she 

18 should not be included in the calculations of number of offspring/female. If neither the male or 

19 female member of the breeding pair dies, but produce no offspring, the number of offspring in 

20 the data record should be recorded as 0 (zero).

21 3.1.3 Statistical Analyses

22 The MTTT collects quantitative data for more than 20 endpoints (Annex 4 – Attach23 ment E) that are typically collected in spreadsheets and then transferred to and analyzed with a 

24 statistical software package. Some of the endpoints are recorded per mysid or composite of 

25 mysids (e.g., day 0 weight), some are recorded per breeding pair, and some are recorded per 

26 basket (i.e., replicate tank). Any endpoints that are significantly affected by the test chemical 

27 should be reported as such.

28 Normality should be assessed using the Shapiro-Wilk test (Shapiro and Wilk 1965). 

29 Screened potential outliers are included in the tests. Outliers can be tested in one generation 

30 analyses using Tukey’s outlier rule on the untransformed data. Outliers are assessed from 

31 residuals (results of subtracting treatment group means from individual values) to avoid 

32 confounding outliers and treatment effects. Any response more than 1.5 times the interquartile 

33 range above the third quartile (75th percentile) or below the first quartile (25th percentile) is 

34 considered an outlier by Tukey’s rule. All statistical outliers should be screened to determine if 

35 there is a non-biological explanation for the outlier response. Unless there is a valid reason for 

36 exclusion, outliers should be included in data analyses. If data deviate substantially from the 

37 assumption of normality, data may be transformed (e.g., log transformation, square root 

38 transformation, arc sin transformation) and retested. If the normality assumption is accepted for 

39 either the untransformed or the transformed data, heterogeneity of variances among the 

40 concentration groups should be assessed with Levene’s test with absolute residuals (Box 1953). 

41 Unless there is a basis for exclusion, a screened outlier test should be included in the 

42 Levene’s tests. If the normality assumption was invalid for both the untransformed and the 

43 transformed data, heterogeneity of variances among the concentration groups should not be
Invertebrate Two-Generation ISR 18 May 28, 2013

1 assessed, as the nonparametric procedures are appropriate for data analyses. If assumptions of 

2 normality and homoscedascity (i.e., equal statistical variance) are upheld, data from each 

3 generation should be analyzed using an analysis of variance (ANOVA) to determine differences 

4 among treatment and control groups. If one or both tests (i.e., normality, equal statistical 

5 variance) fail, a nonparametric statistic such as a Kruskal-Wallis test should be used to compare 

6 treatment groups. The appropriate multiple comparisons procedure should be selected to 

7 compare each treatment level to the control (e.g., Dunnett’s [Dunnett 1955] procedure for 

8 parametric data, Dunn’s test for nonparametric data. ).

9 Mysid generational data should be analyzed according to the same procedures used for 

10 each generation as described above to compare the effects in the F0 and F1 generations. 

11 Comparisons of the F0 and F1 generations can be performed with a two-way mixed effects 

12 ANOVA model, including generation, concentration group, and the interaction of generation and 

13 concentration as fixed effects and replicate (concentration, generation) as a random effect, 

14 allowing unequal variances between the two generations. It is strongly recommended that a 

15 statistician be involved in both the design and analysis of the test results, since the test guideline 

16 allows for some variation in experimental design.

17 3.2 Summary of the Harpacticoid Copepod Development and Reproduction 

18 Test (HCDRT) Protocol

19 ASTM published a full life cycle test using A. tenuiremis in 2004 (ASTM E2317-04; 

20 ASTM 2004). A first draft of an OECD copepod lifecycle guideline was written in 2003 as a co21 project between UK, Denmark, and Sweden. The harpacticoids Nitocra spinipes and Tisbe 

22 battagliai were included together with the calanoid Acartia tonsa – T. battagliai was later 

23 excluded after prevalidation studies due to difficulties in finding interested laboratories for the 

24 validation studies (OECD 2007). Revised draft guidelines were prepared in 2004, at which point 

25 the harpacticoid and calanoid guidelines were separated. The guidelines were intended for 

26 evaluation of adverse long-term effects (including endocrine disruptive effects) of various types 

27 of chemicals (OECD 2007; OECD 2011). A first interlaboratory validation study was conducted 

28 with the harpacticoids N. spinipes and A. tenuiremis and the calanoid A. tonsa (OECD 2007). 

29 Based on the results of the first interlaboratory validation, it was decided to proceed with a 

30 second interlaboratory validation with A. tenuiremis (OECD 2011). The HCDRT with 

31 A. tenuiremis is described below. The performance of this test is described in Section 4.2.

32 3.2.1 Principle of the Test

33 The HCDRT with A. tenuiremis measures the (sub)chronic effects of chemicals on an 

34 estuarine/marine harpacticoid copepod species under semi-static conditions. The test is initiated 

35 with newly hatched <24 hours old copepods (F0 generation), which are exposed individually in 

36 microwell test chambers (300 μL total volume) to seawater (control) and to the test substance 

37 added to seawater at a range of concentrations. The test duration for the complete life-cycle 

38 method with A. tenuiremis is approximately 25 to 36 days (depending mainly on temperature and 

39 any developmental-rate changes induced by the test chemical of interest), which is sufficient 

40 time for newly-hatched copepods (F0) to develop to the adult stage, be mated, extrude two 

41 clutches of eggs (broods) and produce offspring (F1). Specifically, when the copepods reach 

42 adulthood (around 15 to 18 days for A. tenuiremis at 25oC), the gender of each copepod is 

43 determined by light microscopy. Individual adult male:female mating pairs are then constructed 
Invertebrate Two-Generation ISR 19 May 28, 2013

1 and allowed to mate for 7 to 14 days in new isolated microwell test chambers containing 

2 seawater and the test substance added to seawater at the same range of concentrations as during 

3 maturation/development. The mating pairs are followed until the females have been fertilized

4 and have released at least two broods. Hatching success of the first and second brood should be 

5 determined, and the bioassay is usually terminated upon hatch of the second brood, or on day 36, 

6 whichever occurs sooner. 

7 The biological endpoints measured include survival and development of the early 

8 life-stages, and multiple aspects of copepod reproduction (e.g., intersexuality, mating success, 

9 brood size, abortion rates, number of necrotic eggs, and numbers of viable offspring produced). 

10 Population growth rate and age:stage structure can also be calculated and predicted over multiple 

11 generations using collected lifetable survival, sex-ratio, and fecundity data.

12 An overview of the methodology is provided in Table 3-2. Details regarding test animals 

13 and assay system, test substance concentration selection, test substance administration and 

14 analytical determination, performance of the test and performance criteria, data reporting, and 

15 exposure are provided in Appendix F. Biological endpoints of the HCDRT are described below.

16 Table 3-2. Test Conditions for the Harpacticoid Copepod Development and Reproduction 

17 Test (HCDRT) guideline.

Recommended species Amphiascus tenuiremis

Test type Static renewal 

Water temperature 25 ± 2oC 

Salinity 18-40 o/oo 

Photoperiod 12 h light, 12 h dark

Loading rate 20-40 animals per replicate microplate, each added to individual 

microwells

three replicate test vessels (microplates) per concentrations and 

control(s)

Microwell size 300 μL total volume; 250 μL working volume

Test replicate solution volume 100 mL per concentration per water renewal

Volume exchanges of test solutions The frequency of medium renewal will depend on the stability of the 

test substance, but it should occur at least every third day of the 

test (i.e., provide 90-95% water replacement in each test microwell 

every 72-h or less depending on stability of test substance). If 

concentration of test substance is not expected to remain within ± 

50% of nominal values (i.e., within range 50 – 150 %) over the 

maximum renewal period, more frequent medium renewal should 

be considered. It is recommended that, as a minimum, the highest 

and lowest test concentrations and positive controls (if used) be 

analyzed when freshly prepared at the start of the test and 

immediately prior to each renewal.

Age of test organisms at initiation < 24 hours

No. of treatments  5 (plus appropriate controls)

No. replicates per treatment  3 

No. of copepods per test 

concentration (start)

20-40/replicate; 3 replicates/treatment; 60-120 copepods per test 

concentration

No. of copepod mating pairs per test 

concentration (at maturity)

10-20 mating pairs/replicate (in case of 100% survival and balanced 

sex ratio); 3 replicates/treatment

Feeding regime Feeding at every other microwell water renewal

Individual copepods are fed 2 μl of a 1:1:1 volume mixed-algal cell 

suspension (centrifuged, pelletized and re-constituted in fresh 30‰ 

seawater to 107 cells/mL) of a chlorophyte (e.g., Dunaliella 

tertiolecta), a chrysophyte (e.g., Isochrysis galbana) and a 

cryptophyte (e.g., Rhodomonas salina)

Cell density added to each test microwell at each feeding should 
Invertebrate Two-Generation ISR 20 May 28, 2013

target ~ 2 x 104 cells/microwell. 

Aeration None, but dilution seawater should be aerated for 24 h, then filtered 

at 0.2 μm, and ten mixed with test chemical stocks at appropriate 

volumes and concentrations

Dilution water natural or artificial seawater acceptable to A. tenuiremis; uniform 

quality during test; should not affect test outcome; same source as 

culture water 

test organisms must be cultured or maintained at same salinity (± 3 

o/oo) for at least 7 days before start of test

If natural, must use 0.25 micron-filtered

Must allow satisfactory survival and reproduction in controls

Exposure period approximately 36 days at 25 °C

Primary endpoints Survival (F0, F1 – record by developmental stage)

Sex ratio (F0)

Development rate (from nauplius to copepodite stage and adult 

stage, respectively )

Fertilization success (number of mating pairs able to produce two 

successive broods)

Total mean viable offspring per mating pair (i.e., through two broods

averaged over

 the number of mating pairs created and tested)

Time to production of first brood (time interval between two broods)

Optional endpoints Behavioral (e.g., uncoordinated swimming, loss of equilibrium, 

change in pigmentation, atypical quiescence) 

Time from appearance of first egg sacs to release of first and 

second brood

Number of aborted egg sacs

Number of necrotic/infertile eggs per brood

Morphological abnormalities

Population growth rate and age;stage structure (using lifetable 

survival, sex-ratio and fecundity data)

Test acceptability Average mortality in the control(s) ≤ 25% at end of exposure, with 

no individual control replicate microplate should exceed 30% 

mortality

Dissolved oxygen >70% of saturation throughout exposure

temperature should vary by no more than ± 4oC during the entire 

test with a 36 day mean temperature of ± 2 °C 

pH must not vary more than 0.5 units from the average control pH 

with a 36 day mean pH of 8.0 ± 4

conductivity/salinity must not vary more than 10% from the control 

start value (e.g., 30o/oo ± 3 o/oo)

sex-ratio in F0 -controls should on average range between 0.6 to 1.8 

(± 0.2)

average number of viable offspring produced through two broods in 

the controls should not be less than 8 individuals

average number of days to the first brood in the control(s) should at 

a temperature of 25 °C not exceed the number of days representing 

70% of the total exposure period or 25 days, whichever is less

1 Note:

2 Due to the limited amount of test solution volume (approximately 250 μL) in the HCDRT, it may be necessary to 

3 collect and pool individual microwell volumes within microplates within each treatment at the end of each renewal 

4 period for water quality and test material measurements.

5 3.2.2 Endpoints

6 A detailed description of assay endpoints reflective of reproductive and developmental 

7 impairment in aquatic arthropods, including copepods, is provided in a Detailed Review Paper 

8 on Aquatic Arthropods with an Emphasis on Developmental, Reproductive and Endocrine 

9 Disruptive Effects (OECD 2006). Lifecycle endpoints for the recommended species, 

10 A. tenuiremis, are monitored daily in each microplate using light microscopy. Growth 
Invertebrate Two-Generation ISR 21 May 28, 2013

1 (length/weight) is not recorded in the HCDRT. A number of developmental and reproductive 

2 endpoints are monitored and are discussed below.

3 Survival 

4 Survival of larvae (nauplii), juveniles (copepodites), and adults is checked daily up to and 

5 including the last day of the test. Mortality of mating pairs should be checked and recorded daily. 

6 The average mortality in the control(s) must not exceed 25% at the end of the exposure period 

7 and no individual control replicate microplates should exceed 30% mortality. Stage-specific 

8 survival data can be combined with sex ratio and fecundity data to estimate population growth 

9 rate and age:stage structure (Appendix F). 

10 Rate of Development

11 Various studies on copepods have demonstrated that larval development, which is tightly 

12 linked to the molting process, is highly sensitive to a range of chemicals, including suspected 

13 endocrine disruptors. Adverse effect concentrations in these copepod studies were, in most cases, 

14 lower than adverse effect concentrations for similar endpoints reported for other crustaceans. 

15 However, the mode of action of EDCs on crustaceans remains largely unknown, and very little is 

16 known about the endocrine system of copepods (Kusk and Wollenberger 2007 and references 

17 therein). The mean number of days from brood extrusion to naupliar hatching has been used to 

18 study egg development time in copepods following chemical exposure (Cary et al. 2004). 

19 Development rate can also be used in population growth rate and age:stage structure models 

20 (Appendix F).

21 While growth is not measured in the HCDRT, the rate of development is measured as the 

22 number of days it takes for development of each individually isolated copepod, from a first stage 

23 nauplius to a juvenile copepodite to a reproductively mature adult. 

24 Reproduction

25 Time to first brood release is one of the main endpoints often used in copepod life cycle 

26 tests aimed at identifying effects of environmental pollutants with a suspected biological activity 

27 (e.g., Breitholtz and Bengtsson. 2001; Chandler et al. 2004a). Fecundity (brood size) has also 

28 been evaluated in copepods following chemical exposure (e.g., Bejerano and Chandler 2003; 

29 Hutchinson et al. 1999a; Breitholtz and Wollenberger 2003; Breitholtz et al. 2003). Because it is 

30 an important measure of reproductive success, any reduction in fecundity can be interpreted as 

31 an indication of reproductive toxicity (Khan et al. 1992). However, reductions in fecundity need 

32 to be evaluated carefully to distinguish direct reproductive disruption from indirect effects on

33 fecundity due to growth effects (OECD 2006).

34 The HCDRT evaluates a number of reproductive performance measures, such as sex ratio, 

35 fertilization success (number of mating pairs able to produce two successive broods), time to 

36 production of first brood and time interval between two broods, offspring production through 

37 two broods. Optional reproductive endpoints include number of necrotic or infertile eggs per 

38 brood and number of aborted egg sacs. Note that, reproduction data in the HCDRT are expressed 

39 on a per female or per mating-pair basis. 

40 3.2.3 Statistical Analyses

41 The HCRDT collects quantitative development and reproduction data that are typically 

42 collected in spreadsheets and then transferred to and analyzed with a statistical software package 
Invertebrate Two-Generation ISR 22 May 28, 2013

1 (example of data entry sheets are provided in Annex 3 of HCDRT draft protocol – Appendix F). 

2 OECD 2007; OECD 2011). 

3 If desired, acute mortality data for selected time periods (e.g., ≤96 hours) can be analyzed 

4 based on the methods of Stephan (1977) to calculate median lethal (LC50) concentrations of test 

5 materials. Statistically significant effects on survival of adult females are calculated using a 

6 contingency table procedure, Fisher’s Exact Test (Finney et al. 1963), which should be reported 

7 as a separate endpoint. Those concentrations where mortality is significantly different from the 

8 control are not included in the statistical analysis of the reproduction data. 

9 Development rates, stage-specific mortality, fertilization success, and sex ratios of the F0 

10 may all be analyzed statistically for significant differences relative to controls. Reproductive 

11 endpoints may be analyzed as single end of test measurements or relative to different time 

12 intervals during the exposure. The mean number of F1 offspring produced from the fertilized

13 females (F0) in each replicate is calculated. Generally, the reproduction data are not adjusted for 

14 mortality but are presented on a per female or per mating-pair basis. The mean number of 

15 offspring produced by each control and treatment mating pair is calculated. If an animal dies 

16 after producing young, the total number of offspring produced up to the time of death is used. On 

17 occasions, it may be necessary to make an adjustment for mortality where those animals that died 

18 during the exposure (i.e., before giving birth to any young) are not included in the statistical 

19 analysis. One-way nested analysis of variance (ANOVA) is used to evaluate effects of the test 

20 substance on the reproduction (and other chronic endpoints) of the copepods, and a multiple 

21 comparison procedure (e.g. Dunnett’s test [Dunnet 1955] is used to define the treatment level 

22 resulting in significantly different reproduction from the control (p=0.05). For ANOVA, 

23 individual microwell mating pairs are nested within their particular microplate, which is then 

24 replicated within a given treatment. A nested model is necessary because microwells within a 

25 microplate are not strictly independent of one another.

26 ANOVA is a parametric procedure based on the assumptions that the observations are 

27 independent and normally distributed, and that the variance of the observations is homogeneous 

28 across all toxicant concentrations and the control. These assumptions should be checked prior to 

29 using ANOVA, to determine if they have been met. Non-normal distributed data may be 

30 subjected to normalizing transformations such as log transforms, arc-sine square root transforms, 

31 etc., provided both treatment and control datasets are subjected to the same transformation. Tests 

32 for validating these assumptions are Shapiro-Wilks Test for normality and Bartlett’s Test for 

33 homogeneity of variance (US EPA 1988). If the data do not meet the assumptions for ANOVA, 

34 non-parametric procedures such as Steel’s Many-One-Rank Test or Wilcoxon’s Rank Sum Test 

35 (US EPA 1988) may be more appropriate.

36 If a statistically significant difference in survival, development rate, sex ratio, or 

37 reproduction is detected between the control and solvent control, only the solvent control is used 

38 as the basis for the calculation of results. If no significant differences exist between control and 

39 solvent control data, these may be pooled for comparisons with test substance treatments.

40
Invertebrate Two-Generation ISR 23 May 28, 2013

1 4 DEMONSTRATION AND OPTIMIZATION OF THE MYSID AND 

2 COPEPOD TEST METHODS

3 This section describes results of demonstration and optimization studies that have been 

4 performed in general accordance with the MTTT and HCDRT methods presented in Section 3). 

5 4.1 Mysid Two-Generation Toxicity Test (MTTT) Guideline Performance

6 McKenney (1998) synthesized the results of his earlier studies on the effects of chronic 

7 exposure to various pesticides on A. bahia and concluded that life-cycle endpoints (slowed 

8 juvenile growth, inhibited reproductive capacity, metabolic changes) were more sensitive than 

9 survival. Through these studies, McKenney also demonstrated that mysids are generally more 

10 sensitive than most other aquatic species to chemical contaminants. These early studies are not 

11 summarized here and readers are referred to the original studies (McKenney 1982; McKenney 

12 1986; McKenney and Matthews 1990; McKenney et al. 1991) and several reviews 

13 (Verslycke et al. 2004a; Verslycke et al. 2007; OECD 2006). In a later transgenerational 

14 exposure study with fenoxycarb, McKenney (2005) extended the life-cycle test such that 

15 juveniles, released by adults exposed since <24-hour old juveniles, were reared through 

16 maturation without further exposure. Neither maturation time, sex determination, nor young 

17 production was significantly altered during the life-cycle exposure. However, second-generation 

18 adults, exposed to fenoxycarb only as developing embryos and juveniles, produced fewer young 

19 (at 6 μg fenoxycarb/L) and contained significantly fewer males (at 1 μg fenoxycarb/L). These 

20 findings suggested the need for, at least, two-generational exposure protocols to adequately 

21 predict the potential chronic impact of EDCs (Verslycke et al. 2007; OECD 2006) and led to the 

22 development of a proposed MTTT guideline. A proposed MTTT was submitted to OECD in 

23 2006. This guideline was initially proposed to OECD by US EPA in 2004 to estimate toxicity 

24 associated with endocrine disruption and reproductive fitness for an identified potential EDC 

25 (OECD 2004a). The MTTT test is an extension of previous mysid life-cycle test procedures 

26 produced by ASTM (ASTM E1191) and US EPA (OCSPP 850.1350). The MTTT has been used 

27 in demonstration and optimization studies as described further below. Collectively, these studies 

28 form the basis for the recommended protocol described in Section 3.1.

29 4.1.1 Demonstration Studies Using the 2004 Draft MTTT Guideline

30 First set of demonstration studies

31 A first evaluation of the testing procedures and methods proposed by US EPA to OECD 

32 in the draft guideline for a two-generation mysid test to evaluate suspected endocrine-disrupting 

33 chemicals (OECD 2004a) was conducted through a demonstration study with three chemicals: 

34 fenoxycarb, 3,5-dichlorophenol (3,5-DCP), and fipronil (Putt and Cafarella 2006). These 

35 chemicals each have a uniquely identified mode of action: fenoxycarb, a juvenile hormone 

36 mimic designed to prevent target insects from molting to the adult stage; 3,5-DCP, which inhibits 

37 energy production through uncoupling of oxidative phosphorylation; and fipronil, a central 

38 nervous system disruptor that blocks the passage of chlorine ions through the GABA receptor. 

39 The endpoints examined in the F0 and F1 generations in this first evaluation included 

40 survival, growth (total length, dry weight, growth rate between days 7 and 14), and reproductive 

41 measures (time to maturation, time to first and second brood release, interbrood duration, number 
Invertebrate Two-Generation ISR 24 May 28, 2013

1 of young per female in each of the first two broods, total number of young in first two broods, 

2 sex ratio, and percentage of females that are reproductively active).

3 Performance criteria for judging test data acceptability were: >70% survival of control 

4 animals in all test phases over the duration of the chemical exposure; ≥75% of females in the F0 

5 and F1 controls must produce young; and average number of young produced per control female 

6 in the first two broods combined must be at least 8.

7 To check homogeneity of variances in data obtained for each endpoint, Bartlett’s Test 

8 (for normally distributed data) or Levene’s Test (when data were not normally distributed or 

9 there was no variance in a treatment level) were used. Shapiro-Wilk’s test was used to evaluate 

10 normality of the data distribution. ANOVA was conducted on each data set to determine if a 

11 treatment group in the data set was different from the other groups in that data set. Treatment 

12 level endpoints were compared with the performance of the solvent control data by using 

13 William’s Test and Dunnett’s Test. If necessary, mean values were transformed using square 

14 root, arcsine square root, or log conversion procedures. 

15 Fenoxycarb

16 Detailed results of the demonstration study with fenoxycarb are presented in Putt and 

17 Cafarella (2006) and are summarized here.

18 A 52-day flow-through test was initiated at nominal concentrations of 0 (solvent

19 control - triethylene glycol), 1.0, 2.5, 6.0, 16, and 40 μg active ingredient (a.i.)/L. Analysis of the 

20 test solutions demonstrated that the expected concentration-gradient was generally maintained 

21 during the exposure. Mean measured concentrations of fenoxycarb ranged from 82 to 92% of 

22 nominal. Based on mean measured concentrations, the treatment levels were defined as 2.3, 4.9, 

23 14, and 35 μg a.i./L. Artificial seawater (commercially prepared salt formula added to laboratory 

24 well water) was used as dilution water. Newly prepared batches had a pH range of 7.6 to 8.2 and 

25 a salinity of 20 ± 2‰. 

26 The test substance was introduced into 54-L glass aquaria for a minimum of 48 hours 

27 before the test began to allow the test solution to reach an equilibrium. Three replicate aquaria 

28 were used for each treatment level. Exposure for the F0 mysids began when <24h old individuals 

29 were randomly distributed into the test aquaria from stock cultures. Fifteen individuals were 

30 added to each triplicate chamber within each single treatment exposure aquarium, such that each 

31 treatment group received a minimum of 45 mysids. Three additional groups of 15 mysids were 

32 randomly assigned to growth baskets within each replicate aquarium per treatment. Exposures 

33 were terminated 7 days after the mean day of release of the second brood of the F1 mysids in the 

34 solvent controls.

35 Mysids from both generations (F0 and F1) for all treatments were fed twice daily, except 

36 at test initiation and test termination, where they were fed only once due to the time of test 

37 initiation and termination (i.e., later in the day). For each generation, during the period prior to 

38 pairing (e.g., the first 14 days), at least one of the two daily feedings was with brine shrimp 

enriched with Selco®39 , a supplemental substance high in saturated fatty acids. During the period 

subsequent to pairing (e.g., day 15 through test termination), the Selco®40 enriched brine shrimp 

41 were fed to the test organisms once every other day. Rations for reproduction/growth baskets 

42 (containing 15 mysids each) were approximately 1,800 nauplii/basket on days 1 to 3; 2,250 on 

43 days 4 to 6; 2,700 on days 7 to 9; 3,150 on days 10 to 12; and 3,600 on days >12, while brood 
Invertebrate Two-Generation ISR 25 May 28, 2013

1 cups (containing individually paired mysids) were fed approximately 900 nauplii/cup throughout 

2 the test. All treatments and controls received, as near as reasonably possible, the same ration of 

3 food. 

4 Measured water quality parameters (temperature, pH, and salinity) stayed within the 

5 specified limits (Putt and Cafarella 2006). Dissolved oxygen levels dropped below 60% 

6 saturation in both the controls and treatment levels at the same time, but this reduction is not 

7 expected to have impacted study performance, since all test performance criteria were met. 

8 Results of the demonstration study are presented in Table 4-1. F0 generation mysid length 

9 (day 14) was the most sensitive endpoint (statistically significant effect in 2.5 to 40 μg/L

10 treatments). Length growth rate, time to first brood, number of offspring in the first brood, and 

11 total offspring from the F0 generation were also statistically significantly different from controls

12 in 6, 16, and/or 40 μg/L treatments. Compared to controls, significant increases were observed 

13 for F0 14-day dry weight, dry weight growth rate, and F1 total offspring production at the

14 2.5 μg/L treatment level, but similar results were not observed at any other treatment level. No 

15 other significant F1 generation effects were detected among any of the endpoints measured.

16 Table 4-1. Results of the EPA demonstration study of the MTTT with fenoxycarb.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg a.i./L) 1.0 2.5 6.0 16 40 1.0 2.5 6.0 16 40

Mean Measured Concentrations (μg a.i./L) <LOQ 2.3 4.9 14 35 <LOQ 2.3 4.9 14 35

Day 0 dry weight * - - - - - - N9 - N9 - N8 - N9 - N8

Day 7 dry weight - - - - - - - - N1 - N1 -

Day 14 dry weight - ↑ - - - - N2 - N1 - N0 - N0 - N2

Day 7-14 dry weight growth rate - ↑ - - - - N2 - N1 - N0 - N0 - N2

Day 0 length * - - - - - - N9 - N9 - N9 - N9 - N8

Day 7 length - - - - - - - N1 - N1 - -

Day 14 length - ↓ ↓ ↓ ↓ - N2 - N1 - N0 - N0 - N2

Day 7-14 length growth rate - - ↓ ↓ ↓ - N2 - N1 - N0 - N0 - N2

Day 14 survival (%) - - - - - - - - N2 - -

Time to Maturation - - - - - - - - N2 - -

Sex ratio - - - - - - - - N2 - -

Time to 1st Brood - - - ↑ ↑ - - - N2 - -

Time to 2nd Brood - - - - - - - - N2 - -

Interbrood Duration - - - - - - - - N2 - -

Number 1st Brood - - - ↓ - - - - N2 - -

Number 2nd Brood - - - - - - - - N2 - -

Total Offspring/Female - - ↓ ↓ ↓ - ↑ - N2 - -

% Reproducing Females - - - - - - - - N2 - -

17 [<LOQ] The limit of quantitation was 2 μg a.i./L, thus the lowest treatment level was not analytically verified.

18 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

19 [*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples 

20 from the entire population of <24-hour old mysids used to initiate the test were not related to dose levels 

21 used in the study.

22 [N#] Sample size (n) was 3 for each treatment level or control, except as noted (e.g., for N8 , n = 8).

23 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's, Williams' or 

24 Bonferroni's test (α = 0.05).

25 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's, Williams' or 

26 Bonferroni's test (α = 0.05).

27
Invertebrate Two-Generation ISR 26 May 28, 2013

1 3,5-dichlorophenol

2 Detailed results of the demonstration study with 3,5-dichlorophenol (3,5-DCP) are 

3 presented in Putt and Cafarella (2006) and are summarized here.

4 A preliminary range finding test with 3,5-DCP was conducted using an acute 

5 flow-through test exposing <24 hour mysids to nominal concentrations of 0.13, 0.32, 0.80, 2.0, 

6 and 5.0 mg a.i./L and a solvent (acetone) control. Two test vessels containing 10 mysids each 

7 were established for each test concentration and the solvent control. After 96 hours, 0, 5, 5, 25 

8 and 100% mortality was observed among mysids exposed to the 0.13, 0.32, 0.80, 2.0, and 5.0 mg 

9 a.i./L treatment levels, respectively. Mortality of 5% was observed among mysids exposed to the 

10 solvent control. 

11 Based on the preliminary results, a 50-day flow-through test was initiated at nominal 

12 concentrations of 0 (solvent control; triethylene glycol), 0.013, 0.032, 0.076, 0.20, and 

13 0.50 mg a.i./L. Analysis of the test solutions demonstrated that the expected concentration

14 gradient was generally maintained during the exposure. Mean measured concentrations of

15 3,5-DCP ranged from 56 to 98% of nominal, increasing with increasing concentration. Based on 

16 mean measured concentrations of 3,5-DCP, the treatment levels tested were defined as 0.0072, 

17 0.022, 0.072, 0.19, and 0.49 mg a.i./L. Test procedures, feeding, and statistical data evaluation 

18 procedures were the same as for the fenoxycarb experiment.

19 All measured water quality parameters (temperature, pH, and salinity) stayed within the 

20 specified limits (Putt and Cafarella 2006). Dissolved oxygen levels dropped below 60% 

21 saturation in both the controls and treatment levels at the same time, but this reduction is not 

22 expected to have impacted study performance, since all test performance criteria were met. 

23 Results from the 3,5-DCP demonstration test are presented in Table 4-2. Time to 

24 maturation in the F0 generation was the most sensitive endpoint in the mysid test (statistically 

25 significant effect in 0.032 to 0.5 mg/L treatments). Time to first brood, time to second brood, 

26 interbrood duration, number of offspring in the first and second brood, and total offspring 

27 production in the F0 generation were also sensitive endpoints. Mysid F0 day 7 dry weight did not 

28 demonstrate a dose response; however, the dry weights from the highest (0.5 mg/L) and second 

29 lowest (0.032 mg/L) treatment levels were significantly reduced. Interbrood durations in the two 

30 highest treatment levels (0.20 and 0.50 mg/L) in the F0 exposure were statistically significant 

31 compared to the solvent control. In the F1 generation, significant reductions in total number of 

32 offspring per female were observed in the 0.013, 0.076, and 0.50 mg/L treatment levels. The 

33 percent of reproducing females was also a sensitive endpoint for adverse effects in the F1 

34 generation (0.5 mg/L).

35

36
Invertebrate Two-Generation ISR 27 May 28, 2013

1 Table 4-2. Results of the EPA demonstration study of the MTTT with 3,5-DCP.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg a.i./L) 13 32 76 200 500 13 32 76 200 500

Mean Measured Concentrations (μg a.i./L) 7.2 22 72 190 490 7.2 22 72 190 490

Day 0 dry weight * - - - - - -N8 -N8 -N9 -N9 -N9

Day 7 dry weight - ↓ - - ↓ - - - - -

Day 14 dry weight - - - - - - - - - -N1

Day 7-14 dry weight growth rate - - - - - - - - - -

Day 0 length * - - - - - -N9 -N9 -N9 -N9 -N8

Day 7 length - - - - - - - - - -

Day 14 length - - - - - - - - - -N1

Day 7-14 length growth rate - - - - - - - - - -

Day 14 survival (%) - - - - - - - - - -

Time to Maturation - ↑ ↑ ↑ ↑ - - - - -

Sex ratio - - - - - -N2 -N2 -N2 - -

Time to 1st Brood - - - - ↑ - - - - -

Time to 2nd Brood - - - ↑ ↑ - - - - -

Interbrood Duration - - - ↑ ↑ - - - - -

Number 1st Brood - - - - ↓ - - - - -

Number 2nd Brood - - - ↓ ↓ - - - - -

Total Offspring/Female - - - ↓ ↓ ↓ - ↓ - ↓

% Reproducing Females - - - - - - - - - ↓

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples from 

4 the entire population of <24-hour old mysids used to initiate the test were not related to dose levels used in the 

5 study.

6 [N#] Sample size (n) was 3 for each treatment level or control, except as noted (e.g., for N8 , n = 8).

7 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's or Williams' test (α = 0.05).

8 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's or Williams' or test (α = 

9 0.05).

10

11 Fipronil 

12 Detailed results of the demonstration study with fipronil are presented in Putt and 

13 Cafarella (2006) and are summarized here.

14 A 50-day flow-through test was initiated at nominal concentrations of 0 (solvent control; 

15 triethylene glycol), 1.0, 2.5, 6.0, 16, and 40 ng a.i./L. The two lowest nominal concentration 

16 levels (1.0 and 2.5 ng/L) were not analytically measured during the study since they were below 

17 the limit of quantitation (5 ng/L). Analysis of the remaining test solutions demonstrated that the 

18 expected concentration gradient was generally maintained during the exposure. Based on mean 

19 measured concentrations of fipronil, these treatment levels were defined as 4.5, 16, and 

20 44 ng a.i./L. Test procedures, feeding, and statistical data evaluation procedures were the same as 

21 for the fenoxycarb experiment.

22 All measured water quality parameters (temperature, pH, and salinity) stayed within the 

23 specified limits (Putt and Cafarella 2006). Dissolved oxygen levels dropped below 60% 

24 saturation in both the controls and treatment levels at the same time, but this reduction is not 

25 expected to have impacted study performance since all test performance criteria were met. 

26 The fipronil demonstration results are presented in Table 4-3. F0 generation day 7 length 

27 and time to maturation were the most sensitive endpoints in the mysid two-generation exposure 

28 to fipronil (statistically significant effect in 1 to 40 ng/L treatments). Day 14 length, day 7 to 14 
Invertebrate Two-Generation ISR 28 May 28, 2013

1 length growth rate, and survival on day 14 in the F0 generation were also sensitive endpoints. In 

2 the F1 generation, day 14 survival was the most sensitive endpoint (40 ng/L).

3 Table 4-3. Results of the EPA demonstration study of the MTTT with fipronil.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (ng a.i./L) 1.0 2.5 6.0 16 40 1.0 2.5 6.0 16 40

Mean Measured Concentrations (ng a.i./L) <LOQ <LOQ 4.5 16 44 <LOQ <LOQ 4.5 16 44

Day 0 dry weight * - - - - - -N9 -N9 -N9 -N9 ↓N4

Day 7 dry weight - - - - - - - - - -N0

Day 14 dry weight - - - - - - - - - -N0

Day 7-14 dry weight growth rate - - - - - - - - - -

Day 0 length * - - - - - -N9 -N9 -N9 -N9 -N4

Day 7 length ↓ ↓ ↓ ↓ ↓ - - - - -N0

Day 14 length - - - - ↓ - - - - -N0

Day 7-14 length growth rate - - - - ↓ - - - - -

Day 14 survival (%) - - ↓ ↓ ↓ - - - - ↓

Time to Maturation ↑ ↑ ↑ ↑ ↑ - - - - -

Sex ratio - - - - - - - - - -

Time to 1st Brood - - - - - - - - - -

Time to 2nd Brood - - - - -N2 - - - - -N2

Interbrood Duration - - - - -N2 - - - -N2

Number 1st Brood - - - - - - - - - -

Number 2nd Brood - - - - -N2 - - - - -N2

Total Offspring/Female - - - - - - - - - -

% Reproducing Females - - - - - - - - - -

4 [<LOQ] The limit of quantitation was 5 ng a.i./L, thus the two lowest treatment levels were not analytically verified.

5 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

6 [*] Statistical analysis for mean dry weight and length on day 0 was not performed since the nine subsamples 

7 from the entire population of <24-hour old mysids used to initiate the test were not related to dose levels 

8 used in the study.

9 [N#] Sample size (n) was 3 for each treatment level or control, except as noted (e.g., for N8 , n = 8).

10 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's, Williams' or 

11 Bonferroni's test (α = 0.05).

12 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's, Williams' or 

13 Bonferroni's test (α = 0.05).

14 The following conclusions and recommendations based on the first set of demonstrations 

15 studies were taken from Putt and Cafarella (2006):

16  The F0 and F1 growth and reproduction endpoint data routinely passed tests for 

17 normality and homogeneity, typically without requiring any data transformation

18  Less than 24-hour-old mysid growth endpoints and sex ratio were the only two 

19 general endpoints that did not elicit a statistically significant effect with at least one 

20 chemical 

21  Growth rates were either less sensitive or equally as sensitive as the direct 

22 measurement of either dry weight or length 

23  Mysid length was a more sensitive endpoint than dry weight (i.e., length was 

24 significantly reduced with increasing treatment level in two of three tests – dry weight 

25 was significantly impacted in these tests) and easier to collect, especially with the 

26 smallest mysids. However, the sensitivity of the length endpoint varied for day 7 and 

27 day 14 measurements. Therefore, effects on length on day 7 do not necessarily 

28 translate to effects on length on day 14 and vice versa. Thus, measuring growth 
Invertebrate Two-Generation ISR 29 May 28, 2013

1 (particularly mysid length) at multiple time intervals over the first 14 days of 

2 exposure is important if mysid length can be correlated to potential endocrine effects

3  The proposed endpoints in the mysid two-generation study design appear to be 

4 relevant for identifying effects on growth and reproduction. The 3,5-DCP exposure 

5 clearly demonstrated that the reproductive endpoints are sensitive

6  The study design for the two-generation mysid study does not include any 

7 biochemical, biomarker, or histopathology endpoints. Therefore, a conclusive 

8 determination of mode of action is not possible. However, the growth and 

9 reproductive endpoints evaluated in this demonstration are all relevant endpoints to 

10 evaluate effects on mysid growth and reproduction. This demonstration provided 

11 strong evidence that multiple growth and reproduction endpoint monitoring is 

12 necessary in the final study design to identify the wide range of potential effects from 

13 an array of chemicals. 

14 Second set of demonstration studies

15 A second evaluation of the proposed draft guideline (OECD 2004a) was conducted 

16 through a demonstration study with four chemicals: prochloraz (vertebrate aromatase inhibitor), 

17 flutamide (vertebrate androgen receptor antagonist), ketoconazole (weak vertebrate androgen 

18 receptor agonist), and 4-tert-octylphenol (vertebrate estrogen receptor agonist) (Kropp and 

19 Cullinan 2008). These chemicals had previously been used as reference chemicals in the 

20 validation of several Tier 1 screening assays

21 (http://www.epa.gov/scipoly/oscpendo/pubs/assayvalidation/status.htm). The protocol used in 

22 the demonstration study differed from the proposed 2004 guideline in four ways: (1) the mysids 

23 for each study were fed with Artemia nauplii hatched from cysts that were not obtained from a 

24 reference source (sensu Leger and Sorgeloos 1984), but were from a reliable source that has been 

25 used successfully by the test laboratory in many previous studies; (2) retention baskets used 

26 during the testing were constructed from nylon mesh having a slightly larger mesh opening, 

27 355 μm, than the 210- to 330-μm opening stipulated in the guideline and used in the first set of 

28 demonstration studies; (3) mysid dry weights were measured to the nearest 1 μg instead of the 

29 nearest 0.1 μg; and (4) the draft guideline specifies that females be paired with the largest 

30 suspected males―however, the laboratory used a dissecting microscope to examine all mysids, 

31 except for ovigerous females, to confirm gender and maturity before pairing. None of these 

32 modifications is likely to have had an adverse effect on the study.

33 Prochloraz

34 Detailed results of the demonstration study with prochloraz are presented in 

35 Aufderheide (2008a) and are summarized here.

36 A 21-day flow-through range-finding test was initiated at nominal concentrations of 0 

37 (dilution water control), 1.0, 10, 100, and 1,000 μg prochloraz/L. Survival was the most 

38 consistent indicator of a concentration-dependent response during the range-finding test and the 

39 definitive test concentrations were set at a nominal concentration range starting at approximately 

40 one-third the LC50 value (i.e., 300 μg prochloraz/L) after the initial 14 days of the range-finding 

41 test. The definitive toxicity test was performed at nominal concentrations of 0 (control), 13.0, 

42 21.6, 36.0, 60.0, and 100 μg prochloraz/L with three replicate test chambers for each control and 

43 treatment level. The mysids were initially fed according to the feeding regime specified by the 

44 2004 OECD draft guideline. However, due to poor reproductive activity during the first 27 days
Invertebrate Two-Generation ISR 30 May 28, 2013

1 (i.e., increased time to first brood and low number of young in second brood), feedings were 

2 increased from 900 to 1,350 nauplii per brood cup by increasing the number of feedings from 

3 two to three.

4 All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 

5 stayed within the specified limits (Aufderheide 2008a). As a whole, more than 75% of control 

6 pairs produced at least one brood and more than 70% of control animals survived the first 28 

7 days of testing, thus meeting the guidance-specified acceptability criteria (Aufderheide 2008a). 

8 However, the numbers of F1-generation mysids were not sufficient to allow complete evaluation 

9 of endpoints for that generation. Analytical measurements of prochloraz made during the study 

10 varied and showed occasional overlap of exposure concentrations during the study. Reasons for 

11 this variation in concentrations within dose levels were not identified. The use of a self-starting 

12 siphon for water renewal was identified as a likely cause for nutritional issues by effectively 

13 removing brine shrimp nauplii during draw down, and for loss of animals due to impingement 

14 and mortality. 

15 Results from the prochloraz demonstration test are presented in Table 4-4. All but one of 

16 the variables (i.e., 7-day weight) evaluated during the prochloraz study were nonresponsive as 

17 indicated by signal-to-noise ratios <2 (Kropp and Cullinan 2008). Statistical analysis of the 

18 endpoints based on the Kruskal-Wallis and Dunn’s tests showed no significant differences 

19 between treatment mean responses and control mean responses.

20 Table 4-4. Results of the EPA demonstration study of the MTTT with prochloraz.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (mg/L) 13 21.6 36 60 100 13 21.6 36 60 100

Mean Measured Concentrations (mg/L) 11.1 18.9 32.1 53.9 93 11.1 18.9 32.1 53.9 93

Day 14 survival (%) - - - - - na na na na na

Day 28 survival (%) - - - - - na na na na na

Day 7 length - - - - - na na na na na

Day 7 dry weight - - ↓ - - na na na na na

Day 14 male length - - - - - na na na na na

Day 14 female length - - - - - na na na na na

Day 14 dry weight - - - - - na na na na na

Time to 1st Brood - - - - - na na na na na

Time to 2nd Brood - - - - - na na na na na

Interbrood Duration - - - - - na na na na na

Number 1st Brood - - ↓ ↓ ↓ na na na na na

Number 2nd Brood ↓ - ↓ ↓ - na na na na na

Total Offspring (1st and 2nd broods) ↓ - ↓ ↓ ↓ na na na na na

21 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

22 [na] Not assessed due to limited number of offspring.

23 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05).

24 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05).

25 Dunnett’s test (conducted after removing the block effect), indicated that four endpoints 

26 showed significant differences between the treatment and control responses (the significance of 

27 these responses may have been overestimated due to removal of the block effect, which reduced 

28 the error for testing). Thus, responses of mysids to prochloraz generally were inconclusive 

29 (Kropp and Cullinan 2008). This lack of response may have been partly attributable to the 

30 variation in and overlap among doses to which the mysids were exposed. However, the poor 

31 feeding regime and self-starting siphon issues probably also had an important effect on the study.
Invertebrate Two-Generation ISR 31 May 28, 2013

1 Flutamide

2 Detailed results of the demonstration study with flutamide are presented in Aufderheide 

3 (2008b) and are summarized here. The major modifications from the prochloraz study are an 

4 increase of the feeding regime by about 50% more than the 2004 OECD draft guideline 

5 specifications and the discontinuation of the self-starting siphon when the F0 adults were paired.

6 A 21-day flow-through range-finding test was initiated at nominal concentrations of 

7 0 (dilution water control), 0.00098, 0.0098, 0.097, and 0.98 mg flutamide/L. None of the 

8 biological parameters observed for the range-finding test demonstrated a concentration9 dependent response to the flutamide exposure. The definitive toxicity test was performed at 

10 nominal concentrations of 0 (control), 0.125, 0.250, 0.500, 1.00, and 2.00 mg flutamide/L, as 

11 determined from the range-finding test results, with three replicate test chambers for each control 

12 and treatment level. 

13 All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 

14 stayed within the specified limits (Aufderheide 2008b). More than 70% of control animals 

15 survived the first 28 days of testing, thus meeting the guidance-specified acceptability criteria 

16 (Aufderheide 2008b). However, only 67% (instead of the guidance-specified ≥75%) of control 

17 pairs produced at least one brood. Also, the numbers of F1-generation mysids were not sufficient 

18 to allow evaluation of variables for that generation. Analytical measurements of flutamide made 

19 during the study were stable, and there was no overlap among exposure concentrations during 

20 the study. Average measured concentrations were ≥90% of the nominal values for each dose 

21 level. Three variables evaluated during the flutamide study, 7-day length and 14-day male and 

22 female length, were determined to be responsive (signal-to-noise ratios >2). 

23 Results from the flutamide demonstration test are presented in Table 4-5. Statistical 

24 analysis of the endpoints based on the Kruskal-Wallis and Dunn’s tests showed no significant 

25 differences between treatment mean responses and control mean responses. Dunnett’s test

26 (conducted after removing the block effect), indicated that four endpoints showed significant 

27 differences between the treatment and control responses (the significance of these responses may 

28 have been overestimated due to removal of the block effect, which reduced the error for testing). 

29 Three of the four treatment responses were at the highest dose level, which suggests a dose 

30 response. Thus, the responses of mysids to flutamide probably should be considered as generally 

31 inconclusive (Kropp and Cullinan 2008). The lack of response during the flutamide study may 

32 have been partly attributable to inadequate diet.

33 Ketoconazole

34 Detailed results of the demonstration study with ketoconazole are presented in Hicks 

35 (2008b) and are summarized here. The major modifications from the prochloraz and flutamide 

36 studies are a further increase of the feeding regime to three ad libitum feedings per day 

37 (i.e., about 6,500 nauplii per day). F1 mysids received a slightly larger ration. Additionally, the 

38 brine shrimp used each day were enhanced with a mixture of three commercially available 

39 supplements: Lifeline Natural Health Enrichment HUFA, AlgaMac-3000, and AlgaMac-Enhance. 

40 The self-starting siphon was also no longer used. 

41
Invertebrate Two-Generation ISR 32 May 28, 2013

1 Table 4-5. Results of the EPA demonstration study of the MTTT with flutamide.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (mg/L) 0.125 0.25 0.5 1.0 2.0 0.125 0.25 0.5 1.0 2.0

Mean Measured Concentrations (mg/L) 0.121 0.225 0.457 0.914 1.8 0.121 0.225 0.457 0.914 1.8

Day 14 survival (%) - - - - - na na na na na

Day 28 survival (%) - - - - - na na na na na

Day 7 length - - - - ↓ na na na na na

Day 7 dry weight - - - - - na na na na na

Day 14 male length - - - - ↓ na na na na na

Day 14 female length - - - - ↓ na na na na na

Day 14 dry weight - - - - - na na na na na

Time to 1st Brood - - - - - na na na na na

Time to 2nd Brood - - - - - na na na na na

Interbrood Duration - - - - ↓ na na na na na

Number 1st Brood - - - - ↓ na na na na na

Number 2nd Brood - - - - ↓ na na na na na

Total Offspring (1st and 2nd broods) - - - - ↓ na na na na na

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [na] Not assessed due to limited number of offspring.

4 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05).

5 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05).

6 A 21-day flow-through range-finding test was initiated at nominal concentrations of 

7 0 (dilution water control), 1.0, 10, 100, and 1,000 μg ketoconazole/L. Survival was the most 

8 consistent indicator of a concentration-dependent response during the range-finding test and the 

9 definitive test concentrations were set at nominal concentration range starting at approximately 

10 one-third the LC50 value (i.e., 150 μg ketoconazole/L) after 21 days of the range-finding test. 

11 The definitive toxicity test was performed at nominal concentrations of 0 (control), 0 (vehicle 

12 control; approximately 0.05 mL triethylene glycol/L), 3.3, 6.5, 13, 25, and 50 μg ketoconazole/L, 

13 as determined from the range-finding test results, with three replicate test chambers for the 

14 control, vehicle-control, and test-substance treatments. Mysids were fed ad libitum enriched 

15 brine shrimp nauplii at least three times daily, as detailed in Hicks (2008b). 

16 All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 

17 stayed within the specified limits (Hicks 2008b). In addition, F0 and F1 survival, percentage of F0 

18 and F1 control and vehicle-control female mysids that produced young, and average total young 

19 per control and vehicle-control female in the first two broods met the guidance-specified 

20 acceptability criteria, which are ≥75%, ≥70%, and 8, respectively (Hicks 2008b). Analytical 

21 measurements of ketoconazole made during the study were relatively variable, but there was no 

22 overlap among exposure concentrations during the study. Average concentrations were much 

23 less than the nominal values for each dose level. 

24 The demonstration study results for ketoconazole are presented in Table 4-6. Three F0-

25 generation variables evaluated during the ketoconazole study, 7-day length and weight and total 

26 number of young, were determined to be responsive (signal-to-noise ratios >2). One 

27 F1-generation variable, number of total young in the second brood, was determined to be 

28 responsive. Statistical analysis of the endpoints based on the Kruskal-Wallis and Dunn’s tests 

29 showed that the F1-generation number of young in the second brood for the level 3 dose 

30 (8.64 μg/L) was significantly different from that in the control treatment. Dunnett’s test

31 (conducted after removing the block effect), indicated that the F1-generation number of young in 

32 the second brood for the level 3, 4, and 5 doses were significantly different from that in the 
Invertebrate Two-Generation ISR 33 May 28, 2013

1 control treatment. Dunnett’s test also indicated that six additional endpoints showed significant 

2 differences between the treatment and control responses. Three of these, F0 number of young in 

3 the first and second broods and total number of young, showed significant differences at the 

4 highest dose level. The fourth, F0-generation 7-day weight, showed significant differences at the 

5 dose levels 4 and 5. The Dunnett’s test may have slightly over-estimated the number of 

6 significant responses; thus the dose-dependent response to ketoconazole may have occurred for 

7 five endpoints, four of which measured reproductive responses (i.e., total young 1st brood F0, 

8 total young 2nd brood F0, total young F0, and total young 2nd brood F1) (Kropp and 

9 Cullinan 2008).

10 Table 4-6. Results of the EPA demonstration study of the MTTT with ketoconazole.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) 3.3 6.5 13 25 50 3.3 6.5 13 25 50

Mean Measured Concentrations (μg/L) 2.15 4.19 8.64 17.3 33.7 2.15 4.19 8.64 17.3 33.7

Day 14 survival (%) - - - - - - - - - -

Day 28 survival (%) - - - - - - - - - -

Day 7 length - ↑ - - - - - - - -

Day 7 dry weight ↓ - ↑ ↑ ↑ - - - - -

Day 14 male length - - - - - - - - - -

Day 14 female length - - - - - - - - - -

Day 14 dry weight - - - - - - - - - -

Time to 1st Brood - - - - - - - - - -

Time to 2nd Brood - - - - - - - - - -

Interbrood Duration - - - - - - - - - -

Number 1st Brood - - - - ↓ - - - - -

Number 2nd Brood - - - - ↓ ↓ - ↓ ↓ ↓

Total Offspring (1st and 2nd broods) - - - - ↓ - - ↓ - -

11 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

12 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05).

13 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05).

14 4-tert-Octylphenol

15 Detailed results of the demonstration study with 4-tert-octylphenol are presented in Hicks

16 (2008a) and are summarized here. The major modifications from the prochloraz and flutamide 

17 studies are a further increase of the feeding regime to three ad libitum feedings per day 

18 (i.e., about 5,200 nauplii per day). F1 mysids received a slightly larger ration. Additionally, the 

19 brine shrimp used each day were enhanced with a mixture of three commercially available 

20 supplements: Lifeline Natural Health Enrichment HUFA, AlgaMac-3000, and AlgaMac-Enhance. 

21 The self-starting siphon was also no longer used. 

22 A 21-day flow-through range-finding test was initiated at nominal concentrations of 

23 0 (dilution-water control), 1.0, 10, 100, and 1,000 μg 4-tert-octylphenol/L. Survival was the most 

24 consistent indicator of a concentration-dependent response during the range-finding test, and the 

25 definitive test concentrations were set at nominal concentration range, starting at approximately 

26 one-third the 96-hour LC50 for the range-finding test (i.e., 502 μg 4-tert-octylphenol/L × 0.3, 

27 where 0.3 is the correction factor used to account for approximately 30% test-substance 

28 recoveries during the range-finding test). The definitive toxicity test was performed at nominal 

29 concentrations of 0 (control), 0 (vehicle control; approximately 0.025 mL triethylene glycol/L), 

30 3.3, 6.5, 13, 25, and 50 μg 4-tert-octylphenol/L, as determined from the range-finding test results, 

31 with three replicate test chambers for the control, vehicle-control, and test-substance treatments. 
Invertebrate Two-Generation ISR 34 May 28, 2013

1 All measured water quality parameters (temperature, dissolved oxygen pH, and salinity) 

2 stayed within the specified limits (Hicks 2008a). In addition, F0 and F1 survival, percentage of F0 

3 and F1 control and vehicle-control female mysids that produced young, and average total young 

4 per control and vehicle-control female in the first two broods met the guidance-specified 

5 acceptability criteria, which are ≥75%, ≥70%, and 8, respectively (Hicks 2008a). Analytical 

6 measurements of 4-tert-octylphenol made during the study were highly variable. There was some 

7 overlap among exposure concentrations during the study, but this occurred only between the two 

8 lowest dose levels. Average concentrations were much less than the nominal values for each 

9 dose level. 

10 The demonstration study results for 4-tert-octylphenol are presented in Table 4-7. Four 

11 F0-generation variables evaluated during the 4-tert-octylphenol study, 7-day length and weight 

12 and 14-day female length and weight, were determined to be responsive. Statistical analysis of 

13 the endpoints based on the Kruskal-Wallis and Dunn’s tests showed no significant differences 

14 between treatment mean responses and control mean responses. Dunnett’s test (conducted after 

15 removing the block effect) indicated that nine endpoints showed significant differences between 

16 the treatment and control responses (the significance of these responses may have been 

17 overestimated due to removal of the block effect, which reduced the error for testing). Thus, due 

18 to the likely overestimation of significant responses, the results from the test with

19 4-tert-octylphenol generally should be interpreted with caution (Kropp and Cullinan 2008).

20 A number of conclusions and recommendations based on the second set of 

21 demonstrations studies are provided in Kropp and Cullinan (2008) and are discussed further in 

22 Section 4.1.2.

23 Table 4-7. Results of the EPA demonstration study of the MTTT with 4-tert-Octylphenol.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) 3.3 6.5 13 25 50 3.3 6.5 13 25 50

Mean Measured Concentrations (μg/L) 2.18 4.87 9.15 17.9 41.0 2.18 4.87 9.15 17.9 41.0

Day 14 survival (%) - - - - - - - - - na

Day 28 survival (%) - - - - - - - - - na

Day 7 length - - - - ↓ - - - - na

Day 7 dry weight - - - - ↓ - - - - na

Day 14 male length - - - - ↓ - - - - na

Day 14 female length - - - - ↓ - - - - na

Day 14 dry weight (male and female) - - - - ↓ - - - - na

Time to 1st Brood - - - - - - - - - na

Time to 2nd Brood - - - - - - - - - na

Interbrood Duration - - - - - - - - - na

Number 1st Brood - - - - ↓ - - - - na

Number 2nd Brood - - - - - - - - - na

Total Offspring (1st and 2nd broods) - - - - ↓ - - - - na

Number reproducing females (1st brood) - - - - ↓ - - - - na

Number reproducing females (2nd brood) - - - - ↓ - - - - na

24 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

25 [na] Not assessed due to limited number of offspring.

26 [↑] Indicates a statistically significant increase compared to control, based on Dunnett's test (α = 0.05).

27 [↓] Indicates a statistically significant decrease compared to control, based on Dunnett's test (α = 0.05).
Invertebrate Two-Generation ISR 35 May 28, 2013

1 4.1.2 Optimization Studies Using the 2004 Draft Guideline

2 A third evaluation of the testing procedures and methods proposed by USEPA to OECD 

3 in the draft guideline for a two-generation mysid test to evaluate suspected endocrine-disrupting 

4 chemicals (OECD 2004a) was conducted through an optimization study with four chemicals: 

5 lindane (vertebrate aromatase inhibitor), prochloraz (vertebrate aromatase inhibitor), atrazine

6 (weak vertebrate androgen and estrogen receptor agonist), and perfluorodecanoic acid (interacts 

7 with thyroid homeostasis) (Kropp et al. 2009). The protocol used in the optimization study 

8 differed from the 2004 proposed guideline in eight ways: 

9 1. The Artemia nauplii were not from a reference source (sensu Léger and 

10 Sorgeloos 1984), but were from a reliable source that has been successful in previous 

11 studies

12 2. Mysids during the definitive test were fed ad libitum Artemia nauplii enriched with a 

13 mixture of commercially available products (e.g., Salt Creek HUFA Enrich, 

14 AlgaMac-3000, and AlgaMac-Enhance) 

15 3. Self-starting siphons were not used as part of the exposure system design 

16 4. The retention baskets were prepared with a slightly larger mesh, 355-μm mesh 

17 instead of the 210 to 330-μm mesh specified by the guideline 

18 5. The dry weights of the mysids were recorded to the nearest 1 μg instead of 0.1 μg 

19 6. Instead of pairing females with the largest suspected males, all mysids except for the 

20 obviously ovigerous females were isolated and sex and maturity (i.e., male, 

21 non-ovigerous female, or immature) were confirmed with the aid of a dissecting 

22 microscope

23 7. The F0-mysid exposure was initiated with at least 40 post-larval mysids per replicate, 

24 where each of the two retention baskets contained at least 20 post-larval mysids 

25 8. The number of F0-mysid brood pairs was targeted to be 10. 

26

27 Items 1 through 6 were previously utilized in the demonstration studies with 

28 ketoconazole and 4-tert-octylphenol and were shown to be test design improvements. Items 7 

29 and 8 were not used during the demonstration studies but were incorporated into the optimization 

30 study to facilitate initiation of the F1-mysid exposure.

31 Lindane

32 Detailed results of the optimization study with lindane are presented in Hicks (2009a) and 

33 summarized here.

34 A 21-day, flow-through, range-finding test was conducted at nominal concentrations of 

35 0 (dilution-water control), 0.19, 0.38, 0.75, 1.5, and 3.0 μg a.i./L. The estimated LC50 values for 

36 days 4, 7, 14, and 21 were 2.2, 1.6, 1.2, and 1.0 μg a.i./L. Survival was the most consistent 

37 indicator of a concentration-dependent response during the range-finding test. The highest 

38 definitive test concentration selected for the lindane definitive exposure was one fifth the 

39 96-hour LC50 for the range-finding test (i.e., 0.4 μg/L). Nominal concentrations were 0 (control), 

40 0.025, 0.05, 0.10, 0.20, and 0.4 μg a.i./L. Analytical measurements of lindane made during the 

41 study decreased during the course of the study (measured concentrations were 0.0182, 0.0375, 

42 0.0808, 0.159, and 0.327 μg/L). Reasons for this decline in concentrations within dose levels 

43 were not identified. All test acceptability criteria were met. 
Invertebrate Two-Generation ISR 36 May 28, 2013

1 The optimization study results for lindane are presented in Table 4-8. All of the variables 

2 evaluated during the lindane study were nonresponsive (signal-to-noise ratios <2). Statistical 

3 analyses of the endpoints based on the Kruskal-Wallis and Dunn’s tests showed that there were 

4 few significant differences between treatment mean responses and control mean responses: the 

5 F0 (F0 14-day Male Length) was less than controls at 0.4 μg/L, the F1 generation (F1 interbrood 

6 duration) was longer than controls at 0.4 μg/L, and the total number of young per F1 females was 

7 greater than controls at 0.20 μg/L.

8 Table 4-8. Results of the EPA optimization study of the MTTT with lindane.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (ng a.i./L) 25 50 100 200 400 25 50 100 200 400

Mean Measured Concentrations (ng a.i./L) 18.2 37.5 80.8 159 327 18.2 37.5 80.8 159 327

Day 13 survival - - - - - - - - - -

Day 29 survival - - - - - - - - - -

Day 43 survival nr nr nr nr nr - - - - -

Day 7 length - - - - - - - - - -

Day 7 dry weight - - - - - - - - - -

Day 14 male length - - - - ↓ - - - - -

Day 14 female length - - - - - - - - - -

Day 14 dry weight (male and female) - - - - - - - - - -

Time to 1st Brood - - - - - - - - - -

Time to 2nd Brood - - - - - - - - - -

Interbrood Duration - - - - - - - - - ↑

Number 1st Brood - - - - - - - - - -

Number 2nd Brood - - - - - - - - - -

Total Offspring (1st and 2nd broods) - - - - - - - - ↑ -

Number reproducing females (1st brood) - - - - - - - - - -

Number reproducing females (2nd brood) - - - - - - - - - -

9 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

10 [nr] Not reported for the F0 generation.

11 [↑] Indicates a statistically significant increase compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

12 [↓] Indicates a statistically significant decrease compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

13 Prochloraz

14 Detailed results of the optimization study with prochloraz are presented in Hicks (2009c)

15 and are summarized here.

16 The highest definitive test concentration selected for the prochloraz exposure was based 

17 on the results of the range-finding test and definitive exposure conducted as part of the 

18 demonstration study (see Section 4.1.1). The highest prochloraz concentration tested during the 

19 demonstration study was about one-third the 14-day LC50 (100 μg/L), but this was increased to 

20 200 μg/L for the demonstration study. Analytical measurements of prochloraz made during the 

21 study generally were stable and there was no overlap among exposure concentrations during the 

22 study. Average measured concentrations were ≥90% of the nominal values for each dose level. 

23 All test acceptability criteria were met. 

24 Results from the prochloraz optimization test are presented in Table 4-9. Two 

25 F0-generation variables evaluated during the prochloraz study, number of days to first and second 

26 brood, were responsive (signal-to-noise ratios >2). Four F1-generation variables, number of days 

27 to the first and second brood, interbrood duration, and total young in the second brood, were 

28 determined to be responsive. Statistical analyses of the endpoints was based on the 

29 Kruskal-Wallis and Dunn’s tests .
Invertebrate Two-Generation ISR 37 May 28, 2013

1 Table 4-9. Results of the EPA optimization study of the MTTT with prochloraz.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg a.i./L) 25 50 100 200 400 25 50 100 200 400

Mean Measured Concentrations (μg a.i./L) 12.5 25.3 46.2 94.2 188 12.5 25.3 46.2 94.2 188

Day 13 survival - - - - - - - - - -

Day 30 survival - - - - - - - - - -

Day 45 survival nr nr nr nr nr - - - - -

Day 7 length - - - - - - - - - -

Day 7 dry weight - - - - - - - - - -

Day 14 male length - - - - - - - - - -

Day 14 female length - - - - - - - - - -

Day 14 dry weight (male and female) - - - - - - - - - -

Time to 1st Brood - - - - ↑ - - - - ↑

Time to 2nd Brood - - - - ↑ - - - - ↑

Interbrood Duration - - - - - - - - - ↑

Number 1st Brood - - - - - - - - - -

Number 2nd Brood - - - - - - - - - ↓

Total Offspring (1st and 2nd broods) - - - - - - - - - -

Number reproducing females (1st brood) - - - - - - - - - -

Number reproducing females (2nd brood) - - - - - - - - - -

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [nr] Not reported for the F0 generation.

4 [↑] Indicates a statistically significant increase compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

5 [↓] Indicates a statistically significant decrease compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

6 Atrazine

7 Detailed results of the optimization study with atrazine are presented in Hicks (2009b)

8 and are summarized here.

9 The highest definitive test concentration selected for the atrazine definitive exposure was 

10 one-fifth the 96-hour LC50 from a range-finding test. Survival was the most consistent indicator 

11 of a concentration-dependent response during the atrazine range-finding test, but the number of 

12 young per female also appeared to be a consistent indicator of a concentration-dependent 

13 response. Analytical measurements of atrazine made during the study were stable and there was 

14 no overlap among exposure concentrations during the study. Average measured concentrations 

15 were ≥90% of the nominal values for each dose level. All test acceptability criteria were met. 

16 Results from the atrazine optimization test are presented in Table 4-10. One variable 

17 evaluated during the atrazine study, F1 total number of young, was responsive (signal-to-noise 

18 ratios >2). Statistical analyses of the endpoints based on the Kruskal-Wallis and Dunn’s tests 

19 showed that there were no significant differences between treatment mean responses and control 

20 mean responses.

21 Perfluorodecanoic acid (PFDA)

22 Detailed results of the optimization study with PFDA are presented in Hicks (2009a) and 

23 are summarized here.

24 The highest concentration selected for the PFDA definitive exposure was based on these 

25 range-finding test results: 1) the effects observed on number of young per female where a 61% 

26 reduction occurred at the 0.65 μg/L nominal treatment and a 36% reduction at the 0.10 μg/L 

27 nominal treatment; and 2) the measured concentrations were 16% of nominal in the 0.10 μg/L 

28 treatment and 51% of nominal in the 1.3 μg/L treatment. The highest PFDA definitive test 
Invertebrate Two-Generation ISR 38 May 28, 2013

1 nominal concentration selected by interpolating from the range-finding test measured 

2 concentrations was approximately one-hundredth the 96-hour LC50, or 50 μg/L, with four lower 

3 concentrations arranged in a geometric series and a separation factor of approximately 2.5 to 

4 bracket any biological effect. Analytical measurements of PFDA made during the study were 

5 highly variable (CVs > 17%). There was no overlap among exposure concentrations during the 

6 study, but the average concentrations of all dose levels were 56% to 67% of the nominal values. 

7 All test acceptability criteria were met. 

8 Table 4-10. Results of the EPA optimization study of the MTTT with atrazine.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg a.i./L) 13 25 50 100 200 13 25 50 100 200

Mean Measured Concentrations (μg a.i./L) 11.7 22.7 45.1 93.6 193 11.7 22.7 45.1 93.6 193

Day 13 survival - - - - - - - - - -

Day 30 survival - - - - - - - - - -

Day 43 survival nr nr nr nr nr - - - - -

Day 7 length - - - - - - - - - -

Day 7 dry weight - - - - - - - - - -

Day 14 male length - - - - - - - - - -

Day 14 female length - - - - - - - - - -

Day 14 dry weight (male and female) - - - - - - - - - -

Time to 1st Brood - - - - - - - - - -

Time to 2nd Brood - - - - - - - - - -

Interbrood Duration - - - - - - - - - -

Number 1st Brood - - - - - - - - - -

Number 2nd Brood - - - - - - - - - -

Total Offspring (1st and 2nd broods) - - - - - - - - - -

Number reproducing females (1st brood) - - - - - - - - - -

Number reproducing females (2nd brood) - - - - - - - - - -

9 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

10 [nr] Not reported for the F0 generation.

11 [↑] Indicates a statistically significant increase compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

12 [↓] Indicates a statistically significant decrease compared to control, based on Kruskal-Wallis/Dunn's test (α = 

13 0.05).

14 Results from the PFDA optimization test are presented in Table 4-11. All of the variables 

15 evaluated during the PFDA study were nonresponsive (signal-to-noise ratios <2). Statistical 

16 analyses of the endpoints [conducted by Kropp and Cullinan (2009)] based on the 

17 Kruskal-Wallis and Dunn’s tests (of grouped treatment mean responses and control mean 

18 responses) showed only two significant differences (F0 14-day Female Weight and F0 Days to 

19 First Brood). No statistical differences were noted among individual treatment groups and 

20 control groups for any endpoint.

21 4.1.3 Lessons Learned from Demonstration and Optimization Studies Using the 2004 

22 Draft MTTT Guideline

23 A number of recommended modifications to the 2004 draft MTTT guideline were 

24 suggested by Kropp and Cullinan (2008) and Kropp et al. (2009) based on the results of the 

25 demonstration and optimization studies discussed in the previous sections, and they are repeated 

26 below. These recommendations were considered when modifying the 2004 proposed OECD 

27 protocol and draft the recommended MTTT guideline presented in Section 3.1.
Invertebrate Two-Generation ISR 39 May 28, 2013

1 Table 4-11. Results of the EPA optimization study of the MTTT with perfluorodecanoic acid.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg a.i./L) 1.3 3.2 8 20 50 1.3 3.2 8 20 50

Mean Measured Concentrations (μg a.i./L) 0.871 1.94 5.13 11.2 33.3 0.871 1.94 5.13 11.2 33.3

Day 13 survival - - - - - - - - - -

Day 30 survival - - - - - - - - - -

Day 43 survival nr nr nr nr nr - - - - -

Day 7 length - - - - - - - - - -

Day 7 dry weight - - - - - - - - - -

Day 14 male length - - - - - - - - - -

Day 14 female length - - - - - - - - - -

Day 14 dry weight (male and female) -* -* -* -* -* - - - - -

Time to 1st Brood -* -* -* -* -* - - - - -

Time to 2nd Brood - - - - - - - - - -

Interbrood Duration - - - - - - - - - -

Number 1st Brood - - - - - - - - - -

Number 2nd Brood - - - - - - - - - -

Total Offspring (1st and 2nd broods) - - - - - - - - - -

Number reproducing females (1st brood) - - - - - - - - - -

Number reproducing females (2nd brood) - - - - - - - - - -

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [nr] Not reported for the F0 generation.

4 [*] Statistical analyses of these endpoints based on the Kruskal-Wallis and Dunn’s tests (α = 0.05) showed that 

5 there were significant differences between all grouped treatment mean responses and control mean responses 

6 (F0 14-day Female Weight and F0 Days to First Brood).

7 [↑] Indicates a statistically significant increase compared to control, based on Kruskal-Wallis/Dunn's test (α = 0.05).

8 [↓] Indicates a statistically significant decrease compared to control, based on Kruskal-Wallis/Dunn's test (α = 

9 0.05).

10

11 Study design and statistical guidance

12 Statistical guidance provided by the 2004 draft MTTT guideline is minimal, essentially 

13 only describing use of ANOVA or Kruskal-Wallis tests and recommending that the laboratory 

14 include a statistician in the process. There is no guidance about the appropriate experimental 

15 design (randomized versus blocked) that should be used. The correct statistical comparisons 

16 when a vehicle control is required are also not addressed. The lack of guidance could cause 

17 confusion over the correct study design, the use of one-tailed or two-tailed tests, and whether the 

18 treatment response should be compared with the dilution-water or vehicle controls for tests that 

19 employed both.

20 The design of the exposure experiment should be a completely randomized design unless 

21 blocking is required. In the case of the latter, blocks should be characterized by the 

22 environmental qualities that make them different and relevant to the experiment. Blocking makes 

23 the analysis of the data more difficult, especially when a nonparametric analysis is required, and 

24 provides a potentially underestimated variance for testing when the blocks are artificial. A 

25 completely randomized design allows statistical comparisons to be run easily, whereas a block 

26 design requires the fitting of a generalized linear model and more statistical expertise. In all cases, 

27 it is important to recognize that the replicates are the treatment tanks, not the individual retention 

28 baskets within each tank. Thus, the analyses should be conducted on the mean responses of the 

29 treatment tanks.

30 Either the Kruskal-Wallis, followed by the Dunn’s test, or the Dunnett’s test is 

31 appropriate to test the treatment responses under this proposed protocol. One advantage of using 

32 the Kruskal-Wallis is that potential non-monotonic effects can be evaluated. When a vehicle 
Invertebrate Two-Generation ISR 40 May 28, 2013

1 control is used, it is appropriate to compare the vehicle-control response to the dilution-water 

2 control response. However, the dilution-water control is used to evaluate the acceptability of the 

3 test and is not used to evaluate the treatment responses. The treatment responses should be 

4 evaluated by comparing them to the vehicle-control responses.

5 Self-starting Siphon

6 All demonstration and optimization studies, for which the use of the self-starting siphon 

7 was discontinued, were completed successfully with no deterioration of water quality. Thus, a 

8 simple overflow drain or similar system that reduces the likelihood of impingement is 

9 recommended as a modification of the proposed guideline. If reference to a self-starting siphon 

10 remains in the guideline, a note of caution that the siphon likely will increase impingement 

11 should be added.

12 Feeding

13 The demonstration study comparisons of the control responses under the different feeding 

14 regimes strongly suggested that mysids should be fed brine shrimp nauplii ad libitum (Kropp and 

15 Cullinan 2008). The comparisons further suggested that the brine shrimp should be enhanced by 

16 exposing them to supplements that provide additional highly unsaturated fatty acids (HUFA), 

17 especially docosahexaenoic acid (DHA), and eicosapentaenoic acid (EpA), both of which are 

18 relatively poor in natural strains of Artemia (Hanaee et al. 2005). Hanaee et al. (2005) found that 

19 enriching Artemia cysts increased the HUFA content about 1.6 times and the DHA and EpA 

20 content by 1.2 and 84 times, respectively. The four demonstration studies described earlier 

21 included a diet of Artemia nauplii that had been enriched with Lifeline Natural Health 

22 Enrichment HUFA, AlgaMac-3000, and AlgaMac-Enhance. The addition of these or similar 

23 supplements to the brine shrimp nauplii is a simple and cost-effective way to help ensure that the 

24 mysid diet is not a confounding factor during the evaluation of potential EDCs.

25 Initial Test Population Size

26 The size of the F0-mysid population used at test initiation during the optimization studies 

27 was increased versus that specified in the proposed guideline and used during the demonstration 

28 studies. The number of F0 mysids used to initiate the optimization study exposures was 20 per 

29 retention basket, 5 more than the guideline-specified number. The targeted number of F0 mysid 

30 pairs for the demonstration study exposures was 10 per replicate, three pairs more than the 

31 guidelines-specified number. The larger initial F0 population increased the population of the F0

32 mysid generation available for pairing. The increased number of F0-mysid pairs improves the

33 probability that a larger population of F1 mysid would be available to initiate the F1-mysid 

34 reproduction and growth retention baskets within a period of two days. Therefore, F1mysids in 

35 the growth retention baskets were available to supplement the F1-mysid pairing procedure, if 

36 necessary, because the age of both sets of mysids was from 12- to 16-days old. 

37 It is recommended that the population size of the F0 mysids be increased in each replicate 

38 to 20 and the target number of F0 mysid pairs to 10. If the initial F0 population is not increased, or 

39 the increased population is not sufficient during some tests (i.e., reproduction is reduced in 

40 response to the chemical being studied), the population of F1 mysids available for the pairing 

41 procedure may not be sufficient to achieve the targeted number of seven F1-mysid pairs.
Invertebrate Two-Generation ISR 41 May 28, 2013

1 Endpoint Clarification

2 Many endpoints are described in the 2004 draft MTTT guideline only by a simple 

3 sentence or phrase and may require additional information to allow consistent determinations to 

4 be made across testing laboratories. A primary example concerns the treatment of mortality that 

5 may arise during the testing, but that might not be exposure-related. For example, guidance could 

6 be included that directs a laboratory not to include mortality from impingement in the survival 

7 endpoint. Other examples are described in the following subsections.

8 Dry Weight

9 The 2004 draft MTTT guideline provides considerable detail directing how the weight 

10 endpoint is to be obtained, which is appropriate. However, it is recommended that the 

11 requirement to measure weight to the “nearest 0.1 μg” be changed to the “nearest 1 μg.” The 

12 guideline instructions for dry weight measurements should be expanded to specify that weights 

13 of males, females, and immature individuals should be measured and recorded. Data analyses 

14 should then be conducted for each gender.

15 Dead Young

16 Direction is needed to specify how dead young observed in broods produced by F0 and F1

17 mysids should be treated. The guideline should specify that the occurrence and developmental 

18 state of dead young could be determined and recorded, but should not be included in counts of 

19 young per brood even if well-developed.

20 Study Day

21 The Study Day selected to track ALL F1-brood pairs during the demonstration studies 

22 was the age of the youngest mysid on the day following pairing, which may have been 1 to 2 

23 days younger than the oldest. However, during the optimization studies, the tracking of ALL 

24 F1-brood pairs used a continuation of the day following initiation of the test, where study day 0 

25 was test initiation (i.e., start of F0-mysid exposure). Criteria for the selection of the first study 

26 day for F1 mysids following pairing need to be considered and defined in future guidance 

27 documents. The tracking of each F1-mysid brood cup individually would add adversely to the 

28 complexity of the study and data evaluation/interpretation.

29 F0 and F1Exposure Duration

30 The 2004 draft MTTT guideline does not clearly define the F0 and F1 exposure durations, 

31 or state that a brood pair can be terminated at the time of second brood release. If the intention of 

32 the test design is to collect only data up to and including the day of second brood release by a 

33 given brood pair, the guideline should clearly state that the collection of survival and 

34 reproduction data beyond the day of second brood release is not required. Therefore, an adult 

35 pair could be terminated at the time of second brood release or at 7 days after the median day of 

36 second brood release in the control treatment. The guideline also should define how control37 treatment mysids without a mate (i.e., one of the two adults has died) are treated for calculation 

38 of the median day of second brood release and when treatment-exposed mysids without a mate 

39 and not producing a second brood are terminated.

40 Total Young Reproduction Data

41 The 2004 draft MTTT guideline describes the total young endpoint as the “Number of 

42 young per female in each of the first two broods (F0, F1)” but provides no further guidance about 
Invertebrate Two-Generation ISR 42 May 28, 2013

1 how this number is calculated. Throughout the guidance document, number of young is 

2 expressed as young per female, but in this case it is not. During the optimization study, total 

3 young was evaluated as number per female, but the total was calculated two ways. For the first 

4 calculation, the reproduction data for all females were treated equally (i.e., reproduction data for 

5 females that die before producing a second brood or females not producing a second brood will 

6 not be omitted). For the second, only the reproduction data for females that produced a second 

7 brood were included. Both sets of calculations were evaluated statistically and the results 

8 occasionally differed. The guideline should clarify which approach meets the goals of the testing

9 so that the endpoint will be evaluated consistently by various testing laboratories.

10 Time to Maturation

11 The 2004 draft MTTT guideline lists Time to maturation, appearance of marsupial pouch 

12 (F0, F1)” as one of the primary data types to be collected. During the optimization studies, these 

13 data were collected and reported, but they were not evaluated statistically. This endpoint is 

14 somewhat redundant with the time to first brood, which probably is a better indication of any 

15 delay in reproduction caused by exposure to a test chemical. It is recommend that Time to 

16 maturation be dropped as an endpoint.

17 F0 and F1 Mysid Survival

18 Mysid survival was evaluated at multiple time-points during the demonstration and 

19 optimization studies. The time-points were days 14 (or the day of pairing) and 28 (or test 

20 termination) for both generations. However, the 2004 draft MTTT guideline does not clearly 

21 identify how survival should be evaluated, which could cause confusion about the point(s) at 

22 which survival is to be evaluated. Several factors may complicate interpretation:

23 The number of mysids surviving at pairing day may be fewer than the number surviving 

24 at termination because mysids from the growth basket have been used to achieve the desired 

25 number of pairs (e.g., 11 mysids were present in a reproduction basket on day 13 but after 

26 pairing there were 14 mysids present because three mysids from the growth basket were used to 

27 obtain the targeted number of pairs). In this case, evaluation of survival of all replicates and 

28 treatments at the earliest day of pairing and again at termination of the mysid generation allows

29 survival to be evaluated at two time points. This method accounts for differences in mysid 

30 populations at the start of each survival period.

31 Pairing of replicate(s) or treatment(s) mysids could occur on different days, particularly 

32 for F1 mysids. For example, during a study, 7 groups may be paired 13 days after initiation, 

33 whereas three groups may be paired 14 days after initiation. During the demonstration and 

34 optimization studies, survival on the pairing day achieved by most mysids, day 13 for the 

35 example provided, was evaluated.

36 If the proposed guideline is revised so that pairs can be terminated as soon as they release 

37 their 2nd brood, the guideline should specify whether survival in this case should be treated as 

38 survival at termination or as survival at a particular time point (i.e., Day 28).

39 Reproduction Output as Number of Offspring/Female/Day

40 The 2004 draft MTTT guideline states that “Daily assessment of survival throughout the 

41 test is necessary to provide a basis for expression and interpretation of reproductive output, that 

42 is, number of offspring/female/d.” The value of expressing and interpreting reproductive output 

43 as ”daily number of young per female” is not clear. ”Daily” reproductive output does not truly 
Invertebrate Two-Generation ISR 43 May 28, 2013

1 reflect mysid reproduction cycles and therefore may not be appropriate. An appropriate measure 

2 of reproductive output is already obtained by scoring and evaluating the number of young per 

3 female in each of the first two broods. Therefore, the requirement for expression and 

4 interpretation of reproductive output as number of offspring/female/day could be eliminated.

5 Dose Determination and Power

6 The power of a test is defined as the probability of correctly rejecting the null hypothesis 

7 (H0). Achieved statistical power is a function of the coefficient of variation (CV) of the 

8 measured response, the number of replicates, and the percentage difference in the mean 

9 responses observed. In general, sets of observations with large CVs have little power to detect 

10 differences between means that differ by less than 20%. An increase in the number of replicates 

11 used during a test is required to either increase the power of the test for a given level of detection 

12 or to decrease the difference between means that is to be detected for a given power 

13 (Snedecor and Cochran 1980). The CVs observed during the optimization studies for most of the 

14 control data were less than 20% (Kropp et al. 2009). If a 10% difference between the control and 

15 treatment means had occurred, the F0 and F1 measures of length and time to each brood would 

16 have had a power greater than 80% for each chemical. If a 20% difference between the control 

17 and treatment means would have occurred, it would have provided at least 80% power for the 

18 additional measures of the number of young for the F0 and F1 generations for each chemical. 

19 However, most of the percentage differences that were observed were less than 10% 

20 (Kropp et al. 2009). The number of replicates can be increased to detect smaller percentage 

21 differences between the control and treatment means. However, these smaller differences may 

22 not be biologically relevant. Also, an increase in the number of replicates increases the cost of 

23 the laboratory element of the study. The intent of the study should balance the power with the 

24 ability to detect biologically relevant differences. The chemical dosing should be selected to 

25 achieve relevant treatment-related differences.

26 The general lack of treatment-related differences during the optimization study could 

27 have resulted from selected doses that were too low to bracket any biological effect that may 

28 have been suggested by the range-finding tests. These results indicate the difficulties 

29 encountered at selecting definitive test concentrations for a two-generation test when the 

30 objective is to establish a no-observed and lowest-observed effect concentration for both 

31 generations. The maximum chemical concentrations used in these studies were calculated as 

32 percentages of the LC50 (depending on the chemical, one-hundredth to one-third of the LC50) 

33 estimated in the range-finding test. However, during the definitive exposures, the minimum 

34 survival for both generations across all chemicals was 88%. During the range-finding tests for 

35 the demonstration study, only the highest doses had an effect greater than 50% mortality. Higher 

36 chemical concentrations during this study could have created larger percentage differences with 

37 only a slight increase in mortality. The usefulness of a 96-hour LC50 percentage (i.e., one-fifth) 

38 to select the highest definitive exposure concentration is questionable and may need to be further 

39 supported by a longer-term range-finding test (e.g., 14 to 28 days). Setting a level of acceptable 

40 mortality after 14 days of exposure at 20%, for example, and estimating the associated LC20 as 

41 the maximum dose in the definitive test may be a better procedure for determining the exposure 

42 series. This would not guarantee but should increase the likelihood of observing greater, and 

43 biologically relevant, percentage differences between means in the two-generation testing

44 scenarios.
Invertebrate Two-Generation ISR 44 May 28, 2013

1 4.2 Proposed Harpacticoid Copepod Development and Reproduction Test 

2 Protocol Performance

3 A copepod life-cycle toxicity test protocol was previously established for Amphiascus 

4 tenuiremis by the American Society for Testing and Materials (ASTM E2317-04). In addition, a

5 draft guideline was proposed to OECD in 2004 to evaluate effects of chemicals on A. tenuiremis 

6 (OECD 2007). This draft guideline was used in two rounds of OECD validation studies as 

7 described below. In addition, numerous articles have been published based on the draft guideline 

8 as described below. Given that A. tenuiremis was the only species that was retained for the 

9 second OECD interlaboratory validation, further details regarding the performance of lifecycle 

10 tests with other calanoid and harpaticoid species are not summarized here, but have been 

11 described in detail elsewhere (e.g., Gourmelon and Ahtiainen 2007; Kusk and 

12 Wollenberger 2007; OECD 2006; OECD 2007; OECD 2011 and references therein).

13 4.2.1 Demonstration and Optimization Studies Using Proposed HCDRT Protocol

14 Fipronil

15 A series of studies were conducted using the A. tenuiremis life-cycle test with the GABA16 inhibitor insecticide fipronil (Cary et al. 2004; Chandler et al. 2004a,b). Chandler et al. (2004a) 

17 reported on the development and demonstration of the microplate-based life-cycle developmental 

18 and reproduction assay. In the demonstration test, nominal concentrations of fipronil (0, 0.22, 

19 0.36, and 0.60 μg/L) were prepared in seawater and copepods were exposed throughout the 

20 embryo, nauplius, copepodite, and adult stages (16 to 17 days egg to egg). Measured 

21 concentrations (0.16, 0.22, and 0.42 μg/L) were within 68% of nominal and water quality 

22 parameters met standard test guidelines. 

23 Results from the fipronil study are presented in Table 4-12. Copepod survival was >90% 

24 during the test, and concentrations of ≥0.22 μg/L significantly delayed development from 

25 copepodite to adult. Reduced egg extrusion and reproductive failure were significantly at 

26 ≥0.22 μg/L fipronil. A three-generation population growth model also indicated a decline in 

27 population size relative to controls at 0.16 μg/L (Chandler et al. 2004a). 

28 Table 4-12. Results of the Chandler et al. demonstration study of the HCDRT with fipronil.

Endpoint Test Concentration

Nominal Concentrations (μg/L) 0.22 0.36 0.60

Mean Measured Concentrations (μg/L) 0.16 0.22 0.42

Survival (Day 12) - - -

Survival (Day 21) - - -

Development to copepodite (days) - - -

Development to adult (days) - ↑ ↑

Time to egg extrusion after mating ↑ ↑ -

Incidence of egg production (% gravid females) - ↓ ↓

Population growth rate ↓ ↓ ↓

29 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

30 [↑] Indicates a statistically significant increase compared to control.

31 [↓] Indicates a statistically significant decrease compared to control.

32 This study demonstrated that A. tenuiremis could be cultured and reared and development 

33 and reproduction could be assessed using the life-cycle assay. Additional studies successfully

34 applied this life-cycle test to evaluate population growth following exposure to fipronil and its 
Invertebrate Two-Generation ISR 45 May 28, 2013

1 degradation products (Chandler et al. 2004b) and to evaluate sex-linked reproductive effects 

2 (Cary et al. 2004). In another follow-up study, this life-cycle test was used to demonstrate the 

3 effect of dissolved organic matter (DOM) on the toxicity of fipronil to A. tenuiremis (Bejarano et 

4 al. 2005). In this study, the presence of DOM enhanced the acute toxicity of fipronil and 

5 mitigated the acute and chronic effects of chlorothalonil and chlorpyrifos. Thus, the authors 

6 suggested that seawater-only exposure (without DOM) may overestimate acute and chronic 

7 pesticide effects under realistic conditions.

8 Atrazine

9 The A. tenuiremis life-cycle test (2.5 generations) was conducted with nominal 

10 concentrations of 0, 2.5, 25, and 250 μg/L of the triazine herbicide atrazine (Bejarano and 

11 Chandler 2003). Measured concentrations were <0.1, 3.5, 30.3, and 246.6 μg/L and average 

12 water quality parameters were pH = 8.25, salinity 30‰, and dissolved oxygen = 97%. Survival 

13 rates for all treatments was ≥95%. 

14 Results from the atrazine study are presented in Table 4-13. No significant effects were 

15 observed in the F0 generation on development, sex ratios, hatching success, reproduction, and 

16 malformation following atrazine exposure. The number of viable offspring per female (F0) was 

17 significantly reduced at 250 μg/L and estimated population production (using the Leslie matrix 

18 model) was significantly reduced at 25 and 250 μg/L. No significant effects were observed for F1

19 survival, development, sex ratio, or hatching success. However, the number of viable offspring, 

20 malformations, and population production were affected at all test concentrations. In addition, 

21 reproductive failure (F1) was observed at 25 and 250 μg/L. The results of the study provided a 

22 successful demonstration of the copepod life-cycle test and allowed for population growth 

23 modeling. 

24 Table 4-13. Results of the Bejarano and Chandler demonstration study of the HCDRT with

25 atrazine.

Endpoint F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) 2.5 25 250 2.5 25 250

Mean Measured Concentrations (μg/L) 3.5 30.3 246.6 3.5 30.3 246.6

Survival to adulthood - - - - - -

Developmental delays - - - - - -

Sex ratio - - - - - -

Developmental time - - - - - -

Hatching success - - - - - -

Viable offspring per female - - ↓ ↓ ↓ ↓

Reproductive failure - - - - ↑ ↑

Copepod malformations - - - ↑ ↑ ↑

Estimated population production - ↓ ↓ ↓ ↓ ↓

26 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

27 [↑] Indicates a statistically significant increase compared to control.

28 [↓] Indicates a statistically significant decrease compared to control.

29 3,5-dichlorophenol (DCP)

30 The OECD (2007) performed a prevalidation study following draft test guidelines using 

31 two harpacticoid copepods (N. spinipes and A. tenuiremis) and one calanoid copepod (A. tonsa). 

32 Multiple laboratories conducted the developmental and reproduction test using 3,5-DCP to 

33 evaluate the performance of the test methods and sensitivity of the test species. Nominal 

34 3,5-DCP concentrations were 0, 6.6, 20, 60, 180, and 540 μg/L. 
Invertebrate Two-Generation ISR 46 May 28, 2013

1 Results from the DCP study are presented in Table 4-14. In the developmental phase of 

2 the harpacticoid life-cycle test, mortality, development rate, and sex ratio were assessed. The 

3 developmental data were consistent among the laboratories, indicating lowest observed effective

4 concentrations (LOECs) between 6.6 and 20 μg/L. The reproduction data were also consistent for 

5 two of the three endpoints for the harpacticoid test with LOECs between 6.6 and 540 μg/L. 

6 Mortality, development, and reproduction were also monitored for the calanoid copepod tests. 

7 Results for development and reproduction endpoints were variable (LOECs ranging from 6.6 to 

8 540 μg/L) for the calanoid test and two labs found developmental endpoints to be most sensitive 

9 while the third lab found reproduction to be most sensitive. One of the labs did not meet the 

10 acceptability criteria for calanoid developmental data, limiting the comparability of the data.

11 Table 4-14. Results of the OECD demonstration study of the HCDRT with 3,5-DCP.

Endpoint Lab Test Concentration

Nominal Concentrations (μg/L) 6.6 20 60 180 540

Nitrocra spinipes (Harpacticoid copepod)

Time (days) to copepodite ITM - - ↑ ↑ ↑

IVL - ↑ ↑ ↑ ↑

Toxicon - ↑ ↑ - -

Time (days) to adult ITM ↑ ↑ - ↑ ↑

IVL - - ↑ ↑ -

Toxicon - - - - ↑

Sex ratio* ITM - - - - -

Toxicon - - - - -

Fertilization success (proportion with two clutches)* ITM ↑ - - ↓ ↓

Toxicon - - - ↓ ↓

Total viable offspring* ITM - - - - -

Toxicon - - - - -

Time to first clutch* ITM - - ↑ ↑ -

Toxicon - - ↑ ↑ -

Time between first and second clutch* ITM - - - - -

Toxicon - - - - -

Amphiascus tenuiremis (Harpacticoid copepod)

Time (days) to copepodite NOAA ↑ - - - ↑

BEI ↑ - ↑ ↑ ↑

Time (days) to adult NOAA - - - - ↑

BEI - - ↑ ↑ -

Sex ratio NOAA - - - - -

BEI - - - - -

Fertilization success (proportion with two clutches)* NOAA - - - - ↓

Total viable offspring* NOAA - - - - -

Time to first clutch* NOAA - - ↑ ↑ -

Time between first and second clutch* NOAA - - - - -

Acartia tonsa (Calanoid copepod)

Larval development ratio (F0) DHI - - - - ↓

DTU ↓ - - - ↓

OPUS ↓ ↓ ↓ ↓ ↓

Larval development ratio (F1) DHI - - - ↓ ↓

DTU - - - - ↓

OPUS - ↓ ↓ - -

Mean length* DHI - - - ↑ -

DTU - - - - ↓

Mean egg production DHI ↑ ↑ - ↑ -

DTU ↓ ↓ ↓ ↓ ↓

OPUS - - - - ↓

12 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

13 [*] Endpoint not evaluated by one of the labs (IVL, BEI, or OPUS)

14 [↑] Indicates a statistically significant increase compared to control.

15 [↓] Indicates a statistically significant decrease compared to control.
Invertebrate Two-Generation ISR 47 May 28, 2013

1 Following this series of tests, OECD (2007) provided several recommendations for future 

2 development of copepod life-cycle tests. The use of A. tenuiremis was recommended for the full 

3 life-cycle test due to its shorter testing time and better reproductive output and success, which 

4 increases study power. The longer test duration necessary for N. spinipes and large 

5 interlaboratory variability for A. tonsa suggested that these species would be less practical 

6 alternatives. In addition, the labs experienced difficulty with maintaining 3,5-DCP 

7 concentrations during the test, and thus recommended exploring other reference substances.

8 4.2.2 Lessons Learned from Demonstration and Optimization Studies Using Proposed 

9 HCDRT Protocol

10 Several observations were provided following the demonstration and optimization studies 

11 using multiple species of copepods (OECD 2007; Kusk and Wollenberger 2007). These 

12 recommendations ultimately led to the decision to proceed with OECD inter-laboratory 

13 validation testing with only A. tenuiremis. There were several key differences among the 

14 copepod species tested that were recognized during the demonstration phases. The test time to 

15 run the harpacticoid full lifecycle test using N. spinipes was over 40 days, while the test time for 

16 A. tenuiremis was approximately 25 days. This indicated that A. tenuiremis is more appropriate 

17 to use for full life cycle testing to reduce duration and costs (OECD 2007). The number of 

18 replicates in the harpacticoid tests is high (at least 48 specimens per concentration), which allows 

19 for strong statistical power (Kusk and Wollenberger 2007). In addition, the harpacticoid tests 

20 focus on reproduction and development, while the calanoid test focuses more on development of 

21 early life stages (Kusk and Wollenberger 2007). Demonstration tests also indicated that calanoid 

22 tests were more variable than harpacticoid tests. Finally, a standard ASTM method was already 

23 available to draw upon for the A. tenuiremis (ASTM 2004). Based on these factors, A. tenuiremis 

24 was recommend for use in the development of a full life-cycle test (OECD 2007). OECD (2011) 

25 completed further interlaboratory studies with A. tenuiremis, which are summarized in Section 5.

26
Invertebrate Two-Generation ISR 48 May 28, 2013

1 5 INTERLABORATORY EVALUATION OF MYSID AND COPEPOD TEST 

2 METHODS

3 Interlaboratory evaluations of the MTTT and the HCDRT were conducted to: (1) evaluate 

4 the value added of the MTTT over the existing mysid life cycle test; (2) compare the sensitivity 

5 of the mysid and copepod multi-gen methods; (3) evaluate the practical transferability of the 

6 mysid and copepod multi-gen methods; and (4) evaluate the quantitative reproducibility of the 

7 mysid and copepod multi-gen methods. 

8 The following laboratories participated in the interlaboratory validation:

9  Labs performing the MTTT: Smithers Viscient (SMV), University of North Texas 

10 (UNT), and Wildlife International, Ltd. (WLI). 

11  Labs performing the HCDRT: Block Environmental Services (BES), Brixham 

12 Environmental Laboratory (BEL), and Centre for Environment, Fisheries & 

13 Aquaculture Science (Cefas). 

14 The following chemicals were used in the interlaboratory validation: lindane

15 (vertebrate aromatase inhibitor), vinclozolin (vertebrate androgen receptor antagonist), and 

16 4-tert-octylphenol (vertebrate estrogen receptor agonist). The specifics for each study, as well as 

17 the overall Quality Assurance Project Plan (QAPP) and Study Plans are described in the

18 individual laboratory study reports, which are presented in Appendices C and D (Block and 

19 Bradbury 2012a,b; Hala and Garcia 2012a,b; Krueger et al. 2012; Le Page and Brown 2011; Lee 

20 2012; Veauvy et al. 2012). 

21 Following completion of all studies, endpoint data were re-analyzed by Battelle using a 

22 common statistical analysis plan. Results of this evaluation were reported in intra-laboratory 

23 analysis reports for each study (Appendix G). The intra-laboratory evaluation was composed of 

24 two parts: (1) a comparison between the test chemical concentration groups and the concurrent 

25 control groups; and (2) a comparison of the two generations to assess the benefits of a 

26 two-generation study as compared to a one-generation life cycle study. 

27 In addition, an inter-laboratory analysis Appendix C was conducted by Battelle and 

28 consisted of three parts: (1) a comparison of the control group results to assess the range of 

29 variation within studies, among studies within laboratories, and among laboratories; (2) a power 

30 analyses based on the control group results to potentially identify endpoints that can be detected 

31 with high power; and (3) a comparison of the treatment group-control group results for the key 

32 parameters. The inter-laboratory analysis was carried out to answer a series of questions raised 

33 by USEPA

34 1. Is the behavior of control values similar among labs?

35 2. Are dose-response relationships similar among labs?

36 3. For the same chemical, is there value added by extending the test beyond one 

37 generation?

38 Results of the statistical re-analysis are incorporated into the following discussions of the 

39 results of the MTTT test (i.e., results and statistical findings presented below are based on the 

40 statistical re-analysis of the data by Battelle [Appendix C] and differ, in some instances, from 

41 statistical findings in the individual study reports). No statistical re-analysis was conducted for 
Invertebrate Two-Generation ISR 49 May 28, 2013

1 the HCDRT studies. As a result, the HCDRT results are summarized from the original laboratory 

2 study reports.

3 5.1 Lindane

4 Lindane, an organochlorine insecticide, was tested in three different laboratories using 

5 the MTTT and the HCDRT with A. tenuiremis, for a total of six experiments. The results of the 

6 lindane inter-validation study are described in individual laboratory study reports (Appendices C 

7 and D) and are summarized below. Results from the statistical re-analysis by Battelle are 

8 presented in Appendix C and are incorporated below.

9 5.1.1 Exposure Levels

10 MTTT

11 Three laboratories participated in the lindane interlaboratory study of the MTTT: WLI, 

12 SMV, and UNT. Nominal lindane exposure concentrations for all laboratories were 0, 0.032, 

13 0.08, 0.20, 0.5, and 1.250 μg/L. The mean measured concentrations from each of the studies

14 ranged from 67% to 147% of the nominal levels. Measured concentrations were variable over the 

15 test duration and generally greater than 70% of nominal concentrations. Mean exposure 

16 concentrations are presented in Table 5-1 and Table 5-2. Temperature, dissolved oxygen 

17 concentration, pH and salinity were measured daily in each experiment (each treatment and 

18 control group) throughout the exposure period. All water quality parameters were generally 

19 within acceptable limits for mysid survival, growth, and reproduction, with minor exceptions for 

20 water temperature at SMV and WLI. These minor deviations from the guideline test conditions 

21 did not negatively impact the results or interpretation of the studies.

22 HCDRT

23 Three laboratories participated in the EPA-led lindane interlaboratory study of the 

24 HCDRT: BEL, Cefas, and BES. Nominal lindane exposure concentrations were 0, 2.5, 5, 10, 15, 

25 and 20 μg/L. Time-weighted mean measured concentrations were within acceptable limits and 

26 ranged between 72 and 88% of nominal concentrations. Mean exposure concentrations are 

27 presented in Table 5-3. Water quality parameters were within the guideline acceptability criteria 

28 (dissolved oxygen >70%, temperature 25 ± 2 o C., pH of 8.0 ± 0.4, and salinity of 30 ± 3‰) with 

29 minor exceptions that were determined to not affect the test performance at any of the 

30 laboratories.

31 In addition to the EPA-led interlaboratory study, four laboratories participated in an 

32 OECD interlaboratory evaluation of the HCDRT (OECD 2011). Since the OECD method forms 

33 the basis of the method evaluated by EPA, the results from those experiments are presented here 

34 as further validation of this protocol. The four laboratories (USC, NOAA, Stockholm, and 

35 Uppsala) examined the same nominal lindane concentrations (0, 2.5, 5, 10, 15, and 20 μg/L). 

36 Each of the laboratories experienced some difficulties with achieving the desired concentrations 

37 due to loss of lindane in solution (loss rates from 5 to 50%) or in the case of USC and Uppsala 

38 exceeding the nominal concentrations by 50 to 150% for some treatments. Measured 

39 concentrations from each lab are presented in Table 5-4. Most labs were able to maintain 

40 physical parameters within guideline recommendations and requirements; however, data were 

41 not always recorded and reported consistently across labs.

42
Invertebrate Two-Generation ISR 50 May 28, 2013

1 Table 5-1. Survival and growth results of the EPA interlaboratory validation study of the 

2 MTTT with lindane.

Endpoint Laba F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 0.032 0.08 0.20 0.50 1.25 0.032 0.08 0.20 0.50 1.25

Mean Measured Concentrations (μg/L) SMV 0.030 0.10 0.17 0.38 1.10 0.030 0.10 0.17 0.38 1.10

UNT 0.032 0.090 0.23 0.59 1.47 0.032 0.090 0.23 0.59 1.47

Survival Rate (Day 7) SMV - - - - - - - - - -

UNT - - - - - - - - - ↘

Survival Rate (Day 14) SMV - - - - - - - - - -

UNT - - - - - - - - - ↓

Survival (Termination) SMV - - - - - - - - - -

UNT - - - - - - - - - -

Reproduction Survival Rate

(Day 7)

SMV - - - - - - - - - -

UNT - - - - - - - - - ↓

Reproduction Survival Rate

(Day 14)

SMV - - - - - - - - - -

UNT - - - - - - - - - ↓

Reproduction Survival Rate

(Termination)

SMV - - - - - - - - - ↘

UNT - - - - ↘ - - - - -

Day 0 Dry Weight (F1 only) SMV not applicable - - - - -

UNT - - - - -

Day 7 Dry Weight SMV - - - - - - - - - -

UNT - - - - ↑ - - - - -

Day 14 Dry Weight SMV - - ↗ ↗ ↗ - ↓ ↓ - -

UNT - - - - - - - - - -

Termination Dry Weight SMV - - - ↑ - - - - - -

UNT - - - - - - - - - -

Day 0 Length (F1 only) SMV not applicable - - - - -

UNT - - - - -

Day 7 Length SMV - - - - - - - - - ↓

UNT - - - ↑ ↑ - - - ↗ ↗

Day 14 Length SMV ↑ ↑ ↑ ↑ ↑ - ↓ ↓ - -

UNT - ↗ ↗ ↗ ↗ - ↓ - - -

Termination Length SMV - - - ↑ - - - ↓ ↑ -

UNT - - - - - - - - - ↑

3 [a] Statistics were not performed (and data are not presented) for the results from WLI due to due to a lack of 

4 organisms in the negative control group and due to insufficient numbers of offspring produced by the F0 adults.

5 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

6 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re-analysis 

7 of test data (Battelle 2013).

8 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical re9 analysis of test data (Battelle 2013).

10 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical re11 analysis of test data (Battelle 2013).

12 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re13 analysis of test data (Battelle 2013).

14

15

16

17
Invertebrate Two-Generation ISR 51 May 28, 2013

1 Table 5-2. Reproduction results of the EPA interlaboratory validation study of the MTTT with 

2 lindane.

Endpoint Laba F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 0.032 0.08 0.20 0.50 1.25 0.032 0.08 0.20 0.50 1.25

Mean Measured Concentrations (μg/L) SMV 0.030 0.10 0.17 0.38 1.10 0.030 0.10 0.17 0.38 1.10

UNT 0.032 0.090 0.23 0.59 1.47 0.032 0.090 0.23 0.59 1.47

Time to Maturation (females) SMV - - - - - - - - - -

UNT - - - ↘ ↘ - - - - -

Number Offspring/Female (1st Brood) SMV - - - ↗ ↗ - - ↓ ↓ -

UNT - - - - ↑ - - - - -

Number Offspring/Female (2nd Brood) SMV - - - - - - - ↓ - -

UNT - - - - - - - - - -

Total Offspring/Female (2 Broods) SMV - - - ↑ - - ↓ ↓ - -

UNT - - - - - - - - - -

Days to 1st Brood Release SMV - ↓ - ↓ ↓ ↑ ↑ ↑ - -

UNT - - - ↘ ↘ - - - - -

Days to 2nd Brood Release SMV - ↓ - ↓ - - ↑ - - -

UNT - - - - ↓ - - - - -

Inter-Brood Duration SMV - - - - - - - - - -

UNT - - - - - - - - - -

Total Reproductive Days for the First Two 

Broods

SMV - ↓ - ↓ - - ↑ - - -

UNT - - - - - - - - - -

#Offspring/Female Per Reproduction Day for 

the First Two Broods

SMV - - - ↗ ↗ - ↓ ↓ - -

UNT - - - - - - - - - -

% Reproducing Females SMV - - - - - - - - - -

UNT - - - - - - - - - -

Sex ratio (males/females) SMV - - - - - - - - - -

UNT - - - - - - - - - -

3 [a] Statistics were not performed (and data are not presented) for the results from WLI due to due to a lack of 

4 organisms in the negative control group and due to insufficient numbers of offspring produced by the F0 adults.

5 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

6 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re-analysis 

7 of test data (Battelle 2013).

8 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical re9 analysis of test data (Battelle 2013).

10 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical re11 analysis of test data (Battelle 2013).

12 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re13 analysis of test data (Battelle 2013).

14

15

16
Invertebrate Two-Generation ISR 52 May 28, 2013

1 Table 5-3. Results of the EPA interlaboratory validation study of the HCDRT with lindane.

Endpoint Lab Test Concentration

Nominal Concentrations (μg/L) All 2.5 5 10 15 20

Mean Measured Concentrations (μg/L) BEL 2.1 3.6 7.2 11 17

BES 2.1 3.8 8.7 12.6 16.2

Cefas 1.4 3.0 5.5 8.6 11

Survival (Termination) BEL - - - ↓ ↓

BES - - - ↓ ↓

Cefas - - - - -

Development to copepodite BEL - - ↓ ↓ ↓

BES - ↑ ↑ ↑ ↑

Cefas - - ↑ ↑ ↑

Development to adult BEL - ↓* ↓* ↓* ↓*

BES - ↑ ↑ ↑ ↑

Cefas - - ↑ ↑ ↑

Sex ratio BEL - - - - -

BES - - - - -

Cefas - - - - -

Time to 1st brood BEL - - - - -

BES - - - - -

Cefas - - - - ↑

Time to 2nd brood BEL - - - - -

BES - - - - -

Cefas - - - - -

Number of offspring in 1st brood BEL - - - - -

BES - - - - ↓

Cefas ↓ ↓ ↓ ↓ ↓

Number of offspring in 2nd brood BEL - - - - -

BES - - - ↓ ↓

Cefas - - - ↓ ↓

Mating pair reproduction success BEL - - - - -

BES - - ↓ ↓ ↓

Cefas - - ↓ ↓ ↓

Number nauplii hatched over two clutches BEL - - - - -

BES - - - ↓ ↓

Cefas ↓ ↓ ↓ ↓ ↓

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [↑] Indicates a statistically significant increase compared to control.

4 [↓] Indicates a statistically significant decrease compared to control.

5 * The endpoint for development time to adult is considered unreliable because of the misidentification of 

6 copepodites as adults as discussed in the report and therefore the overall NOEC and LOEC for this study has 

7 been based on development to copepodite.

8

9
Invertebrate Two-Generation ISR 53 May 28, 2013

1 Table 5-4. Results of the OECD interlaboratory validation study of the HCDRT test with 

2 lindane.

Endpoint Lab Test Concentration

Nominal Concentrations (μg/L) All 2.5 5 10 15 20

Mean Measured Concentrations at 72 hours (μg/L) USC 3.7 5.2 25.3 22.8 22.4

NOAA 3.1 4.3 7.3 31.3 24.5

Stockholm 1.5 2.6 4.3 10.1 11.5

Uppsala 16.3 8.6 53.8 25.6 51.1

Survival (Termination) USC - - ↓ - ↓

NOAA - ↓ ↓ ↓ ↓

Stockholm - - - - ↓

Uppsala - - ↓ - -

Development to copepodite USC - - ↑ ↑ ↑

NOAA - ↑ - ↑ ↑

Stockholm - ↑ - ↑ ↑

Uppsala - - - - -

Development to adult USC - - ↑ ↑ ↑

NOAA - ↑ - ↑ ↑

Stockholm - ↑ - ↑ ↑

Uppsala - - - ↑ ↑

Sex ratio (F:M) USC - - - - -

NOAA - ↑ ↑ ↑ ↑

Stockholm - - - - -

Uppsala - - - - -

Mating pair reproduction success USC - - - ↓ ↓

NOAA - - ↓ ↓ -

Stockholm - - ↓ - ↓

Uppsala [a] [a] [a] [a] [a]

Number nauplii hatched over two clutches USC - - - ↓ ↓

NOAA - - ↓ ↓ ↓

Stockholm - - - - ↓

Uppsala - ↓ - - -

Population Growth Rates USC - na ↓ na ↓

NOAA - na ↓ na ↓

Stockholm - na ↓ na ↓

Uppsala - na - na -

3 [-] Indicates an endpoint was measured, but was not statistically significantly different from control. Note that time 

4 to 1st and 2nd brood and number of offspring in 1st and 2nd Brood were not reported.

5 [↑] Indicates a statistically significant increase compared to control.

6 [↓] Indicates a statistically significant decrease compared to control.

7 [a] The mating success criterion of >70% was not met at the Uppsala lab.

8 [na] Not assessed - population growth rate was only calculated for the 2.5, 10, and 20 μg/L treatments.

9 [nr] Not reported – data for this parameter not reported in the OECD interlaboratory report.

10 5.1.2 Sensitive Endpoints

11 MTTT

12 All laboratories observed a number of significant effects as indicated by the statistical 

13 re-analysis (Appendix G), primarily in the F0 generation (see Table 5-1 and Table 5-2 above). 

14 Control survival in the WLI study was below acceptability criteria; therefore these data were not 

15 included in the statistical reanalysis by Battelle. As a result, endpoint data as reported in the 

16 original WLI study report are not summarized here.

17 Survival endpoints were not significantly affected in the F0 generations. A decreasing 

18 trend (at the highest concentration) for reproduction survival rate (F0 at termination) was 
Invertebrate Two-Generation ISR 54 May 28, 2013

1 observed in the UNT test. In the F1 generation, significant decreases in reproduction survival rate 

2 (day 7 and 14) and total survival rate (day 7 and 14) were found in the highest concentration 

3 group in the UNT test. Only terminal reproduction rate was found to be significant in the SMV 

4 test with a decreasing trend for the highest concentration tested. Several growth endpoints were 

5 significantly increased as compared to the control in the F0 generation but were variably 

6 significantly increased or decreased as compared to the control in the F1 generation (Table 

7 5-1 above). Fecundity endpoints (measured as the number of offspring) were variable among 

8 UNT and SMV, and consistent dose-response relationships were not apparent. Sex ratio was 

9 unaffected at all test concentrations and in both generations. A decreasing trend was observed for 

10 the two highest concentration groups (UNT) for time to maturity in the F0 generation. Time to 

11 first and second brood were significantly reduced compared to the control in the F0 generation in 

12 several concentration groups at both SMV and UNT. However, the F1 time to first and second 

13 brood increased compared to the control in several concentration groups at SMV and not at UNT. 

14 Overall, the most consistent endpoints in the lindane inter-laboratory studies were the growth

15 endpoints (length). 

16 All biological acceptability criteria were met during the tests at UNT and SMV. At WLI, 

17 more than 75% of F0 females produced young. However, only 50% of the control F1 females 

18 produced young, even though more than 75% of F1 females in two of the treatment groups 

19 produced young. Further, the F1 generation in the controls did not produce an average combined 

20 total number of young in the first two broods ≥8 (i.e., seven were produced per female, and so 

21 the criterion was missed by 13%). Due to the lack of organisms in the WLI test, statistics were 

22 not performed.

23 HCDRT

24 HCDRTs conducted by BES and Cefas met acceptability criteria; however, the tests 

25 conducted by BEL were considered unreliable. BEL experienced practical difficulties carrying 

26 out the copepod test due to issues with the identification of copepodite maturation and 

27 determination of sex. The poor reliability and problems regarding the quality of the data have 

28 been acknowledged and for these reasons, particular care should be taken when drawing 

29 conclusions from the BEL reproduction data. 

30 Copepod survival was significantly affected at 15 and 20 μg/L as reported by BEL and 

31 BES, while no significant change in survival was noted by Cefas. Lindane had a significant 

32 inhibitory effect on copepod development into both copepodite and adult life-stages (Table 5-3

33 above). Specifically, lindane significantly increased the time to develop from the naupliar to the 

34 copepodite life-stages and also significantly increased the time necessary for exposed copepods 

35 to mature into sexually mature adults (at concentrations as low as 2.5 μg/L). Sex ratio was not 

36 significantly affected during any of the tests. Lindane was observed to have a significant effect 

37 on copepod reproduction, with a reduction in the percentage of successful mating pairs and a 

38 reduction in the total number of viable eggs and hatched nauplii. These results were consistent in 

39 the two labs with reliable results (BES and Cefas). The lowest concentration at which lindane 

40 affected reproductions was 2.5 μg/L (nominal).

41 Biological results from the OECD interlaboratory study are presented in Table 5-4 above. 

42 Survival was significantly reduced at lindane concentrations of 10, 15, and 20 μg/L at one or

43 more labs, respectively. Three of four participating laboratories reported consistent 

44 concentration-dependent delays in development rates, consistent naupliar-specific mortality, 
Invertebrate Two-Generation ISR 55 May 28, 2013

1 consistent mating-success depressions, and consistent reduction of fecundity (OECD, 2011). 

Interlaboratory comparisons of lindane life table data using the Leslie matrix model12

3 (Leslie 1945) produced consistent patterns of response at 10 and 20 μg/L, indicating significant 

4 reductions in population growth rates. Results for time to first and second brood and number per 

5 brood were not reported in the OECD interlaboratory study and thus are not comparable to the 

6 EPA EDSP interlaboratory studies.

7 5.1.3 Conclusions

8 MTTT

9 The interlaboratory results for two of the three labs (UNT and SMV) were within 

10 acceptable test method guidelines, while one lab experienced poor performance in the F1 

11 generation (WLI). In general, labs demonstrated the ability to perform the test according to the 

12 proposed method. Lindane exposure significantly affected growth and reproduction in the mysid,

13 primarily in the F0 generation; however, endpoint results were generally variable between 

14 laboratories and between generations (Table 5-1 and Table 5-2 above).

15 HCDRT

16 The HCDRT interlaboratory validation study with lindane was conducted by seven 

17 laboratories (three in an EPA-led study and four in an OECD-led study). All laboratories 

18 generally were able to perform the test within acceptable guidelines with some difficulties in 

19 producing consistent lindane test concentrations, differentiating age differences for 

20 developmental endpoints, and achieving the desired mating success criterion. Due to these issues, 

21 OECD (2011) recommended that further work be conducted to address the proficiency, 

22 manpower, and training of laboratories performing the test, continue validation of the guideline 

23 to identify preferred endpoints, and obtain additional information on the basic biology of the test 

24 species. However, despite some of these difficulties, most labs detected delays in development 

25 and reduced fecundity at the higher test concentrations (Table 5-3 and Table 5-4 above). 

26 No consistent biological responses were observed between the MTTT and the HCDRT. 

27 The test concentrations in the MTTT (highest test concentration of 1.25 μg/L) were all below 

28 those tested in the HCDRT (lowest test concentration of 2.5 μg/L).

29 5.2 Atrazine

30 Atrazine was tested in three laboratories using only the HCDRT with A. tenuiremis, for a

31 total of three experiments. The results of the atrazine interlaboratory validation study are 

32 described in OECD (2011) and summarized below.

33 Three laboratories participated in the OECD (2011) atrazine interlaboratory study of the 

34 HCDRT: USC, NOAA, Stockholm. Nominal atrazine exposure concentrations were 0, 26, 43, 72, 

35 120, and 200 μg/L. Measured concentrations were within 50% of nominal concentrations, and 

36 atrazine was stable with relatively little loss from initial measured concentrations. Mean 

37 exposure concentrations are presented in Table 5-5. Most labs were able to maintain physical 

38 parameters within guideline recommendations and requirements; however, physical and copepod 

39 response data were not always recorded and reported consistently across labs.

 

1 The Leslie matrix model is a discrete, age-structured model used to estimate population growth based on fertility 

and survival rates.
DRAFT

Invertebrate Two-Generation ISR 56 May 28, 2013

1 Table 5-5. Results of the OECD interlaboratory validation study of the HCDRT with atrazine.

Endpoint Lab Test Concentration

Nominal Concentrations (μg/L) All 26 43 72 120 200

Mean Measured Concentrations at 72 hours (μg/L) USC 31.1 48.8 94.1 159.5 208.5

NOAA 31.5 54.9 93.6 162.2 210.4

Stockholm 36.1 63.9 88.6 144.9 221.0

Survival (Termination) USC - - - - -

NOAA - - - - -

Stockholm - - - - -

Development to copepodite USC - - - - -

NOAA - - - - -

Stockholm - - - - -

Development to adult USC - - - - -

NOAA - - - - -

Stockholm - - - - -

Sex ratio (F:M) USC - - - - -

NOAA - - - - -

Stockholm - - - - -

Mating pair reproduction success USC - - - - -

NOAA - - - - -

Stockholm - - - - -

Number nauplii hatched over two clutches USC - - - ↓ ↓

NOAA - - - ↓ ↓

Stockholm - - - - -

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control. Note that time 

3 to 1st and 2nd brood and number of offspring in 1st and 2nd Brood were not reported.

4 [↑] Indicates a statistically significant increase compared to control.

5 [↓] Indicates a statistically significant decrease compared to control.

6 [nr] Not reported – data for this parameter not reported in the OECD interlaboratory report.

7 5.2.1 Sensitive Endpoints

8 Control survival was within the guideline criterion of >80% for all labs except the carrier 

9 solution control from the Stockholm lab. Stockholm attributed the normal-control result to non10 random selection of control nauplii at experimental set up, and low stock culture densities prior 

11 to the beginning of the test. Survival, development, and reproduction results are presented in 

12 Table 5-5. No labs found concentration dependency or treatment-specific significant differences 

13 in mortality, development rate, sex ratios, or mating success at any test concentration or for any 

14 life-stage relative to controls (i.e., nauplius, copepodite, or adult). However, the number of viable 

15 offspring produced through two clutches or broods at 120 and 200 ug/L atrazine were 

16 significantly depressed relative to controls (USC and NOAA labs). All labs achieved >70% 

17 mating pair success in controls except for Stockholm, which achieved 44.2% success. Stockholm 

18 normal control matings could not be completed because that set of microplates was accidentally 

19 dropped prior to clutch extrusion. Solvent control matings that were successful by Stockholm 

20 produced two-brood clutch sizes of ~10 offspring, which was close to the mean response seen in 

21 all atrazine treatments, and within the guideline two-brood clutch-size criterion of > 8 viable 

22 offspring in controls.

23 5.2.2 Conclusions

24 The HCDRT interlaboratory evaluation with atrazine was conducted by three laboratories. 

25 Two of three laboratories were able to perform the test within acceptable guidelines. The 
DRAFT

Invertebrate Two-Generation ISR 57 May 28, 2013

1 biological results for most endpoints were consistent across labs and showed a 

2 concentration-dependent decrease in number of nauplii hatched over two clutches (Table 5-5).

3 5.3 Vinclozolin

4 Vinclozolin was tested using the MTTT at UNT and the HCDRT at BES, for a total of 

5 two experiments. The results of the study are described in the laboratory test reports (Hala and 

6 Garcia 2012a; Block and Bradbury 2012b) and summarized below. Results from the statistical 

7 re-analysis of the MTTT data produced at UNT by Battelle are presented in Appendix C and are 

8 incorporated below.

9 5.3.1 Exposure Levels

10 MTTT

11 One laboratory (UNT) tested the effects of vinclozolin using the MTTT assay. Nominal

12 test concentrations were 0, 25, 50, 100, 200, and 600 μg/L. The time-weighted average 

13 concentrations of vinclozolin in the exposure tanks were < 0.00156 (control), 8.2, 24.8, 37.3, 

14 119.5 and 402.5 μg/L (Table 5-6 and Table 5-7). The measured values were 33 to 67% of 

15 nominal concentrations. Water quality parameters were measured throughout the mysid test and 

16 reported as: dissolved oxygen was ≥ 6.9 mg/L, temperature ranged from 25 to 27°C, pH ranged 

17 from 7.8 to 8.5, and light intensity ranged from 33 to 98 foot-candles. All water quality 

18 parameters were within acceptable limits for mysid survival, growth and reproduction.

19 Table 5-6. Survival and growth results of the EPA validation study of the MTTT with 

20 vinclozolin.

Endpoint Lab F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 25 50 100 200 600 25 50 100 200 600

Mean Measured Concentrations (μg/L) UNT 8.2 24.8 37.3 119.5 402.5 8.2 24.8 37.3 119.5 402.5

Survival (Day 7) UNT - - - - ↘ - - - - -

Survival (Day 14) UNT - - - - ↘ - - - ↓ -

Survival (Termination) UNT - - - - - - - - - -

Reproduction Survival Rate (Day 7) UNT - - - - ↘ - - - - -

Reproduction Survival Rate (Day 14) UNT - - - - - - - - - ↘

Reproduction Survival Rate (Termination) UNT - - - - - - - - - -

Day 0 Dry Weight (F1 only) UNT not applicable - - - - -

Day 7 Dry Weight UNT - - - - - - - - - -

Day 14 Dry Weight UNT - - - - ↓ - - - - ↓

Termination Dry Weight UNT - - - - ↘ - - - - -

Day 0 Length(F1 only) UNT not applicable - - - - ↗

Day 7 Length UNT - - - - - - - - - -

Day 14 Length UNT ↑ - ↑ - - - - - - -

Termination Length UNT - - - - - - ↗ ↗ ↗ ↗

21 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

22 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re23 analysis of test data (Battelle 2013).

24 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical re25 analysis of test data (Battelle 2013).

26 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical re27 analysis of test data (Battelle 2013).

28 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical re29 analysis of test data (Battelle 2013).

30
DRAFT

Invertebrate Two-Generation ISR 58 May 28, 2013

1 Table 5-7. Reproduction results of the EPA validation study of the MTTT with vinclozolin.

Endpoint Lab F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 25 50 100 200 600 25 50 100 200 600

Mean Measured Concentrations (μg/L) UNT 8.2 24.8 37.3 119.5 402.5 8.2 24.8 37.3 119.5 402.5

Time to Maturation (females) UNT - - - - - - - - - -

Number Offspring/Female (1st Brood) UNT - - - - - - - - - -

Number Offspring/Female (2nd Brood) UNT - - - - - - - - - ↘

Total Offspring/Female (2 Broods) UNT - - - - - - - - - ↘

Days to 1st Brood Release UNT - - - ↗ ↗ - ↑ - - -

Days to 2nd Brood Release UNT - - - - - - - - - ↗

Inter-brood duration UNT - - - - - - - - - ↑

Total Reproductive Days for the First 

Two Broods

UNT - - - - - - - - - -

#Offspring/Female Per Reproduction Day 

for the First Two Broods

UNT - - - - - - - - - -

% Reproducing Females UNT - - - - ↘ - - - - -

Sex ratio (males/females) UNT - - - - ↑ - - - - -

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re-analysis 

4 of test data (Battelle 2013).

5 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical 

6 re-analysis of test data (Battelle 2013).

7 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical 

8 re-analysis of test data (Battelle 2013).

9 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical 

10 re-analysis of test data (Battelle 2013).

11 HCDRT

12 Vinclozolin concentrations of 12.96, 21.6, 36, 60, and 100 μg/L were tested in one 

13 laboratory (BES). The chemical analysis of vinclozolin test concentrations showed that 

14 concentrations of the test substance were all below the laboratory reporting limit (Table 5-8). A 

15 review of the procedure for preparing the test solutions indicated that the solutions were prepared 

16 from the primary stock solutions properly. Moreover, the primary stock solutions used to prepare 

17 the test concentrations were measured to be within 10 percent of the expected concentration of 

18 200 mg/L. A review of the sample handling following collection indicated that samples were 

19 submitted to the laboratory outside the recommended holding time. These data suggest that test 

20 chemical was likely lost through adsorption onto test chambers, sample storage bottles, as well as 

21 through natural degradation processes prior to analysis.

22 5.3.2 Sensitive Endpoints

23 MTTT

24 The vinclozolin mysid two-generation toxicity test conducted by UNT met all control 

25 acceptability criteria. Endpoint responses are summarized in Table 5-6 and Table 5-7. Survival 

26 endpoints for F0 generation were generally not significantly different from the control (based on 

27 ANOVA between group means), although some survival endpoints showed a decreasing trend 

28 for the highest concentration group (based on a linear trend test across treatment groups). 

29 A significant reduction in survival was found at the second highest concentration for the F1 

30 generation for day 14 survival rate. Growth, measured as dry weight, was reduced compared to 

31 the control in both generations. However, growth measured as length increased as compared to 

32 the control in several exposure groups and in both generations (Table 5-6). 
DRAFT

Invertebrate Two-Generation ISR 59 May 28, 2013

1 Table 5-8. Results of the EPA validation study of the HCDRT with vinclozin.

Endpoint Lab Test Concentration

Nominal Concentrations (μg/L) All 12.96 21.60 36 60 100

Mean Measured Concentrations (μg/L) BES <DL <DL <DL <DL <DL

Survival (Termination) BES - - - - -

Development to copepodite BES - - - - -

Development to adult BES - - - - -

Sex ratio BES - - - - -

Time to 1st brood BES - ↑ ↑ - -

Time to 2nd brood BES - - - - -

Number of offspring in 1st brood BES - - - - -

Number of offspring in 2nd brood BES - - - - -

Mating pair reproduction success BES - - - - -

Number nauplii hatched over two clutches BES - - - - -

2 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

3 [↑] Indicates a statistically significant increase compared to control.

4 [↓] Indicates a statistically significant decrease compared to control.

5 The time to brood release showed an increasing trend in both generations in the highest 

6 exposure groups. Sex ratio (male/female) was significantly increased compared to the control in 

7 the F0 generation in the highest exposure group, but was unaffected in F1. A decreasing trend in 

8 reproduction performance (% of females reproducing in the F0 generation) was observed at the 

9 highest concentration, but was unaffected in the F1 generation. Fecundity showed a decreasing 

10 trend, which was most apparent for the highest concentration group in the F1 generation (Table 

11 5-7).

12 HCDRT

13 All water quality and biological performance criteria were met for this study. Vinclozolin 

14 did not affect copepod survival at any exposure level by day 16 of the test, with the survival rate 

15 not significantly reduced further by test termination on day 36. Similarly, the developmental 

16 endpoints tested were also not significantly affected, as vinclozolin did not significantly affect 

17 the time for A. tenuiremis nauplii to mature into the copepodite stage and subsequent 

18 transformation to the adult stage at test concentrations up to 100 μg/L. Vinclozolin had no effect 

19 on gender assignment for all concentrations tested. Vinclozolin did, however, significantly delay 

20 the time for mated females to extrude their first brood at the 21.6 and 36 μg/L exposure levels, 

21 although the time for females to extrude their second brood was not delayed at any exposure 

22 level tested (see Table 5-8). Vinclozolin did not significantly reduce or increase the number of 

23 hatched offspring at any test concentration, although an appreciable decrease in hatched 

24 offspring in the 100 μg/L treatment was noted. Lastly, with regards to reproductive endpoints, 

25 fertilization success was not significantly affected for any test treatment when compared to the 

26 carrier control. An appreciable decrease (not significant) in the number of successful mating 

27 pairs was noted in organisms exposed vinclozolin concentrations of 21.6, 36, and 60 μg/L. 
DRAFT

Invertebrate Two-Generation ISR 60 May 28, 2013

1 5.3.3 Conclusions

2 MTTT

3 The MTTT conducted by UNT was within acceptable test method guidelines. Exposure 

4 to vinclozolin significantly affected a number of growth (decreased weight, increased length) and 

5 reproduction (increased sex ratio in F0, decreased fecundity in F1, decreased number of actively 

6 reproducing females in F0, increased interbrood duration in F1) parameters in both generations, 

7 whereas survival endpoints were generally not significantly affected (compared to controls). The 

8 results indicate that UNT successfully conducted the MTTT test with minimal deviation from the 

9 test guideline.

10 HCDRT

11 The HCDRT conducted by BES was within acceptable test method guidelines. However, 

12 since all measured vinclozolin concentrations were below the laboratory reporting limit, results 

13 were reported on a nominal basis. Vinclozolin did not affect any of the copepod survival and 

14 development endpoints at any exposure level. However, since the true exposure concentrations 

15 are uncertain, these results should be interpreted with caution. Of the measured reproduction 

16 endpoints, only the time to first brood was significantly increased at 21.6 and 36 μg/L, but not at 

17 higher exposure levels. 

18 Vinclozolin exposure resulted in significant reproductive delays (measured as time to 

19 first brood release) at some treatment levels in both copepod and mysid tests; however, 

20 consistent concentration-response relationships were not observed in either test.

21 5.4 4-tert-octylphenol

22 4-tert-octylphenol was tested in two different laboratories using the MTTT method. 

23 Copepod interlaboratory studies were not conducted with this chemical. The results of the 

24 interlaboratory validation study are described in the individual laboratory study reports 

25 (Claude et al. 2012; Lee 2012a,c). Results from the statistical re-analysis of the MTTT data 

26 produced at SMV and WLI by Battelle are presented in Appendix C and are incorporated below.

27 5.4.1 Exposure Levels

28 Nominal 4-tert-octylphenol exposure concentrations were 0.26, 0.64, 1.6, 4.0, and 

29 10 μg/L for both laboratories (SMV and WLI). Analysis of the test solutions throughout the 

30 exposure period demonstrated that the expected concentration-gradient was generally maintained 

31 during the exposure. Mean measured concentrations of 4-tert-octylphenol ranged from 98 to 150%

32 (SMV) and 74 to 127% (WLI) of nominal. The mean measured concentrations of 

33 4-tert-octylphenol (as active ingredient) for each lab are presented in Table 5-9 and Table 5-10. 

34 Based on the results, it was determined that the appropriate quality control was maintained 

35 during the analyses of the 4-tert-octylphenol exposure solution samples in both laboratories. The 

36 water quality parameters of both labs remained within tolerance limits as defined in the test 

37 protocol.

38
DRAFT

Invertebrate Two-Generation ISR 61 May 28, 2013

1 Table 5-9. Reproduction results from the EPA validation study of the MTTT using 

2 4-tert-Octylphenol.

Endpoint Lab F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 0.26 0.64 1.6 4.0 10 0.26 0.64 1.6 4.0 10

Mean Measured Concentrations (μg/L) SMV 0.36 0.98 2.0 3.9 11 0.36 0.98 2.0 3.9 11

WLI 0.33 0.69 1.4 3.2 7.2 na na na na na

Time to Maturation (females) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Number Offspring/Female (1st Brood) SMV - ↓ ↓ - - - - - - ↘

WLI - - - - - na na na na na

Number Offspring/Female (2nd Brood) SMV - - - ↓ - - - - - -

WLI ↓ ↓ ↓ ↓ - na na na na na

Total Offspring/Female (2 Broods) SMV ↓ ↓ - ↓ ↓ ↑ - - - -

WLI - - ↓ ↓ - na na na na na

Days to 1st Brood Release SMV ↓ - - - - - - - - -

WLI - - - - - na na na na na

Days to 2nd Brood Release SMV - - - - - - - - - -

WLI - - - - - na na na na na

Inter-brood duration SMV ↑ - - ↑ - - - - - -

WLI - - - - - na na na na na

Total Reproductive Days for the First Two Broods SMV - - - - - - - - - -

WLI - - - - - na na na na na

#Offspring/Female Per Reproduction Day for the 

First Two Broods

SMV - - - - - - - - - -

WLI - - - - - na na na na na

% Reproducing Females SMV - - - - - - - - - -

WLI - - - - - na na na na na

Sex ratio (males/females) SMV - - - - - - - - - -

WLI - - - - - na na na na na

3 [na] Not assessed due to insufficient production of live young to continue an F1 generation in all treatment groups.

4 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

5 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re-analysis of test data 

6 (Battelle 2013).

7 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical re-analysis of test data 

8 (Battelle 2013).

9 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical re-analysis of test 

10 data (Battelle 2013).

11 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statisticalre-analysis of test 

12 data (Battelle 2013).

13

14 5.4.2 Sensitive Endpoints

15 4-tert-octylphenol exposure at SMV indicated that the F0 generation appeared to be more 

16 sensitive (ten endpoints affected) than the F1 generation (four endpoints affected). A summary of 

17 the responses for each endpoint in each generation is presented in Table 5-9 and Table 5-10. The 

18 most sensitive endpoints were those associated with fecundity (reduced number of offspring in 

19 broods 1 and 2 and increased interbrood duration) and growth (increased weight and length), 

20 whereas survival endpoints were not significantly affected in both generations. The results from 

21 WLI similarly indicated that the most sensitive endpoints were reproduction (reduced number of 

22 offspring in broods 1 and 2) and growth (reduced weight and increased length at day 14), 

23 whereas survival endpoints were unaffected. The reproductive effects apparent in the F0 

24 generation (WLI) resulted in insufficient production of live young to continue an F1 generation 

25 in all treatment groups. Therefore, statistical analysis was not performed on the exposed groups 

26 of the F1 generation (WLI), and only the F1 control group was evaluated and found to perform 

27 within test acceptability criteria.
DRAFT

Invertebrate Two-Generation ISR 62 May 28, 2013

1 Table 5-10. Survival and growth results from the EPA validation study of the MTTT using 

2 4 tert-Octylphenol.

Endpoint Lab F0 (Parental) F1 (1st Generation)

Nominal Concentrations (μg/L) All 0.26 0.64 1.6 4.0 10 0.26 0.64 1.6 4.0 10

Mean Measured Concentrations (μg/L) SMV 0.36 0.98 2.0 3.9 11 0.36 0.98 2.0 3.9 11

WLI 0.33 0.69 1.4 3.2 7.2 na na na na na

Survival (Day 7) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Survival (Day 14) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Survival (Termination) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Reproduction Survival Rate (Day 7) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Reproduction Survival Rate (Day 14) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Reproduction Survival Rate (Termination) SMV - - - - - - - - - -

WLI - - - - - na na na na na

Day 0 Dry Weight (F1 only) SMV not applicable - - - - -

WLI na na na na na

Day 7 Dry Weight SMV - - - ↗ ↗ - - - - -

WLI - ↓ - - - na na na na na

Day 14 Dry Weight SMV ↑ - - - ↑ - - - - -

WLI - - - ↘ ↘ na na na na na

Termination Dry Weight SMV - - - - - - - - - -

WLI - - - - - na na na na na

Day 0 Length(F1 only) SMV not applicable - - - - -

WLI na na na na na

Day 7 Length SMV ↑ ↑ ↑ ↑ ↑ ↑ - - ↑ -

WLI - - - ↘ ↘ na na na na na

Day 14 Length SMV ↗ ↗ ↗ ↗ ↗ - - - ↗ ↗

WLI ↑ ↑ ↑ ↑ ↑ na na na na na

Termination Length SMV - - ↘ ↘ ↘ - - - - -

WLI - - - - - na na na na na

3 [na] Not assessed due to insufficient production of live young to continue an F1 generation in all treatment groups.

4 [-] Indicates an endpoint was measured, but was not statistically significantly different from control.

5 [↑] Indicates a statistically significant increase compared to control (ANOVA F-test), based on statistical re-analysis 

6 of test data (Battelle 2013).

7 [↓] Indicates a statistically significant decrease compared to control (ANOVA F-test), based on statistical 

8 re-analysis of test data (Battelle 2013).

9 [↗] Indicates a statistically significant increasing concentration trend (Linear trend test), based on statistical 

10 re-analysis of test data (Battelle 2013).

11 [↘] Indicates a statistically significant decreasing concentration trend (Linear trend test), based on statistical 

12 re-analysis of test data (Battelle 2013).

13 5.4.3 Conclusions

14 The interlaboratory results for the SMV and WLI labs demonstrated that the test was 

15 performed successfully and within acceptable test method guidelines, except for the F1 

16 generation from WLI. At WLI, the reproductive effects apparent in the F0 generation resulted in 

17 insufficient production of live young to continue an F1 generation in all treatment groups. Several 

18 growth and reproduction endpoints in the F0 generation were found to be sensitive in both 

19 laboratories, but the responses were not fully consistent between laboratories (i.e., weight and 

20 length effects were inconsistent, whereas fecundity effects were consistent). The reduction in 

21 fecundity in the F0 generation was the most consistent effect observed in both laboratories. 
DRAFT

Invertebrate Two-Generation ISR 63 May 28, 2013

1 4-tert-octylphenol appears to result in increased growth as compared to the control, although 

2 growth endpoint variability was observed between both studies.

3
DRAFT

Invertebrate Two-Generation ISR 64 May 28, 2013

1 6 DISCUSSION AND SUMMARY OF THE MTTT AND HCDRT METHODS

2 6.1 Reproducibility of the Test Methods

3 MTTT

4 McKenney first demonstrated transgenerational effects in A. bahia using a 

5 two-generation exposure protocol (Section 4.1). These studies led to the development of a 

6 proposed MTTT guideline (OECD, 2004a). The MTTT guideline was used in demonstration and 

7 optimization studies using a number of representative endocrine-active chemicals in two 

8 different laboratories (Section 4.1). Subsequently, the MTTT was used in an inter-laboratory 

9 validation study consisting of six studies carried out at four laboratories with three representative 

10 endocrine-active chemicals (i.e., lindane, vinclozolin, and 4-tert-octylphenol; Section 5). Data for 

11 25 different endpoints (8 growth parameters, 11 reproduction parameters, and 6 survival 

12 parameters) collected during the inter-laboratory validation study were re-analyzed to evaluate 

13 the reproducibility of the recommended MTTT included as Appendix C. Results of the MTTT 

14 validation studies can be summarized as follows:

15  Except for minor deviations from the guidelines test conditions, the different 

16 laboratories were able to successfully execute the proposed MTTT. In one study, one

17 laboratory was unable to achieve sufficient reproduction in the controls to complete 

18 the second generation within the guidelines acceptability criteria (i.e., WLI in the 

19 lindane study).

20  Large laboratory-to-laboratory variability was observed in the reproduction and 

21 growth endpoint data collected for the control groups. Specifically, the coefficients of 

22 variability (CVs) across laboratories and studies in the control groups in the F0

23 generation exceeded 20% for 7 out of 11 reproduction endpoints and five out of eight

24 growth endpoints (Table 6-1). Conversely, the CVs across laboratories and studies in 

25 the control groups in the F0 were below 20% for all six survival endpoints. There is 

26 no clear consensus on what constitutes an acceptable inter-laboratory CV; however, 

27 desired maximum CVs of 20 and 30% have been used in the past (e.g., Moore 

28 et al., 2000). While there is a positive association between the F0 and F1 CVs, 

29 particularly for the reproduction and survival parameters, the CVs in the F1

30 generation tended to exceed those for the F0 generation for the same parameter (Table 

31 6-1).

32  Significant differences were observed in the technical proficiency (as estimated by 

33 the variability of the endpoint measurements in the control groups) among labs that 

34 participated in the inter-laboratory validation study, indicating difficulties in 

35 transferability of the MTTT between these labs. 

36  Significant variability was observed within laboratories between studies for the 

37 reproductive and survival endpoints in the control groups, whereas that variability 

38 was smaller for the growth endpoints (length and weight).

39  Where the same chemical was tested in two laboratories (i.e., lindane and 

40 4-tert-octylphenol), dose-response relationships were generally not consistent (based 

41 on comparisons of significantly affected endpoints at each treatment level) among the 

42 laboratories. Day 14 F0 length was the only growth parameter with consistently 

43 detected significant increases at 0.08 μg lindane/L or above at two labs. Number of 

44 offspring, days to first and second brood, and length at day 14 provided the most 
DRAFT

Invertebrate Two-Generation ISR 65 May 28, 2013

1 consistent response in the inter-laboratory studies (see summary tables in Section 5). 

2 Number of offspring in brood 2 and total number of offspring in two broods were the 

3 only two endpoints that showed a significant trend across 4-tert-octylphenol 

4 concentration groups.

5  Growth endpoints were generally affected at lower concentrations than reproductive 

6 endpoints in the inter-laboratory validation studies.

7  Endpoint responses between the F0 and F1 generations were not consistent and were 

8 significantly different in several instances.

9 Table 6-1. Ranges of coefficient of variation (CV) in the F0 and F1 generations based on six 

10 studies carried out at four laboratories with three chemicals (see Section 5).

Category Parameter CV – F0 CV – F1

Growth Day 0 Length 12.589 18.446

Day 14 Length 12.632 23.558

Day 14 Dry Weight 14.643 16.468

Termination Length 21.725 18.067

Termination Dry Weight 26.105 25.123

Day 7 Length 28.817 16.204

Day 0 Dry Weight 37.991 39.547

Day 7 Dry weight 84.464 18.223

Reproduction Total Reproductive Days for the First Two Broods 4.252 8.651

#Offspring/Female per Reproduction Day for the First Two Broods 4.343 7.951

Time to Maturation 12.229 22.588

% Reproducing Females 13.127 1.408

Sex Ratio (males/females) 21.837 38.342

Days to 2nd Brood Release 24.899 13.868

Days to 1st Brood Release 25.550 15.457

Inter-Brood Duration 25.656 7.679

Number Offspring/Female (2nd Brood) 31.483 64.164

Number Offspring/Female (2 Broods) 33.032 57.475

Number Offspring/Female (1st Brood) 35.188 68.846

Survival Reproduction Survival Rate (Day 7) 1.912 3.729

Survival Rate (Day 7) 2.719 5.284

Survival Rate (Day 14) 5.254 3.061

Survival rate (Termination) 6.857 11.602

Reproduction Survival Rate (Day 14) 10.143 7.324

Reproduction Survival Rate (Termination) 19.978 22.154

11 HCDRT

12 Chandler and colleagues conducted a range of studies with the harpacticoid copepod A.

13 tenuiremis, which served as initial development studies for the HCDRT (Section 4.2). A copepod 

14 life-cycle toxicity test protocol was established for A. tenuiremis by the American Society for 

15 Testing and Materials in 2004 (ASTM E2317-04). In addition, a draft guideline was proposed to 

16 OECD in 2004 to evaluate effects of chemicals on A. tenuiremis (OECD, 2007). This draft 

17 guideline was used in two rounds of OECD validation studies (OECD 2007, 2011) and an EPA18 led inter-laboratory validation study (Section 5) and results can be summarized as follows:

19  Based on the prevalidation study (OECD 2007), in which two harpacticoid (N. 

20 spinipes and A. tenuiremis) and one calanoid copepod (A. tonsa) were tested, the 

21 A. tenuiremis assay was recommended for further validation due to its shorter test 

22 time, better reproductive output and success, its focus on reproductive and 
DRAFT

Invertebrate Two-Generation ISR 66 May 28, 2013

1 developmental endpoints, its greater statistical power due to high replication, and its

2 availability as an existing ASTM method.

3  Lindane was tested at the same concentrations in three laboratories as part of the 

4 EPA-led interlaboratory validation study and in four laboratories as part of the OECD 

5 validation study. Laboratories in the OECD validation study had difficulties obtaining 

6 the desired chemical concentrations. While most of the seven test laboratories were 

7 able to perform the test within guideline recommendations and requirements, data 

8 were not always recorded and reported consistently among laboratories. For example, 

9 one of the labs in the OECD validation study experienced difficulties with the 

10 identification of copepodite maturation and determination of sex. Lindane generally 

11 caused reduced survival, developmental delay, and reduced fecundity, although 

12 differences were observed in the concentrations at which these effects were reported 

13 by different labs. In addition, some labs did not find the same endpoints to be 

14 significantly affected. Due to these issues, OECD recommended that further work be 

15 conducted to address the proficiency, manpower, and training of laboratories 

16 performing the test, continue validation of the guideline to identify preferred 

17 endpoints, and obtain additional information on the basic biology of the test species.

18  Atrazine was tested in three laboratories as part of the OECD interlaboratory 

19 validation study. One of the laboratories was not able to achieve the required mating 

20 success in the controls; however the other two labs were able to perform the test 

21 within acceptable guidelines and reported consistent biological responses 

22 (i.e., reduced number of nauplii hatched over two clutches in two highest test 

23 concentrations with none of the other endpoints significantly affected).

24  Only one laboratory tested vinclozolin as part of the EPA validation study. While the 

25 test was conducted within test method guidelines, all measured vinclozolin 

26 concentrations were undetected.

27 6.2 Data Interpretation

28 MTTT

29 The MTTT is proposed as a Tier 2 test to be used in EPA’s EDSP. If Tier 2 testing is 

30 indicated based on results of Tier 1 screening, EPA generally expects that the summary narrative 

31 and characterization would not only explain the basis of the conclusions regarding potential 

32 interactions with the estrogen (E), androgen (A), and/or thyroid (T) hormonal pathways, but also 

33 provide a discussion on why additional data are needed for assessing potential adverse outcomes 

34 via potential endocrine disruption (US EPA 2011). 

35 While vertebrate-type steroids have been detected in several crustaceans, including 

36 mysids (Verslycke et al., 2002), their endogenous origin is questioned in many cases (LaFont, 

37 2000). Crustaceans are believed to lack E, A, and T hormone axes. In addition, little is known 

38 about the potential of chemicals that may affect the E, A, and T hormone axes through any 

39 known modes of action (i.e., chemicals that are expected to be identified though Tier 1 screening) 

40 to interfere with invertebrate hormone axes or the endpoints measured in the MTTT. While dose 

41 response information can be obtained for the growth and reproductive endpoints evaluated in the 

42 MTTT, the study design for the MTTT does not include any biochemical, biomarker, or 

43 histopathology endpoints. Therefore, a specific mode of action determination is not possible, and 
DRAFT

Invertebrate Two-Generation ISR 67 May 28, 2013

1 would require additional endpoint measurements or be based on extrapolation of a documented 

2 mode of action in other arthropods. From a risk assessment perspective, the distinction between 

3 endocrine or metabolic toxicity based on sound mechanistic evidence is not crucial. This 

4 distinction is probably less crucial than identifying the most sensitive endpoints that can lead to 

5 long-term impacts at the population level (Verslycke et al. 2007).

6 Dose-response information, such as LOEC, NOEC, and ECx information for the 

7 biological endpoints included in the MTTT, should be collected since such data will be required 

8 to support chemical risk assessment. In addition, common population modeling approaches can 

9 be employed based on the data obtained in the MTTT to estimate population-level effects 

10 (Raimondo and McKenney 2005a). 

11 HCDRT

12 The HCDRT is proposed as an option or alternative Tier 2 test to be used in EPA’s EDSP. 

13 Crustaceans, including copepods, are believed to lack E, A, and T hormone axes. In addition, 

14 little is known about why chemicals that may affect the E, A, and T hormone axes through any 

15 known modes of action (i.e., chemicals that would be identified in Tier 1) may interfere with 

16 invertebrate hormone axes or the endpoints measured in the HCDRT. Nevertheless, it must be 

17 assessed whether a vertebrate-active endocrine active substance may affect an invertebrate to 

18 complete a comprehensive assessment of potential risks.

19 Dose-response information, including LOEC, NOEC, and ECx information for the 

20 biological endpoints included in the HCDRT, will be required to support subsequent risk 

21 assessment. In addition, the survival, sex ratio, and fecundity data obtained in the HCDRT can be 

22 used to calculate and predict population growth rate (e.g., intrinsic rate of natural increase 

23 [rm or λ] and age:stage structure. Such modeling may produce additional valuable data for 

24 interpreting data collected in the HCDRT and potentially extrapolating results to 

25 population-level effects (OECD [158] 2011).

26 6.3 Strengths and Limitations of Each Test

27 6.3.1 Strengths and Limitations of the MTTT

28 The recommended MTTT describes a two-generation toxicity test with an estuarine 

29 mysid that considers growth, survival, and reproductive endpoints in parents and offspring as an 

30 integrated measure of toxicant exposure. The test is intended to serve as a higher-tier test with an 

31 aquatic arthropod for collecting definitive concentration-response information on adverse effects 

32 suitable for ecological risk assessment. As with any toxicological assay, strength and limitations 

33 must be recognized and considered when evaluating data and drawing conclusions. Significant 

34 advantages, limitations, and potential further refinements of the MTTT are summarized below.

35 Advantages of the MTTT

36  Many environmental testing laboratories have established experience with A. bahia

37 due to its long-standing use as a standard test model for regulatory toxicity testing. 

38 Laboratories were generally able to successfully perform the MTTT within the 

39 recommended acceptability criteria.

40  Mysids have been used as a test model for evaluating reproductive toxicity for several 

41 decades and an increasing body of literature is available regarding their use for 
DRAFT

Invertebrate Two-Generation ISR 68 May 28, 2013

1 evaluating endocrine toxicity, including the availability of mechanistic endpoints 

2 (e.g., vitellin, ecdysteroid receptor gene expression). 

3  The test can be conducted in continuous or intermittent flow, which has significant 

4 advantages with respect to ensuring consistent water quality and maintaining desired 

5 chemical exposure concentrations. 

6  The control variability of the survival and several of the reproduction (i.e., days to 

7 brood release and offspring production) and growth (i.e., length) endpoints, especially 

8 in the F0 generation, indicate that these should be able to detect significant adverse 

9 effects with adequate statistical power.

10  Common population modeling approaches can be employed based on the data 

11 obtained in the MTTT to estimate population-level effects (Raimondo and 

12 McKenney 2005a). 

13 Limitations of the MTTT

14  A large number of endpoints are included and there is some redundancy between 

15 endpoints (e.g., growth is evaluated by both weight and length). In addition, several 

16 endpoints require animals to be sacrificed, resulting in reduced replication for other 

17 endpoints.

18  Several aspects of the MTTT, such as the collection of newly hatched offspring, 

19 pairing of mature males/females, etc. are time-consuming and resource-demanding. 

20 Also, care and experience is required to minimize non-treatment related mortality due 

21 to handling or impingements. 

22  Considerable variability was observed in endpoint responses between different 

23 laboratories, resulting in reduced power to detect significant differences. Some of the 

24 variability between different laboratories may be attributable to intrinsic differences 

25 in the laboratory animal populations, small differences among laboratories in feeding 

26 and feed quality, etc. 

27  Some of the endpoints were consistently non-responsive (e.g., sex ratio, time to 

28 maturation), and their value may need to be evaluated further.

29  The addition of a second generation significantly adds to the time and resources 

30 required to perform the MTTT. The value of the additional information obtained from 

31 the second generation was not obvious based on the results of the inter-laboratory 

32 validation studies. As discussed in the potential further refinement section, additional 

33 chemicals may need to be tested before the value of the second generation can be 

34 adequately evaluated.

35  In cases where the test chemical causes reductions in fecundity, it may be difficult to 

36 complete the second generation of the MTTT due to insufficient young.

37  While the MTTT provides general guidance for conducting statistical analysis, more 

38 specific guidance may be needed to ensure consistency. Statistical evaluation of the 

39 many endpoints and the potential for confounding factors (e.g., fecundity is related to 

40 female body size in mysids) requires a qualified statistician.
DRAFT

Invertebrate Two-Generation ISR 69 May 28, 2013

1 Potential further refinements of the MTTT

2 The lack of treatment-related differences could have resulted from selected doses that 

3 were too low to bracket any biological effect that may have been suggested by an acute

4 range-finding test. It may be better to select definitive exposure concentrations based on a 

5 longer-term range-finding test. For example, the guideline provides an alternative of conducting 

6 a pared-down life-cycle range-finding test (e.g., five breeding pairs at three dose levels + 

7 controls carried through 21 days). The value of the latter alternative should be evaluated further. 

8 The lack of treatment-related differences could also have resulted from the selection of 

9 test chemicals used in the different validation studies. The selected chemicals were 

10 representative endocrine active chemicals in vertebrates, but may not exhibit endocrine toxicity 

11 in arthropods. Further validation with known arthropod endocrine active chemicals (e.g., insect 

12 growth regulators, ecdysone agonists, etc.) may provide better data for evaluating strengths and 

13 weaknesses of the currently proposed MTTT. Such chemicals are also potentially more likely to 

14 result in transgenerational toxicity.

15 It may be sufficient to use length measurements to evaluate growth, given the difficulty 

16 of accurately measuring changes in weight in mysids. Furthermore, it may be sufficient to 

17 calculate a growth rate based on length at Day 0 and termination. This would reduce the amount 

18 of animals needed for collecting growth endpoint data, resulting in greater replication or 

19 potentially lower loading rates if sufficient statistical power is maintained. It may also be worth 

20 exploring whether non-lethal methods for obtaining length measurements are possible 

21 (e.g., digital imaging and processing).

22 6.3.2 Strengths and Limitations of HCDRT

23 Significant advantages and limitations of the HCDRT are summarized below. 

24 Advantages of HCDRT

25  Relative to other harpacticoid copepod studies in the literature, A. tenuiremis is more 

26 chronically sensitive than all other species published to date where there is 

27 comparative data (ASTM 2004).

28  Laboratories were generally able to successfully culture A. tenuiremis, harvest gravid 

29 copepods, and hatch healthy nauplii needed to conduct the HCDRT. Labs also 

30 generally demonstrated the ability to execute the HCDRT within the stated 

31 performance requirements. 

32  Microplate testing allows for easy (individual-based) tracking of endpoints and high 

33 replication resultin in strong statistical power. 

34  Relatively consistent concentration-dependent delays in development rates, increased 

35 naupliar-specific mortality, mating-success depressions, and reduction of fecundity 

36 were observed during the lindane interlaboratory validation study. 

37  Population modeling may be valuable but is not currently included in guideline.

38  A. tenuiremis has a rapid life cycle (16 to 17 d at 25 ̊C) and can be easily cultured in 

39 water-only and sediment:water matrices.

40 Limitations of the Copepod Test

41  Commercial testing laboratories generally have no prior experience with this species. 

42 Issues of proficiency in the successful application of the test method were identified 
DRAFT

Invertebrate Two-Generation ISR 70 May 28, 2013

1 as an issue with the HCDRT (OECD 2011). For example, some labs had initial 

2 difficulties with the identification of copepodite maturation and determination of sex.

3  Microwell plate design results in difficulties for continuous monitoring of water 

4 quality and difficulties producing consistently accurate and precise measured 

5 concentrations. Consequently, test results need to be reported in nominal 

6 concentrations rather than measured concentrations (OECD 2011). 

7  Absence of effects on most of the endpoints reported in the atrazine study

8 (OECD 2011).

9  Possible lack of sensitivity of the population of A. tenuiremis used in the study

10 (OECD 2011).

11  Influence of mortality (males more sensitive than females) on the sex ratio 

12 (OECD 2011). 

13  Manpower required in the study (50% time of a technician).

14  Limited knowledge of the copepod endocrine system.

15  Small size limits biochemical studies.

16 Potential Further Refinements of the HCDRT

17 Some recommendations for further work were provided by OECD (2011) and are 

18 repeated here:

19  Performing the test method for controls alone to reassure that the test performance 

20 and the validity criteria set out in the HCDRT can be met by the laboratories

21  Need for more information on the basic biology of this test species

22  It would be very useful to have more specific information on how different feeding 

23 levels affect the life history progression of this copepod.

24 6.4 Rationale for the Use of Mysid Versus Copepod Test

25 Currently, the MTTT provides a number of clear advantages over the HCDRT as an 

26 aquatic arthropod test to evaluate chemical effects on invertebrate growth, reproduction, and 

27 survival:

28  While both the MTTT and HCDRT require significant laboratory experience, there is 

29 significantly more experience with the use of mysids as a toxicity test model 

30 compared to copepods

31  There is a large amount of published toxicity data for A. bahia, and available 

32 evidence suggest that mysids are generally more sensitive than other standard toxicity 

33 test species

34  Our understanding of the basic biology of mysids (specifically A. bahia) is 

35 significantly greater than for the proposed harpacticoid copepod species

36  Mysids have been used as a test model for evaluating reproductive toxicity for several 

37 decades, and an increasing body of literature is available regarding their use for 

38 evaluating endocrine toxicity, including the availability of new mechanistic 

39 endpoints (e.g., vitellin, ecdysteroid receptor gene expression)

40  Similar to copepods, mysids are of ecological importance, have a wide geographic 

41 distribution, are available year-round in the field, and can be easily transported and 

42 cultured 
DRAFT

Invertebrate Two-Generation ISR 71 May 28, 2013

1  The MTTT test system allows for continuous or intermittent flow exposures, which 

2 has significant advantages over the static renewal conditions employed in the 

3 HCDRT with respect to ensuring consistent water quality and maintaining desired 

4 chemical exposure concentrations 

5  A more robust inter-laboratory validation study has been conducted with the MTTT 

6 than with the HCDRT where additional validation studies are still underway 

7  The greater body size of mysids makes them more amenable to additional 

8 biochemical/molecular measurements

9  The MTTT includes well-established growth endpoints, which appeared to be among 

10 the most sensitive among the endpoints tested, whereas the HCDRT does not include 

11 growth measurements

12  Although both data obtained through the MTTT and HCDRT can be used for 

13 population modeling, the MTTT has data through two generations.

14

15
DRAFT

Invertebrate Two-Generation ISR 72 May 28, 2013

1 7 REFERENCES

2

3 [Includes works cited and works consulted]

4

5 Andersen, HR, Wollenberger, L, Halling-Sorensen, B, and Kusk, KO. 2001. Development of 

6 Copepod Nauplii to Copepodites—A Parameter for Chronic Toxicity Including Endocrine 

7 Disruption. Environmental Toxicology and Chemistry 20(12): 2821–2829.

8

9 ASTM (American Society for Testing and Materials). 1997. Standard Guide for Conducting 

10 Life-cycle Toxicity Tests with Saltwater Mysids. E 1191-97. American Society for Testing and

11 Materials. West Conshohocken, Pennsylvania.

12

13 ASTM (American Society for Testing and Materials). 2004. Standard Guide for Conducting 

14 Renewal Microplate-Based Life-Cycle Toxicity Tests with a Marine Meiobenthic Copepod. 

15 ASTM Standard No. E2317-04. ASTM, Philadelphia, pp. 1-16.

16

17 Aufderheide, J. 2008a. Prochloraz: Two-Generation Life-Cycle Toxicity Test of the Saltwater 

18 Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by 

19 Battelle-Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, 

20 Columbia, Missouri. USEPA EDSP Task Order 7. ABC Study No. 62295. 101 pages.

21

22 Aufderheide, J. 2008b. Flutamide: Two-Generation Life-Cycle Toxicity Test of the Saltwater 

23 Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by 

24 Battelle-Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, 

25 Columbia, Missouri. USEPA EDSP Task Order 7. ABC Study No. 62300.

26

27 Battelle. 2013. Data Analysis Support for Invertebrate Tier 2 Test and Generation of Integrated 

28 Summary Report. Endocrine Disruptor Screening Program. Task Order No: 2. Statistical Support 

29 for Technical Directive #3. Inter-Laboratory Statistical Analysis. Contract No: EP-W-11-063.

30

31 Bechmann, RK. 1999. Effect of the Endocrine Disrupter Nonylphenol on the Marine Copepod

32 Tisbe battagliai. The Science of the Total Environment. 233(1-3):33-46.

33

34 Bejarano, AC, and Chandler, GT. 2003. Reproductive and Developmental Effects of Atrazine on 

35 the Estuarine Meiobenthic Copepod Amphiascus Tenuiremis. Environmental Toxicology and

36 Chemistry 22(12): 3009–3016. 

37

38 Bejarano, AC, Chandler, GT, and Decho, AW. 2005. Influence of natural dissolved organic 

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

40 fipronil on the meiobenthic estuarine copepod Amphiascus tenuiremis. Journal of Experimental 

41 Marine Biology and Ecology 321: 43–57.

42

43 Bejarano, AC, Chandler, GT, He, L, and Coull, BC. 2006. Individual to population level effects 

44 of South Louisiana crude oil water accommodated hydrocarbon fraction (WAF) on a marine 

45 meiobenthic copepod. Journal of Experimental Marine Biology and Ecology 332: 49–59.

46
DRAFT

Invertebrate Two-Generation ISR 73 May 28, 2013

1 Bengtsson, B-E. 1978. Use of a harpacticoid copepod in toxicity tests. Marine Pollution Bulletin. 

2 9:238-241.

3

4 Block, DS, and Bradbury, N. 2012a. Effect of Lindane on the Survival, Development, and 

5 Reproduction of the Marine Copepod Amphiascus tenuiremis. Draft Report. Sponsored by RTI 

6 International, Research Triangle Institute, North Carolina. Prepared by Block Environmental

7 Services, Pleasant Hill, California. USEPA EDSP Task order 30. Report No. 27569. 127 pages.

8

9 Block, DS, and Bradbury, N. 2012b. Effect of Vinclozolin on the Survival, Development, and 

10 Reproduction of the Marine Copepod Amphiascus tenuremis. Final Report. Sponsored by RTI 

11 International, Research Triangle Park, North Carolina. Prepared by Block Environmental 

12 Services, Pleasant Hill, California. EPA Contract EP-W-06-026. USEPA EDSP Task Order 30. 

13 Report No. 27568. 180 pages.

14

15 Box, GEP. 1953. Non-normality and tests on variances, Biometrika 40, 318-335.

16

17 Breitholtz, M. and Bengtsson, BE. 2001. Oestrogens Have No Hormonal Effect on the

18 Development and Reproduction of the Harpacticoid Copepod Nitocra spinipes. Marine Pollution 

19 Bulletin. 42(10):879-886.

20

21 Breitholtz, M, Wollenberger, L, and Dinan, L. 2003. Effects of Four Synthetic Musks on the Life 

22 Cycle of the Harpacticoid Copepod Nitocra spinipes. Aquatic Toxicology. 63(2):103-118.

23

24 Breitholtz M., Wollenberger, L. 2003. Effects of three PBDEs on development, reproduction and 

25 population growth rate (rm) of the harpacticoid copepod Nitocra spinipes. Aquatic Toxicology. 

26 64:85-96.

27

28 Brown RJ, Rundle SD, Hutchinson TH, Williams TD, Jones MB. 2003. Copepod life-cycle test 

29 and growth model for interpreting the effects of lindane. Aquatic Toxicology. 63:1-11.

30

31 Brown RJ, Rundle SD, Hutchinson TH, Williams TD, Jones MB. 2005. A microplate freshwater 

32 copepod bioassay for evaluating acute and chronic effects of chemicals. Environmental 

33 Toxicology and Chemistry. 24(6):1528-1531.

34

35 Bushong, SJ, Zeigenfuss, MC, Unger, MA, Hall Jr., LW. 1990. Chronic tributyltin toxicity 

36 experiments with the Chesapeake Bay copepod, Acartia tonsa. Environmental Toxicology and 

37 Chemistry. 9:359-366.

38

39 Cary, TL, Chandler, GT, Volz, DC, Walse, SS, and Ferry, JL. 2004. Phenylpyrazole Insecticide 

40 Fipronil Induces Male Infertility in the Estuarine Meiobenthic Crustacean Amphiascus 

41 tenuiremis. Environmental Science and Technology. 38(2):522–528.

42

43 Caspers, N. 1998. No estrogenic effects of bisphenol-A in Daphnia magna STRAUS. Bulltin of 

44 Environmental Contamination and Toxicology. 61:143-148.

45
DRAFT

Invertebrate Two-Generation ISR 74 May 28, 2013

1 Chandler, GT. 2012. Summary USEPA Laboratory Performance Report for the Block 

2 Environmental Services, Inc., Validation of the OECD 2009 Draft “Harpacticoid Copepod

3 Development and Reproduction Test with Amphiascus tenuiremis” using the Reference Test 

4 Compound Vinclozolin. Unpublished review comments to P. Browne, U.S. EPA, from 

5 University of South Carolina.

6

7 Chandler, GT, Cary, TL, Volz, DC, Walse, SS, Ferry, JL, Klosterhaus, SL. 2004a. Fipronil 

8 Effects on Estuarine Copepod (Amphiascus Tenuiremis) Development, Fertility, and 

9 Reproduction: A Rapid Life-Cycle Assay in 96-Well Microplate Format. Environmental 

10 Toxicology and Chemistry. 23(1): 117–124.

11

12 Chandler, GT, Cary, TL, Bejarano, AC, Pender, J, and Ferry, JL. 2004b. Population 

13 Consequences of Fipronil and Degradates to Copepods at Field Concentrations: An Integration 

14 of Life Cycle Testing with Leslie Matrix Population Modeling. Environmental Science and 

15 Technology. 38(23): 6407–6414.

16

17 Chandler, GT, and Green, AS. 1996. A 14-day harpacticoid copepod reproduction bioassay for 

18 laboratory and field contaminated muddy sediments. Techniques in Aquatic Toxicology. Lewis, 

19 Boca Raton, FL, USA, 23-39.

20

21 Chandler, GT and Green, AS. 2001. Development-stage specific life-cycle bioassay for

22 assessment of sediment-associated toxicant effects on benthic copepod production.

23 Environmental Toxicology and Chemistry. 20:171-178.

24

25 Chang, ES. 1993. Comparative Endocrinology of Molting and Reproduction: Insects and

26 Crustaceans. Annual Reviews of Entomology. 38:161-180.

27

28 Chang, ES, Chang, SA, and Mulder, EP. 2001. Hormones in the Lives of Crustaceans: an

29 Overview. American Zoologist. 41(5):1090-1097.

30

31 Cuzin-Roudy, J and Saleuddin, ASM. 1989. The Mysid Siriella armata, a Biological Model for 

32 the Study of Hormonal-Control of Molt and Reproduction in Crustaceans - a Review. 

33 Invertebrate Reproduction and Development. 16(1-3):33-42.

34

35 D’Agostino, A, Finney, C. 1974. The effect of copper and cadmium on the development of 

36 Tigriopus japonicus. In: Pollution and physiology of marine organisms, Academic Press, pp. 

37 445- 463.

38

39 deFur, P L. 2004. Use and role of invertebrate models in endocrine disruptor research and testing. 

40 ILAR journal/National Research Council, Institute of Laboratory Animal Resources. 45(4), 484-

41 493.

42

43 deFur, PL, Crane, M, Ingershold, C, Tattersfield, L. 1999. Endocrine Disruption in Invertebrates: 

44 Endocrinology, Testing and Assessment. Society of Environmental Toxicology and Chemistry, 

45 Pensacola, FL, USA.

46
DRAFT

Invertebrate Two-Generation ISR 75 May 28, 2013

1 Depledge, MH. and Billinghurst, Z. 1999. Ecological Significance of Endocrine Disruption in 

2 Marine Invertebrates. Marine Pollution Bulletin. 39(1-12):32-38.

3

4 Dhadialla, TS, Carlson, GR, and Le, DP. 1998. New insecticides with ecdysteroidal and juvenile 

5 hormone activity. Annual review of entomology. 43(1):545-569.

6

7 Dunnett, C.W. 1955. Journal of American Statistic Association 50, 1096. 

8

9 EDSP. July 16, 2007. Validation of Screening and Testing Assays Proposed for the EDSP. 

10 Draft version 5.4. U.S. Environmental Protection Agency Endocrine Disruptor Screening 

11 Program, available at 

12 http://www.epa.gov/scipoly/oscpendo/pubs/edsp_validation_paper_v%205.4.pdf. 21 pages.

13

14 EDSTAC (Endocrine Disruptor Screening and Testing Advisory Committee). 1998. Final

15 Report. EPA/743/R-98/003. U.S. Environmental Protection Agency. Washington, D.C.

16

17 Environment Agency. 2007. Endocrine disruption horizon scanning: aquatic invertebrates review. 

18 Environment Agency, Bristol, United Kingdom. Science Report: #SC030276/SR1.

19

20 Feder, PI., and Ma, J. 2012a. Endocrine Disruptor Screening Program Technical Directive 03 -

21 Mysid Data Analysis – SMV (Smithers Viscient) Lindane Analysis Issues with Endpoints and 

22 STATCHARRMS Analysis Procedures. Draft Internal Notes on Intra-Laboratory Data Analysis 

23 prepared by Battelle for USEPA EDSP Task Order 2. 4 pages.

24

25 Feder, PI, and Ma, J. 2012b. Endocrine Disruptor Screening Program Technical Directive 03 -

26 Mysid Data Analysis – SMV (Smithers Viscient) Lindane Analysis Data Plots. Draft Intra27 Laboratory Data Analysis prepared by Battelle for USEPA EDSP Task Order 2. 12 pages.

28

29 Feder, PI, and Ma, J. 2012c. Endocrine Disruptor Screening Program Technical Directive 03 -

30 Mysid Data Analysis – SMV (Smithers Viscient) Lindane Analysis Statistical Tables. Draft 

31 Intra-Laboratory Data Analysis prepared by Battelle for USEPA EDSP Task Order 2. 15 pages.

32

33 Feder, PI, and Ma, J. 2012d. Endocrine Disruptor Screening Program Technical Directive 03 -

34 Mysid Data Analysis – SMV (Smithers Viscient) Octylphenol Analysis Statistical Tables and 

35 Data Plots. Draft Intra-Laboratory Data Analysis prepared by Battelle for USEPA EDSP Task 

36 Order 2. 29 pages.

37

38 Finney DJ, Latscha, R, Bennett, BM, Hsu, P. 1963. Tables for testing significance in a 2x2 

39 contingency table. Cambridge University Press, London. 

40

41 FRN (Federal Register Notice). 1998. Endocrine Disruptor Screening Program. Federal Register. 

42 August 11, 1998. 63(154):42852-42855.

43

44 Fockedey, N, Ghekiere, A, Bruwiere, S, Janssen, CR, and Vincx, M. 2006. Effect of salinity and 

45 temperature on the intra-marsupial development of the brackish water mysid Neomysis integer 

46 (Crustacea: Mysidacea). Marine Biology. 148(6):1339-1356.
DRAFT

Invertebrate Two-Generation ISR 76 May 28, 2013

1

2 Ford, AT, Fernandes, TF, Read, PA, Robinson, CD, and Davies, IM. 2004b. The Costs of 

3 Intersexuality: A Crustacean Perspective. Marine Biology. 145:951-957.

4

5 Ford, AT, Fernandes, TF, Rider, SA, Read, PA, Robinson, CD, and Davies, IM. 2004a. 

6 Endocrine Disruption in a Marine Amphipod? Field Observations of Intersexuality and 

7 Demasculinisation. Marine Environmental Research. 58:169-173.

8

9 Gaertner, K, Chandler, GT, Quattro, J, Ferguson, PL, and Sabo-Attwood, T. 2012. Identification 

10 and expression of the ecdysone receptor in the harpacticoid copepod, Amphiascus tenuiremis, in 

11 response to fipronil. Ecotoxicology and Environmental Safety. 76:39–45.

12

13 Ghekiere, A, Fenske, M, Verslycke, T, Tyler, C, Janssen, CR. 2005. Development of a 

14 quantitative enzyme-linked immunosorbent assay to study vitellogenesis in the mysid Neomysis 

15 integer (Crustacea: Mysidacea). Comp. Biochem. Physiol. A. 142(1):43–49.

16

17 Ghekiere, A, Fockedey, N, Verslycke, T, Vincx, M, and Janssen, CR. 2007. Marsupial 

18 development in the mysid< i> Neomysis integer</i>(Crustacea: Mysidacea) to evaluate the 

19 effects of endocrine-disrupting chemicals. Ecotoxicology and environmental safety. 66(1):9-15.

20

21 Ghekiere, A, Verslycke, T, and Janssen, C. 2006a. Effects of methoprene, nonylphenol, and 

22 estrone on the vitellogenesis of the mysid Neomysis integer. General and comparative 

23 endocrinology. 147(2):190-195.

24

25 Ghekiere, A, Verslycke, T, Fockedey, N, and Janssen, CR. 2006b. Non-target effects of the 

26 insecticide methoprene on molting in the estuarine crustacean Neomysis integer (Crustacea: 

27 Mysidacea). Journal of experimental marine biology and ecology. 332(2):226-234.

28

29 Gourmelon A, Ahtiainen J. 2007. Developing test guidelines on invertebrate development and 

30 reproduction for the assessment of chemicals including potential endocrine active substances –

31 The OECD perspective. Ecotoxicology. 16:161-167.

32

33 Hahn, T, Liess, M, and Schulz, R 2001. Effects of the Hormone Mimetic Insecticide 

34 Tebufenozide on Chironomus riparius Larvae in Two Different Exposure Setups. Ecotoxicology 

35 and Environmental Safety. 49(2):171-178.

36

37 Hahn, T, Schenk, K, and Schulz, R. 2002. Environmental Chemicals With Known Endocrine 

38 Potential Affect Yolk Protein Content in the Aquatic Insect Chironomus riparius. Environmental 

39 Pollution. 120(3):525-528.

40

41 Hahn, T, and Schulz, R. 2002. Ecdysteroid Synthesis and Imaginal Disc Development in the 

42 Midge Chironomus riparius as Biomarkers for Endocrine Effects of Tributyltin. Environmental 

43 Toxicology and Chemistry. 21(5):1052-1057.

44

45 Hala, D, and Garcia, S. 2012a. Mysid (Americamsys bahia) Two-Generation Test with Lindane. 

46 Sponsored by RTI International, Research Triangle Park, North Carolina. Performed by 
DRAFT

Invertebrate Two-Generation ISR 77 May 28, 2013

1 University of North Texas, Denton, Texas. USEPA EDSP Task Order 30. Project ID# 100.01. 

2 198 pages.

3

4 Hala, D, and Garcia, S. 2012b. Mysid (Americamsys bahia) Two-Generation Test with 

5 Vinclozolin. Sponsored by RTI International, Research Triangle Park, North Carolina. 

6 Performed by University of North Texas, Denton, Texas. USEPA EDSP Task Order 30. Project 

7 ID# 100.02. 198 pages.

8

9 Hanaee, J, Agh, N., Hanaee, M., Delazar, A., and Sarker, SD. 2005. Studies on the Enrichment 

10 ofArtemia urmiana Cysts for Improving Fish Food Value. Animal Feed Science and Technology. 

11 120:107-112.

12

13 Hicks, GRF, Coull, BC. 1983. The ecology of marine meiobenthic harpacticoid copepods. 

14 Ocenogr. Mar. Biol. Ann. Rev. 21:67-125.

15

16 Hicks, GR, Coull, BC. 1986. Distribution and behaviour of meiofaunal copepods inside and 

17 outside seagrass beds. Mar. Ecol. Prog. Ser. 31:159-170.

18

19 Hicks, S. 2008a. 4-tert-Octylphenol: Two-Generation Life-Cycle Toxicity Test of the Saltwater 

20 Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by 

21 Battelle-Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, 

22 Columbia, Missouri. USEPA EDSP Task Order 7. ABC Study No. 62308. 128 pages.

23

24 Hicks, S. 2008b. Ketoconazole: Two-Generation Life-Cycle Toxicity Test of the Saltwater 

25 Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by 

26 Battelle-Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, 

27 Columbia, Missouri. USEPA EDSP Task Order 7. ABC Study No. 62304. 128 pages.

28

29 Hicks, SL. 2009a. Perfluorodecanoic Acid (PFDA): Two-Generation Life-Cycle Toxicity Test 

30 of the Saltwater Mysid, Americamysis bahia, Conducted under Flow-Through Test Conditions. 

31 Sponsored by Battelle-Pacific Northwest Division, Sequim, Washington. Performed bABC 

32 Laboratories, Columbia, Missouri. USEPA EDSP Task Order 18. ABC Study No. 64079. 146 

33 pages.

34

35 Hicks, SL. 2009b. Atrazine: Two-Generation Life-Cycle Toxicity Test of the Saltwater Mysid, 

36 Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by Battelle37 Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, Columbia, 

38 Missouri. USEPA EDSP Task Order 18. ABC Study No. 64078. 141 pages.

39

40 Hicks, SL. 2009c. Prochloraz: Two-Generation Life-Cycle Toxicity Test of the Saltwater Mysid, 

41 Americamysis bahia, Conducted under Flow-Through Test Conditions. Sponsored by Battelle42 Pacific Northwest Division, Sequim, Washington. Performed by ABC Laboratories, Columbia,

43 Missouri. USEPA EDSP Task Order 18. ABC Study No. 64076. 138 pages.

44

45 Hirano, M, Ishibashi, H, Kim, E-Y, Arizon, K, Iwata, H. 2010. Development of an in vitro 

46 Reporter Gene Assay for Screening the Ecdysone Receptor Agonists in Mysid Crustacean. 
DRAFT

Invertebrate Two-Generation ISR 78 May 28, 2013

1 Interdisciplinary Studies on Environmental Chemistry — Environmental Specimen Bank, Eds., 

2 T. Isobe, K. Nomiyama, A. Subramanian and S. Tanabe, pp. 141–147.

3

4 Hollister, TA, Heitmuller, PT, Parrish, PR, and Dyar, EE. 1980. Studies to Determine 

5 Relationships Between Time and Toxicity of an Acidic Effluent and an Alkaline Effluent to Two 

6 Estuarine Species. In: Aquatic Toxicology. Pp. 251-265. American Society for Testing and 

7 Materials. Philadelphia, Pennsylvania.

8

9 Huberman, A. 2000. Shrimp endocrinology. A review. Aquaculture. 191:191–208.

10

11 Hutchinson, TH, Brown, R, Brugger, KE, Campbell, PM, Holt, M, Lange, R, Mccahon, P,

12 Tattersfield, LJ, and Van Egmond, R. 2000. Ecological Risk Assessment of Endocrine

13 Disruptors. Environmental Health Perspectives. 108(11):1007-1014.

14

15 Hutchinson, TH, Pounds, NA, Hampel, M, and Williams, TD. 1999a. Impact of Natural and 

16 Synthetic Steroids on the Survival, Development and Reproduction of Marine Copepods (Tisbe 

17 battagliai). The Science of the Total Environment. 233(1-3):167-179.

18

19 Hutchinson, TH, Pounds, NA, Hampel, M, and Williams, TD. 1999b. Life-Cycle Studies

20 With Marine Copepods (Tisbe battagliai) Exposed to 20-Hydroxyecdysone and 

21 Diethylstilbestrol. Environmental Toxicology and Chemistry. 18(12):2914-2920.

22

23 Huys, R, Gee, JM, Moore, CG, Hamond, R. 1996. Marine and brackish water harpacticoid 

24 copepods (Part 1). (Huys, R., Gee, J. M., Moore, C. G. and Hamond, R. Eds.), Synopsis of the 

25 British Fauna (New Series), The Dorset Press, Great Britain, ISBN: 1-85153-256-0.

26

27 Ingersoll, CG, Hutchinson, TH, Crane, M, Dodson, S, DeWitt, T, Gies, A, Huet, M-C,

28 McKenney, CL, Oberdörster, E, Pascoe, D, Versteeg, DJ, and Warwick, O. 1999. The

29 Endocrinology of Invertebrates. In: Laboratory toxicity tests for evaluating potential effects of 

30 endocrine disrupting compounds, DeFur, P.L., Crane, M., Ingersoll, C., and Tetterfield, L. eds., 

31 Pp. 107–197. Society of Environmental Toxicology and Chemistry (SETAC) Press. Pensacola, 

32 Florida.

33

34 Johnston NM, Ritz DA. 2001. Synchronous development and release of broods by the swarming 

35 mysids Anisomysis mixta australis, Paramesopodopsis rufa, and Tenagomysis tasmaniae 

36 (Mysidacea: Crustacea). Mar Ecol Prog Ser. 223:225–233.

37

38 Khan A, Barbieri J, Khan S, Sweeney F. 1992. A new short term mysid toxicity test using sexual 

39 maturity as an endpoint. Aquat Toxicol 23:97–105.

40

41 Kropp, RK, and Cullinan, VI. 2008. Evaluation of Proposed Ecotoxicological Testing Guidelines 

42 Using Mysids to Determine Endocrine Disruptive Effects from Chemical Exposure to Prochloraz, 

43 Flutamide, Ketoconazole, and 4-tert-Octylphenol. Washington DC, U.S. Environmental 

44 Protection Agency Task Order 7 Summary Report PNWD-3960 prepared by Battelle-Pacific 

45 Northwest Division. 49 pages.

46
DRAFT

Invertebrate Two-Generation ISR 79 May 28, 2013

1 Kropp, RK, Cullinan, VI, and Hicks, SL. 2009. Evaluation of Proposed Ecotoxicological Testing 

2 Guidelines Using Mysids to Determine Endocrine Disruptive Effects from Chemical Exposure to 

3 Lindane, Atrazine, Prochloraz, and Perfluorodecanoic Acid. Washington DC, U.S. 

4 Environmental Protection Agency Task Order 18 Summary Report prepared by Battelle-Pacific 

5 Northwest Division and ABC Laboratories. 34 pages.

6

7 Krueger, HO, Claude, MD, and Kendall, TZ. 2012. Two-Generation Life-Cycle Toxicity Test 

8 [Lindane] with Mysids (Americamsys bahia) Interlaboratory Comparison. Draft Report. 

9 Washington DC, U.S. Environmental Protection Agency, prepared by Wildlife International, 

10 Easton, Maryland. USEPA EDSP Contract No. EP-W-06-026. Study No. 185A-102-1. 269 pages.

11

12 Kuhn, A, Munns, WR, Champlin, D, McKinney, R, Tagliabue, M, Serbst, J, and Gleason, T. 

13 2001. Evaluation of the Efficacy of Extrapolation Population Modeling to Predict the Dynamics 

14 of Americamysis bahia Populations in the Laboratory. Environmental Toxicology and Chemistry. 

15 20(1):213-221.

16

17 Kuhn, A, Munns, WR, Poucher, S, Champlin, D, and Lussier, S. 2000. Prediction of

18 Population-Level Response from Mysid Toxicity Test Data using Population Modeling

19 Techniques. Environmental Toxicology and Chemistry. 19(9):2364-2371.

20

21 Kusk KO, Petersen S. 1997. Acute and chronic toxicity of tributyltin and linear alkylbenzene 

22 sulfonate to the marine copepod Acartia tonsa. Environmental Toxicology and Chemistry. 

23 16:1629-1633.

24

25 Kusk KO, Wollenberger L. 2007. Towards and internationally harmonized test method for 

26 reproductive and developmental effects of endocrine disrupters in marine copepods. 

27 Ecotoxicology. 16:183-195.

28

29 Lafont, R. 2000. The Endocrinology of Invertebrates. Ecotoxicology. 9(1-2):41-57.

30

31 Le Page, G, and Brown, RJ. 2011. Determination of the effect of lindane on the survival and 

32 reproduction of the marine copepod Amphiascus tenuremis (semi-static procedure with 

33 individually contained animals). Draft Report. Washington DC, U.S. Environmental Protection 

34 Agency, prepared by Brixham Environmental Laboratory, Devon, UK. Study No. 11-0030/B, 

35 Report No. BR0476/B. 48 pages.

36

37 LeBlanc, G. 2007.Crustacean endocrine toxicology: a review.Ecotoxicology. 16:

38 61–81.

39

40 Leger, P, and Sorgeloos, P. 1984. International study on Artemia. XXIX. Nutritional evaluation 

41 of Artemia nauplii from different geographical origin for the marine crustacean Mysidopsis bahia. 

42 Marine Ecology Progress Series. 15:307-309.

43

44 Lee, MR. 2012. Lindane and 4-Tert-octylphenol – Two-Generation Life-Cycle Toxicity Test 

45 with Mysids (Americamsys bahia). Final Report. Sponsored by RTI International, Research 
DRAFT

Invertebrate Two-Generation ISR 80 May 28, 2013

1 Triangle Park, North Carolina. Performed by Smithers Viscient, Wareham, Massachusetts. SMV 

2 Study No. 13888.6109. EPA Contract No. EP-W-06026. 468 pages.

3

4 Leslie, PH. 1945. On the use of matrices in certain population mathematics. Biometrika. 33:183–

5 212.

6

7 Lussier, SM, Gentile, JH, and Walker, J. 1985. Acute and Chronic Effects of Heavy Metals and 

8 Cyanide on Mysidopsis bahia (Crustacea:Mysidacea). Aquatic Toxicology. 7:25-35.

9

10 Lussier, SM, Kuhn, A, and Comeleo, R. 1999. An Evaluation of the Seven-Day Toxicity Test 

11 with Americamysis bahia (Formerly Mysidopsis bahia). Environmental Toxicology and 

12 Chemistry. 18(12):2888-2893.

13

14 McKenney CL Jr. 1982. Interrelationships between energy metabolism, growth dynamics, and 

15 reproduction during the life cycle of Mysidopsis bahia as influenced by sublethal endrin 

16 exposure. In Vernberg WB, Calabrese A, Thurberg FP, Vernberg FJ, eds, Physiological 

17 Mechanisms of Marine Pollutant Toxicity. Academic, New York, NY, USA, pp 447–476.

18

19 McKenney, CL Jr. 1985. Associations Between Physiological Alterations and Population 

20 Changes in an Estuarine Mysid During Chronic Exposure to a Pesticide. In: Marine Pollution 

21 and Physiology: Recent Advances. Pp. 397-418. University of South Carolina Press. Columbia, 

22 S.C.

23

24 McKenney, CL Jr. 1986. Influence of the organophosphate insecticide fenthion on Mysidopsis 

25 bahia exposed during a complete life cycle I. Survival, reproduction, and age-specific growth. 

26 Dis Aquat Organ. 1:131–139.

27

28 McKenney, CL Jr. 1994. Alterations in Growth, Reproduction, and Energy Metabolism of 

29 Estuarine Crustaceans as Indicators of Pollutant Stress. In: Biological Monitoring of the 

30 Environment: A Manual of Methods. Salanki J., Jeffrey, D., and Hughes, G.M. eds., Pp. 111-115. 

31 CAB International. Wallingford, England.

32

33 McKenney, CL Jr. 1996. The Combined Effects of Salinity and Temperature on Various

34 Aspects of the Reproductive Biology of the Estuarine Mysid, Mysidopsis bahia. Invertebrate 

35 Reproduction and Development. 29(1):9-18.

36

37 McKenney, CL Jr. 1998. Physiological dysfunction in estuarine mysids and larval decapods with 

38 chronic pesticide exposure. In: Wells PG, Lee K, Blaise C (eds) Microscale testing in aquatic 

39 toxicology: advances, techniques, and practice. CRC Press, Boca Raton, FL, USA, pp 465–476.

40

41 McKenney CL Jr. 2005. The influence of insect juvenile hormone agonists on metamorphosis 

42 and reproduction in estuarine crustaceans. Integr. Comp. Biol. 45:97–105.

43

44 McKenney, CL Jr., and Celestial, DM. 1996. Modified Survival, Growth and Reproduction in an 

45 Estuarine Mysid (Mysidopsis bahia) Exposed to a Juvenile Hormone Analogue Through a 

46 Complete Life Cycle. Aquatic Toxicology. 35(1):11-20.
DRAFT

Invertebrate Two-Generation ISR 81 May 28, 2013

1

2 McKenney CL Jr, Hamaker TL, Matthews E. 1991. Changes in the physiological performance 

3 and energy metabolism of an estuarine mysid (Mysidopsis bahia) exposed in the laboratory 

4 through a complete life cycle to the defoliant DEF. Aquat Toxicol. 19:123–136.

5

6 McKenney CL Jr, and Matthews E. 1990. Alterations in the energy metabolism of an estuarine 

7 mysid (Mysidopsis bahia) as indicators of stress from chronic pesticide exposure. Mar Environ 

8 Res. 30:1–19.

9

10 Mees, J, Abdulkerim, Z, and Hamerlynck, O. 1994. Life-History, Growth and Production of 

11 Neomysis-integer in the Westerschelde Estuary (SW Netherlands). Marine Ecology Progress 

12 Series. 109(1):43-57.

13

14 Mees, J, Fockedey, N, Dewicke, A, Janssen, CR, and Sorbe, J-C. 1995. Aberrant Individuals of 

15 Neomysis integer and Other Mysidacea: Intersexuality and Variable Telson Morphology. 

16 Netherlands Journal of Aquatic Ecology. 29(2):161-166.

17

18 Moore, CG, and Stevenson, JM. 1991. The Occurrence of Intersexuality on Harpacticoid

19 Copepods and its Relationship with Pollution. Marine Pollution Bulletin. 22:72-74.

20

21 Moore, CG, and Stevenson, JM. 1994. Intersexuality in Benthic Harpacticoid Copepods in the

22 Firth of Forth, Scotland. Journal of Natural History. 28:1213-1230.

23

24 Moore DRJ, Warren-Hicks W, Parkhurst BR, Teed RS, Baird RB, Berger R, Dentone DL, and 

25 Pletl JJ. 2000. Intra- and intertreatment variability in reference toxicant tests: implications for 

26 whole effluent toxicity testing programs. Environ Toxicol Chem 19(1):105-112.

27

28 Mu, XY, and LeBlanc, GA. 2002a. Developmental Toxicity of Testosterone in the Crustacean

29 Daphnia magna Involves Anti-Ecdysteroidal Activity. General and Comparative Endocrinology. 

30 129(2):127-133.

31

32 Mu, XY, and LeBlanc, GA. 2002b. Environmental Antiecdysteroids Alter Embryo Development

33 in the Crustacean Daphnia magna. Journal of Experimental Zoology. 292(3):287-292.

34

35 Mu, XY, and LeBlanc, GA. 2004. Synergistic Interaction of Endocrine-Disrupting Chemicals:

36 Model Development using an Ecdysone Receptor Antagonist and a Hormone Synthesis Inhibitor.

37 Environmental Toxicology and Chemistry. 23(4):1085-1091.

38

39 Nagaraju, GPC, Suraj, NJ, and Reddy, PS. 2003. Methyl Farnesoate Stimulates Gonad

40 Development in Macrobrachium malcolmsonii (H. Milne Edwards) (Decapoda, Palaemonidae).

41 Crustaceana. 76:1171-1178.

42

43 NIEHS. 1997. Validation and Regulatory Acceptance of Toxicological Test Methods, A Report 

44 of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods. 

45 NIH Report 97-3981. Research Triangle Park, NC.

46
DRAFT

Invertebrate Two-Generation ISR 82 May 28, 2013

1 Nimmo, DR, Bahner, LH, Rigby, RA, Sheppard, JM, and Wilson, AJ Jr. 1977. Mysidopsis bahia: 

2 An Estuarine Species Suitable for Life-Cycle Toxicity Tests to Determine the Effects of a 

3 Pollutant. In: Aquatic Toxicology and Hazard Evaluation, Mayer F.L. and Hamelink, J.L. eds. Pp. 

4 109-116. American Society for Testing and Materials. Philadelphia, PA.

5

6 Nimmo, DR, and Hamaker, TL. 1982. Mysids in Toxicity Testing - A Review. Hydrobiologia. 

7 93(1-2):171-178.

8

9 Nimmo, DR, Hamaker, TL, Mathews, E, and Moore, JC. 1981. An Overview of the Acute and 

10 Chronic Effects of 1st and 2nd Generation Pesticides on an Estuarine Mysid Mysidopsis bahia. 

11 In: Biological Monitoring Of Marine Pollutants; Symposium On Pollution And Physiology Of 

12 Marine Organisms. Pp. 3-20. Academic Press, Inc. New York, N.Y.

13

14 Nimmo, DR, Hamaker, TL, and Sommers, CA. 1978. Entire Life-Cycle Toxicity Test using

15 Mysids (Mysidopsis bahia) in Flowing Water. EPA-600/9-78-010. U.S. Environmental

16 Protection Agency, Environmental Research Laboratory, Gulf Breeze, FL. (ERL, GB X107).

17

18 Oberdörster, E, and Cheek, AO. 2001. Gender Benders at the Beach: Endocrine Disruption in 

19 Marine and Estuarine Organisms. Environmental Toxicology and Chemistry. 20(1):23-36.

20

21 OECD (Organization for Economic Cooperation and Development). 1996. Final Report of the 

22 OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative 

23 Toxicological Test Methods.

24

25 OECD (Organization for Economic Cooperation and Development). 2004a. Mysid Two26 Generation [Toxicity] Test Guideline, Draft Proposal for a New Guideline. Organisation for 

27 Economic Co-Operation and Development. 18 pages.

28

29 OECD (Organization for Economic Cooperation and Development). 2004b. Harpacticoid 

30 Copepod Development and Reproduction Test, OECD Draft Guidelines for Testing of Chemicals, 

31 Proposal for a New Guideline. Organisation for Economic Co-Operation and Development. 38 

32 pages.

33

34 OECD (Organization for Economic Cooperation and Development). 2005. Proposal for a New 

35 Guideline: Calanoid Copepod Development and Reproduction Test with Acartia tonsa. OECD 

36 Draft Guidelines for Testing of Chemicals. 39 pp.

37

38 OECD (Organization for Economic Cooperation and Development). 2005a. Guidance Document 

39 on the Validation and International Acceptance of New or Updated Test Methods for Hazard 

40 Assessment. 34.

41

42 OECD (Organization for Economic Cooperation and Development). 2006. Detailed Review 

43 Paper on Aquatic Arthropods in Life Cycle Toxicity Tests with an Emphasis on Developmental, 

44 Reproductive and Endocrine Disruptive Effects. Organisation for Economic Co-Operation and 

45 Development Series on Testing and Assessment Number 55. Environment Directorate, Joint 

46 Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and 
DRAFT

Invertebrate Two-Generation ISR 83 May 28, 2013

1 Biotechnology. ENV/JM/MONO(2006)22. JT03212400. 125 pages. Available at: 

2 http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2006)22&doc

3 language=en.

4

5 OECD (Organization for Economic Cooperation and Development). 2007. Validation Report of 

6 the Full Life-Cycle Test with the Harpacticoid Copepods Nitocra Spinipes and Amphiascus 

7 Tenuriremis and the Calanoid Copepod Acartia Tonsa—Phase 1. Organisation for Economic Co8 Operation and Development Series on Testing and Assessment Number 79. Environment 

9 Directorate, Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, 

10 Pesticides and Biotechnology. ENV/JM/MONO(2007)26. JT03231276. 102 pages. Available at: 

11 http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2007)26&doc

12 language=en.

13

14 OECD (Organization for Economic Cooperation and Development). 2008. Report of the 

15 Validation of an Enhancement of OECD TG 211: Daphnia Magna Reproduction Test. 

16 Organisation for Economic Co-Operation and Development Series on Testing and Assessment 

17 Number 93. Environment Directorate, Joint Meeting of the Chemicals Committee and the 

18 Working Party on Chemicals, Pesticides and Biotechnology. ENV/JM/MONO(2008)20. 

19 JT03249178. 137 pages. Available at: 

20 http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2008)20&doc

21 language=en.

22

23 OECD (Organization for Economic Cooperation and Development). 2009. Harpacticoid 

24 Copepod Development and Reproduction Test with Amphiascus tenuiremis, OECD Draft 

25 Guidelines for Testing of Chemicals, Proposal for a New Guideline. Organisation for Economic 

26 Co-Operation and Development. 26 pages.

27

28 OECD (Organization for Economic Cooperation and Development). 2010a. Mysid Two29 Generation [Toxicity] Test Guideline, Draft Proposal for a New Guideline. Organisation for 

30 Economic Co-Operation and Development. 18 pages.

31

32 OECD (Organization for Economic Cooperation and Development). 2010b. Detailed Review 

33 Paper (DRP) on Molluscs Life-Cycle Toxicity Testing. Organisation for Economic Co-Operation 

34 and Development Series on Testing and Assessment Number 121. Environment Directorate, 

35 Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and 

36 Biotechnology. ENV/JM/MONO(2010)9. JT03284405. 182 pages. Available at: 

37 http://search.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2010)9&docla

38 nguage=en.

39

40 OECD (Organization for Economic Cooperation and Development). 2011. Report of Progress on 

41 the Interlaboratory Validation of the OECD Harpacticoid Copepod Development and 

42 Reproduction Test. Organisation for Economic Co-Operation and Development Series on 

43 Testing and Assessment Number 158. Environment Directorate, Joint Meeting of the Chemicals 

44 Committee and the Working Party on Chemicals, Pesticides and Biotechnology. 

45 ENV/JM/MONO(2011)38. JT03306012. 53 pages. Available at: 

46 http://www.oecd.org/chemicalsafety/testingofchemicals/48542861.pdf.
DRAFT

Invertebrate Two-Generation ISR 84 May 28, 2013

1

2 Oehlmann, J. and Schulte-Oehlmann, U. 2003. Endocrine Disruption in Invertebrates. Pure and 

3 Applied Chemistry. 75(11-12):2207-2218.

4

5 Olmstead, AW and LeBlanc, GA. 2002. Juvenoid Hormone Methyl Farnesoate is a Sex

6 Determinant in the Crustacean Daphnia magna. Journal of Experimental Zoology. 293(7):736-

7 739.

8

9 Putt, AE, and Cafarella, MA. 2006. Mysid (Americamysis bahia) Two-Generation Test 

10 Demonstration [fenoxycarb, fipronil, and 3,5-DCP]. Washington DC, U.S. EPA EDSP Final 

11 Report prepared by Springborn Smithers Laboratory for Battelle Marine Sciences Laboratory. 

12 EPA Contract 68-W-01-023. Work Assignment 5-10. Project 44598. Study Numbers 13784.6104, 

13 6105, and 6106. 242 pages.

14 See 13888.6109F (Two final reports combined in one elec. file).

15

16 Quirt, J, and Lasenby, D. 2002. Cannibalism and ontogenetic changes in the response of the 

17 freshwater shrimp Mysis relicta to chemical cues from conspecific predators. Canadian journal 

18 of zoology. 80(6):1022-1025.

19

20 Raimondo, S, and McKenney, CL. 2005a. Projecting population-level responses of mysids 

21 exposed to an endocrine disrupting chemical. Integrative and Comparative Biology. 45(1):151-

22 157.

23

24 Raimondo, S, and McKenney, CL. 2005b. Projected population‐level effects of thiobencarb 

25 exposure on the mysid, Americamysis bahia, and extinction probability in a concentration‐decay 

26 exposure system. Environmental toxicology and chemistry. 24(3):564-572.

27

28 Renberg, L, Svanberg, O, Bengtsson, B-E, Sundström, G. 1980. Chlorinated guaiacols and 

29 catechols bioaccumulation potential in bleaks (Alburnus alburnus, Pisces) and reproductive and 

30 toxic effects on the harpacticoid Nitocra spinipes (Crustacea). Chemosphere 9, 143-150.

31

32 SAB, SAP. (1999). Review of the Endocrine Disruptor Screening Program by a Joint 

33 Subcommittee of the Science Advisory Board and the Scientific Advisory Panel. U.S. 

34 Environmental Protection Agency. EPA-SAB-EC-99-013.

35

36 Shapiro, SS, and Wilk, MB. 1965. An analysis of variance test for normality (complete samples). 

37 Biometrika, 52(3/4), 591-611.

38

39 Snedecor, GW and Cochran, WG. 1980. Statistical methods, Ames, Iowa: Iowa State University 

40 Press.

41

42 Stephan CE. 1977. Methods for calculating an LC50. Proceedings 1st annual symposium on 

43 aquatic toxicology. Aquatic Toxicology and Hazard Evaluation. Ed: Mayer F L, Hamelink J L, 

44 ASTM, 1977, STP 634, 65-84. 

45
DRAFT

Invertebrate Two-Generation ISR 85 May 28, 2013

1 Stringer, TJ, Glover, CN, Keesing, V, Northcott, GL, and Tremblay, LA. 2012. Development of 

2 a harpacticoid copepod bioassay: Selection of species and relative sensitivity to zinc, atrazine 

3 and phenanthrene. Ecotoxicology and Environmental Safety 80: 363–371. 9 pages.

4

5 Størrup, JG, Richardson, K, Kierkegaard, E, Pihl, NJ. 1986. The cultivation of Acartia tonsa 

6 DANA for use as a live food source for marine fish larvae. Aquaculture. 52:87-96.

7

8 Subramoniam, T. 2000. Crustacean ecdysteroids in reproduction and embryogenesis. 

9 Comparative Biochemistry and Physiology C. 125:135-156.

10

11 Tarkpea, M, Eklund, B, Linde, M, Bengtsson, B-E. 1999. Toxicity of conventional, elemental 

12 chlorine-free, and totally chlorine-free kraft-pulp bleaching effluents assessed by short-term 

13 lethal and sublethal bioassays. Environmental Toxicology and Chemistry. 18(11):2487-2496.

14

15 Tarrant, AM, Behrendt, L, Stegeman, JJ, and Verslycke, T. 2011. Ecdysteroid receptor from the 

16 American lobster (Homarus americanus): EcR/RXR isoform cloning and ligand-binding 

17 properties. General and Comparative Endocrinology. 173(2):346-355.

18

19 Tatarazako, N, Oda, S, Watanabe, H, Morita, M, and Iguchi, T. 2003. Juvenile Hormone

20 Agonists Affect the Occurrence of Male Daphnia. Chemosphere. 53(8):827-833.

21

22 Touart, LW. 1982. The effects of diflubenzuron on molting and regeneration of two estuarine 

23 crustaceans, the mysid shrimp Mysidopsis bahia and the grass shrimp Palaemonetes pugio. 

24 University of West Florida, Pensacola, FL.

25

26 Turpen, S, Hunt, JW, Anderson, BS, and Pearse, JS. 1994. Population Structure, Growth, and 

27 Fecundity of the Kelp Forest Mysid Holmesimysis costata in Monterey Bay, California. Journal 

28 of Crustacean Biology. 14(4):657-664.

29

30 Ustach, JF. 1979. Effects of sublethal oil concentrations on the copepod, Nitocra affinis.

31 Estuaries. 2(4), 273-276

32

33 US EPA. 1988. Short-term methods for estimating the chronic toxicity of effluents and receiving 

34 waters to marine and estuarine organisms. US Environmental Protection Agency, Office of 

35 Research and Development. EPA-600/4-87/028. 

36

37 US EPA. 2006. U.S. EPA Peer Review Handbook. EPA/100/B-06/002.

38

39 US EPA. 2011. Office of Chemical Safety and Pollution Prevention, Endocrine Disruptor 

40 Screening Program. "Weight of Evidence: Evaluating Results of EDSP Tier 1 Screening to 

41 Identify the Need for Tier 2 Testing." 47p.

42

43 Vandenbergh, GF, Adriaens, D, Verslycke, T, and Janssen, CR. 2003. Effects of 17α44 ethinylestradiol on Sexual Development of the Amphipod Hyalella azteca. Ecotoxicology and 

45 Environmental Safety. 54(2):216-222.

46
DRAFT

Invertebrate Two-Generation ISR 86 May 28, 2013

1 Veauvy, C, Roberts, E, and Lyons, BP. 2012. Determination of the effect of lindane on the 

2 survival and reproduction of the marine copepod Amphiascus tenuremis (semi static procedure 

3 with individually contained animals). Final Report. Sponsored by U.S. EPA through RTI 

4 International, Research Triangle Park, North Carolina. Prepared by Cefas Weymouth Laboratory, 

5 Weymouth, Dorset, UK. Protocol No. P0603. Contract Report C5069. 130 pages.

6

7 Verslycke T, De Wasch K, De Brabander HF, Janssen CR. 2002 Testosterone metabolism in the 

8 estuarine mysid Neomysis integer (Crustacea; Mysidacea): identification of testosterone 

9 metabolites and endogenous vertebrate-type steroids. Gen Comp Endocrinol. 126:190–199.

10

11 Verslycke, T, Fockedey, N, McKenney, CL, Roast, SD, Jones, MB, Mees, J, Janssen, CR. 2004a. 

12 Mysid crustaceans as potential test organisms for the evalution of environmental endocrine 

13 disruption: a review. Environ Toxicol Chem. 23(5):1219–1234.

14

15 Verslycke, T, Poelmans, S, De Wasch, K, De Brabander, HF, Janssen, CR. 2004b. Testosterone 

16 and energy metabolism of Neomysis integer following exposure to endocrine disruptors. Environ 

17 Toxicol Chem. 23(5):1289–1296.

18

19 Verslycke T, Poelmans S, De Wasch K, Vercauteren J, Devos C, Moens L, Sandra P, De 

20 Brabander HF, Janssen CR. 2003a. Testosterone metabolism in the estuarine mysid Neomysis 

21 integer (Crustacea: Mysidacea) following tributyltin exposure. Environ Toxicol Chem. 22:2030–

22 2036.

23

24 Verslycke T, Vangheluwe M, Heijerick D, De Schamphelaere K, Van Sprang P, Janssen CR.

25 2003b. The toxicity of metal mixtures to the estuarine mysid Neomysis integer under changing 

26 salinity. Aquat Toxicol. 64:307–315.

27

28 Verslycke T, Ghekiere A, Raimondo S, Janssen C. 2007. Mysid crustaceans as standard models 

29 for the screening and testing of endocrine-disrupting chemicals. Ecotoxicology. 16: 205-219.

30

31 Voipio, A. (ed.) 1981. The Baltic Sea. Elsevier, Amsterdam.

32

33 Watts, MW, Pascoe, D, and Carroll, K. 2001. Survival and Precopulatory Behaviour of

34 Gammarus pulex Exposed to Two Xenoestrogens. Water Research. 35(10):2347-2352.

35

36 Watts, MW, Pascoe, D, and Carroll, K. 2002. Population Responses of the Freshwater

37 Amphipod Gammarus pulex (L.) to an Environmental Oestrogen 17α-ethinyloestradiol.

38 Environmental Toxicology and Chemistry. 21(2):445-450.

39

40 Wilson, EO (ed.). 1988. Biodiversity. National Academy Press. Washington DC.

41

42 Winkler G, Greve W. 2002. Laboratory studies of the effect of temperature on growth, moulting 

43 and reproduction in the co-occurring Mysids Neomysis integer and Praunus flexuosus. Mar Ecol 

44 Prog Ser 235:177-188.

45
DRAFT

Invertebrate Two-Generation ISR 87 May 28, 2013

1 Yokota H, Sudo Y, Yakabe Y. 2005. Development of an in vitro binding assay with ecdysone 

2 receptor of mysid shrimp. In: Proceedings of the 26th annual meeting of the society of 

3 environmental toxicology and chemistry, Baltimore, MD.

4

5 Zou, EM. 2005. Impacts of xenobiotics on crustacean molting: the invisible endocrine disruption. 

6 Integr Comp Biol. 45:33–38.

7

8
DRAFT

Invertebrate Two-Generation ISR 88 May 28, 2013

1 8 LIST OF APPENDICES

2

3 APPENDIX A. VALIDATION OF SCREENING AND TESTING ASSAYS 

4 PROPOSED FOR THE EDSP (DRAFT JULY 2007)

5 APPENDIX B. DETAILED REVIEW PAPER ON AQUATIC ARTHROPODS IN 

6 LIFE CYCLE TOXICITY TESTS (OECD NO. 55, JULY 2006)

7 APPENDIX C. REPORTS ON THE INTER-LABORATORY VALIDATION 

8 TRIALS FOR THE MITT

9 C.1 Inter-laboratory Statistical Analysis [Mysid Data Across All Studies], 2013

10

11 C.2 Mysid Study with Vinclozolin (100.02), University of North Texas, 2012

12

13 C.3 DRAFT Mysid Study with Lindane (100.01), University of North Texas, 2012

14

15 C.4 Mysid Studies with Lindane and 4-tert-Octylphenol (185A-102-1, -2A), Wildlife 

16 International, 2012

17

18 C.5 Mysid Studies with Lindane and 4-tert-Octylphenol (13888.6109, 6111), Smithers 

19 Viscient, 2012

20

21 APPENDIX D. REPORTS ON THE INTER-LABORATORY VALIDATION 

22 TRIALS FOR THE HCDRT

23 D.1 Report of Progress on the Interlaboratory Validation of the OECD Harpacticoid Copepod

24 Development and Reproduction Test (OECD No. 158, August 2011)

25

26 D.2 Validation Report of the Full Life Cycle Test with the Harpacticoid Copepods Nitocra 

27 spinipes and Amphiascus tenuiremis and the Calanoid Copepod Acartia tonsa (OECD 

28 No. 79, August 2007)

29

30 D.3 Copepod Study with Lindane (27569), Block Environmental Services, 2012

31

32 D.4 Copepod Study with Vinclozolin (27568), Block Environmental Services, 2012

33

34 D.5 Copepod Study with Lindane (BR0476/B), Brixham, 2011

35
DRAFT

Invertebrate Two-Generation ISR 89 May 28, 2013

1 D.6 Copepod Study with Lindane (C5069), Cefas, 2011

2

3 APPENDIX E. MYSID TWO-GENERATION TEST GUIDELINE (DRAFT 

4 PROPOSAL FOR A STANDARDIZED PROTOCOL AS AN IN VIVO TIER 2 

5 TEST), APRIL 2013

6 APPENDIX F. HARPACTICOID COPEPOD DEVELOPMENT AND 

7 REPRODUCTION TEST WITH AMPHIASCUS TENUIREMIS (STANDARDIZED 

8 OECD DRAFT PROTOCOL FOR THE HCDRT AS A POTENTIAL 

9 ALTERNATIVE TO THE MTTT), MAY 2009

10 APPENDIX G. INTRA-LABORATORY STATISTICAL ANALYSIS REPORTS 

11 FOR TWO-GENERATION INVERTEBRATE STUDIES

12 G.1 Intra-laboratory statistical analysis for Mysid Lindane Study at Smithers Viscient, 2013

13

14 G.2 Intra-laboratory statistical analysis for Mysid Octylphenol Study at Smithers Viscient, 

15 2013

16

17 G.3 Intra-laboratory statistical analysis for Mysid Lindane Study at University of North Texas, 

18 2013

19

20 G.4 Intra-laboratory statistical analysis for Mysid Vinclozolin Study at University of North 

21 Texas, 2013

22

23 G.5 Intra-laboratory statistical analysis for Mysid Octylphenol Study at Wildlife International, 

24 2013